Pharmacological effects and underlying mechanisms of LBPs
A number of preclinical and a few clinical studies on the pharmacological activities and possible mechanisms of LBPs have been reported in the literature (Tables 1 and 2). LBPs exhibit a wide array of therapeutic/medicinal effects on aging, fatigue, cancer, colitis, stroke, diabetes, Alzheimer’s disease (AD), and glaucoma in different animal models.

Anti-aging and antioxidant effects
Goji berries have long been used in Oriental medicine as a potent anti-aging agent. Aging is a progressive deterioration of physiological function that impairs the ability of an organism to maintain homeostasis and consequently increases the organism’s susceptibility to disease and death.10 The aging of the immune system (immunosenescence) is associated with dramatic reduction in immune responsiveness as well as functional dysregulation. This translates into less-effective innate and adaptive immune responses, increased reactivity against self-antigens (autoimmunity), and decreased incidences of infectious diseases and cancer.11 Oxidative damage of biomolecules increases with age and is postulated to be a major causal factor of various degenerative disorders.12,13 Oxidative stress is a condition under which increased production of free radicals, reactive species (including singlet oxygen and reactive lipid peroxidation products, such as reactive aldehydes and peroxides), and oxidant-related reactions occur, which result in cellular and organ damage. Free radical scavengers or antioxidants play an important role in retarding biological aging. Consequently, the concept of anti-aging by antioxidants such as LBPs has been supported by a line of evidence. Oxidative stress has been incriminated as one of several mechanisms that induce toxic effects in different organs due to enhanced production of oxygen free radicals and is considered as a major risk factor that contributes to increased lipid peroxidation and reduced antioxidants in aging and aging-related degenerative diseases.12,13

Zebrafish
Cellular senescence can be triggered by a number of factors including aging, DNA damage, oncogene activation, and oxidative stress. Senescence represents a stress response in which cells withdraw from the cell cycle and lose the capability to proliferate in response to growth factors or mitogens. Senescent cells show increased expression of recognized biomarkers of senescence, including staining for β-galactosidase at pH of 6.0 (senescence-associated-β-gal [SA-β-gal]), decreased replicative capacity, and increased expression of p53, p21, p16, and other cyclin-dependent kinase inhibitors, such as p27 and p15.14 p53, a tetrameric transcription factor and tumor suppressor, regulates cell-cycle control, DNA repair, apoptosis, cellular senescence, and cellular stress responses. p53 can promote or inhibit senescence.14 p21 is the first identified downstream target of p53, and it is an essential mediator of p53-dependent cell-cycle arrest. In a recent study, Xia et al15 explored the mechanisms of action of LBPs by phenotypic and SA-β-gal assays, evaluated the survival rates in vivo, and determined expression profiling of genes related to the p53 signaling pathway in a zebrafish model. Zebrafish embryos were continuously exposed to various concentrations of LBPs (1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, and 4.0 mg/mL) for 3 days. The results of fluorescent acridine orange and SA-β-gal staining indicated that cell apoptosis and senescence mainly occurred in the head at 24 hours and 72 hours post-fertilization. In addition, resistance to replicative senescence was observed at low doses of LBPs, especially at the 3.0 mg/mL concentration.15 Furthermore, the expression of genes that relate to aging, such as p53, p21, and Bax, was decreased, while Mdm2 (a p53-specific E3 ubiquitin ligase acting as the principal cellular antagonist of p53) and telomerase reverse transcriptase genes were upregulated by LBPs. The results indicate that the beneficial effects of LBPs on cell apoptosis and aging might be mediated by the p53-mediated signaling pathway (Figure 2).

Mice and rats
The effect of LBPs on age-induced oxidative stress in different organs of aged (20 months) Kunming mice was investigated by Li et al.16 LBPs were extracted from Goji fruits purchased from Jinghe County herb market, Xinjiang, People’s Republic of China, and the amount of the polysaccharides was found to be 97.54% by phenol-sulfuric acid method. The mice were treated with 200 mg/kg, 350 mg/kg, or 500 mg/kg body weight LBPs by gastric gavage for 30 days. The study showed that increased endogenous lipid peroxidation, and decreased antioxidant activities in the lungs, liver, brain, and heart, as assessed by superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant capacity (TAOC), and immune function as determined by measuring thymus and spleen index, phagocytic index, and phagocytic activity were observed in aged mice and restored to normal levels in LBP-treated mice.16 The level of lipofuscin, an important marker for oxidative damage, in various organs was increased in aged mice and suppressed by LBPs. In contrast, the level of malondialdehyde (MDA) in blood and other organs was significantly increased in aged mice compared to young mice, and the high level of MDA was brought down by LBP treatment.16 The inhibitory effect of LBPs on lipid peroxidation in aged mice might be, at least in part, attributed to the influence on the antioxidant enzymes and non-enzymatic system. These findings demonstrate that LBPs can promote the antioxidative enzymes and immune functions that are suppressed in aging and thereby decrease the risk of lipid peroxidation accelerated by age-induced generation of free radicals.
Li17 also reported that administration of 50 mg/kg, 100 mg/kg, or 200 mg/kg body weight LBPs by oral gavage for 30 days restored abnormal oxidative capacity to almost normal levels in streptozotocin-induced diabetic Wistar rats. LBPs were extracted from dry fruits of Goji berries in boiling water. The polysaccharides consisted of D-rhamnose, D-xylose, D-arabinose, D-fucose, D-glucose, and D-galactose with molar ratio of 1:1.07:2.14:2.29:3.59:10.06 and linked together by β-glycosidic linkages. Diabetes was induced by a single intraperitoneal injection of 50 mg/kg body weight streptozotocin. Results showed that the activities of blood and liver antioxidant enzymes (SOD, GPx, CAT, and glutathione reductase [GR]) and glutathione (GSH) level in diabetic rats were significantly decreased, and MDA level significantly increased compared to normal control rats. Administration of 50–200 mg/kg LBPs for 30 days significantly increased the activities of these antioxidant enzymes and decreased MDA level in diabetic rats compared to the model group.17
The modulatory effect of LBPs on oxidative stress was also investigated, in Kunming mice fed with high-fat diet for 2 months, by Wu et al.18 Mice were treated orally with 50 mg/kg, 100 mg/kg, or 150 mg/kg body weight of LBPs once every day for 2 months. The results showed that the activities of blood and hepatic antioxidant enzymes (SOD, GPx, and CAT) and the GSH level in model mice significantly decreased, and blood and hepatic MDA and nitric oxide (NO) levels significantly increased compared to normal control mice. Administration of LBPs dose-dependently and significantly increased the activities of antioxidant enzymes and decreased MDA level in mice compared to the model group.18
Niu et al19 explored the modulatory effect of LBPs on exercise-induced oxidative stress in skeletal muscles in male Wistar rats. The exhaustive exercise experimental rats underwent a 30-day exhaustive exercise program. Rats were also treated orally with 100 mg/kg, 200 mg/kg, or 300 mg/kg body weight LBPs once per day for 1 month. This model of experimental exhaustive exercise promoted oxidative stress in skeletal muscle tissues of rats, with decreased muscle glycogen content, decreased SOD and GPx activity, and increased MDA level and creatine kinase (CK) activity in skeletal muscle tissues of exhaustive exercise animals.19 The results showed that LBPs administration dose-dependently decreased the oxidative stress induced by the exhaustive exercise with increased SOD and GPx activity and reduced MDA level in skeletal muscles.19
Some metabolic products that can damage cell member lipid are generated during the process in which galactose is reduced into galactitol, such as the increases of lipid peroxidation and lipofuscin, finally leading to body aging.20 Continuous injection of D-galactose in animals will inevitably cause glucose metabolism disorder, thereby causing abnormal metabolism of heart, liver, kidney, brain, and other important organs. D-galactose-induced mouse-aging model has been used to test the anti-aging capacity of LBPs. Results showed that LBPs increased SOD, CAT, and GPx levels in blood and reduced MDA level. They also improved SOD activity of skin and reduced MDA content of skin.21 A similar effect was observed with LBPs in another study.22 Their mechanism may be related to the alleviation of glucose metabolism disorder and the resistance of the generation of lipid peroxide and other substances, which damage cell membrane lipids. Another study investigated the inhibiting effects of LBPs on non-enzymatical glycation in D-galactose-induced mouse-aging model.23 The lymphocyte proliferation and interleukin (IL)-2 activity, learning and memory abilities, and SOD activity of erythrocytes were enhanced by LBPs.23

Human umbilical vein endothelial cells
Liu et al24 examined the effects of LBPs on angiotensin II-induced senescence of human umbilical vein endothelial cells (HUVECs) and the role of p53 and p16 in such effects. HUVECs were treated with 1×106 mM angiotensin II to induce cell senescence, which was identified using SA-β-gal staining. Flow cytometry was used for analyzing the cell cycle changes, and the cell viability was assessed. LBPs treatment of angiotensin II-exposed cells resulted in decreased β-gal-positive cells with a reduction in G0/G1 phase cells and an increase in S phase cells.24 It also increased the cell viability and significantly decreased the expression levels of p53 and p16 (both tumor suppressors and senescence regulators) in HUVECs. These results demonstrate that LBPs can delay angiotensin II-induced aging of HUVECs possibly by downregulating the expression of p53 and p16. The p16-mediated senescence acts through the retinoblastoma pathway inhibiting the action of the cyclin-dependent kinases leading to G1 cell cycle arrest.25 Retinoblastoma is maintained in a hypophosphorylated state resulting in the inhibition of transcription factor E2F1.

Clinical studies in healthy volunteers
Amagase and Nance26 performed a randomized, double-blind, placebo-controlled, clinical study to investigate the general effects of use of a standardized L. barbarum juice (GoChi) for 14 days in healthy Chinese subjects. GoChi was produced from fresh ripe LBFs grown in the People’s Republic of China. As a finished product, the juice contains 1,632 mg/day serving (120 mL, ie, 13.6 mg/mL) of LBPs. The placebo matched the color, flavor, and taste of GoChi in a formulation of sucralose (10 mg), artificial fruit flavor (30 mg), citric acid (60 mg), and caramel color (12 mg) in 30 mL of purified water. The effects of GoChi were examined by questionnaire subjective ratings (0–5) of general feelings of well-being, neurologic/psychologic traits, gastrointestinal, musculoskeletal, and cardiovascular complaints as well as any adverse effects. Body weight, body mass index, blood pressure, pulse rate, and visual acuity were also measured before and after consuming 120 mL of GoChi/day or placebo control solution.26 Significant differences between day 1 and day 15 were found in the GoChi group (n=16) with increased ratings for energy level, athletic performance, quality of sleep, ease of awakening, ability to focus on activities, mental acuity, calmness, and feelings of health, contentment, and happiness. GoChi also significantly reduced fatigue and stress, and improved regularity of gastrointestinal function. In contrast, the placebo group (n=18) showed only two significant changes (heartburn and happiness).26 No significant changes in musculoskeletal or cardiovascular complaints were observed in either group. All parametric data (body weight, etc) were not significantly different between groups or between day 1 and day 15 for either group. These results clearly indicate that daily consumption of GoChi for 14 days increases subjective feelings of general well-being and improves neurologic/psychologic performance and gastrointestinal functions.
Amagase et al27 further conducted a randomized, double-blind, placebo-controlled clinical study to examine the antioxidant effects of GoChi in healthy Chinese adults living in Hunan province, People’s Republic of China. In the study, 50 Chinese healthy adults aged 55–72 years were recruited and treated with Goji juice containing 13.6 mg/mL LBPs at a dose of 120 mL/day or placebo (n=25 each group).27 In vivo antioxidant markers including serum levels of SOD, GPx, and lipid peroxidation (indicated by the level of MDA) were determined before and after GoChi or placebo consumption for 30 days. The results showed that GoChi consumption significantly increased serum SOD level by 8.4% and GPx by 9.9%, whereas MDA was significantly decreased by 8.7%.27 There were no dropouts during this 30-day trial. After GoChi or placebo consumption, no abnormalities were seen in subjects’ energy, urine, stools, or other examined physical parameters. These data indicate that chronic GoChi is well tolerated in humans and can promote antioxidant capacity in humans via upregulating antioxidative enzymes.
Four randomized, blind, placebo-controlled clinical trials were pooled to identify the general effects of oral consumption of 120 mL/day GoChi.28 A questionnaire consisting of symptoms graded 0–5 was given to the participants. For each question, the score changes in the questionnaire between pre- and post-intervention were summarized by the standardized mean difference and associated standard error of the mean to perform the meta-analysis. The change was also characterized into a binary outcome, improved or not, to derive odds ratio (OR) and associated standard error of the mean derived by a binary outcome using the Mantel–Haenszel method. The meta-analysis and heterogeneity were evaluated with the R program using the rmeta package. In total, 161 participants (18–72 years old) were included in the meta-analysis. Compared with the placebo group (n=80), the GoChi-treated group (n=81) showed significant improvements in weakness, stress, mental acuity, ease of awakening, shortness of breath, focus on activity, sleep quality, daydreaming, and overall feelings of health and well-being under a random effects model.28 A fixed effects model showed additional improvements in fatigue, depression, circulation, and calmness. The OR indicated significantly higher chance to improve fatigue, dizziness, and sleep quality.28 Three studies had statistically significant heterogeneity in procrastination, shoulder stiffness, energy, and calmness. The meta-analysis confirmed the various health-promoting effects of GoChi in humans.

Summary of the anti-aging and antioxidative effectsof LBPs
In summary, LBPs have shown potent anti-aging and antioxidant activities (Figure 3). They increase SOD, GPx, CAT, and GR activities, thereby inhibiting oxidative stress-induced damage. LBPs ameliorate oxidative stress-induced cellular apoptosis. They can delay angiotensin II-induced aging of HUVECs by downregulating the expression of p53 and p16. In the ischemia/reperfusion (I/R) injuries to heart, LBPs significantly decreased the myocardium lactate dehydrogenase (LDH) level and increased Na+/K+-ATPase and Ca2+-ATPase activities. LBPs ameliorate oxidative stress-induced cellular apoptosis by downregulating Bax and upregulating Bcl-2.