INTRODUCTION Wheatgrass is young grass, most often of the common wheat plant Triticum aestivum. In the 1930s, Dr. Charles Schnabel, who has been called the "father of wheatgrass", began documenting a wide range of its health benefits, based on his observations in animals and humans. Schnabel patented a dietary supplement derived from the dehydration of young wheatgrass shoots, which was marketed until the 1950s. In the 1970s, Ann Wigmore renewed the popularity of wheatgrass. Based on her personal health experience, Wigmore wrote books and lectured on the benefits of wheatgrass, as part of a raw/living food diet. Only some of the many claimed health benefits of wheatgrass have been scientifically investigated. While a number of components of wheatgrass have demonstrated positive effects, the added value encompassed by the substance as an entity is not clear. We start this review with an examination of the qualities of the components of wheatgrass, followed by a discussion of the forms of wheatgrass and their characteristics. Next, we review the evidence for the health benefits of wheatgrass, from in vitro *Address correspondence to this author at the Integrated Oncology and Palliative Care Unit, Rambam Health Care Campus, POB 9602, Haifa 31096, Israel; Tel: 972-4-7773128; Fax: 972-4-7773168; E-mail: g_barsela@rambam.health.gov.il experiments, experiments in animals, and clinical trials.

INTRODUCTION Wheatgrass is young grass, most often of the common wheat plant Triticum aestivum. In the 1930s, Dr. Charles Schnabel, who has been called the "father of wheatgrass", began documenting a wide range of its health benefits, based on his observations in animals and humans. Schnabel patented a dietary supplement derived from the dehydration of young wheatgrass shoots, which was marketed until the 1950s. In the 1970s, Ann Wigmore renewed the popularity of wheatgrass. Based on her personal health experience, Wigmore wrote books and lectured on the benefits of wheatgrass, as part of a raw/living food diet. Only some of the many claimed health benefits of wheatgrass have been scientifically investigated. While a number of components of wheatgrass have demonstrated positive effects, the added value encompassed by the substance as an entity is not clear. We start this review with an examination of the qualities of the components of wheatgrass, followed by a discussion of the forms of wheatgrass and their characteristics. Next, we review the evidence for the health benefits of wheatgrass, from in vitro *Address correspondence to this author at the Integrated Oncology and Palliative Care Unit, Rambam Health Care Campus, POB 9602, Haifa 31096, Israel; Tel: 972-4-7773128; Fax: 972-4-7773168; E-mail: g_barsela@rambam.health.gov.il experiments, experiments in animals, and clinical trials.

 While healing qualities have also been attributed to the topical application of wheatgrass extract [1], the scope of this review is limited to oral administration. At the end, we raise issues to be considered in the clinical realm and in future research. COMPONENTS OF WHEATGRASS AND THEIR HEALTH BENEFITS Chlorophyll, which has been referred to as "living food" and as "green blood", is the primary nutrient in fresh wheatgrass. In addition, flavonoids, a large variety of vitamins including vitamins C and E, choline, minerals, enzymes, indoles, and a number of amino acids are considered to be responsible for the health benefits claimed. The alkaline pH of 7.4 is also considered an asset. Among the qualities attributed to chlorophyll are the promotion of cancer prevention, protection against side effects of cancer treatment, and contribution to a positive hematological status. Extracts from wheat sprout roots and leaves were shown to inhibit metabolic activity of carcinogens, and chlorophyll was identified as the active substance [2]. Chorophyllin, a derivative of chlorophyll, was shown to protect mitochondria against oxidative damage [3] and to induce the activity of mammalian phase 2 proteins that protect cells against oxidants and electrophiles [4]. This 2 Mini-Reviews in Medicinal Chemistry, 2015, Vol. 15, No. 0 Bar-Sela et al. anti-oxidant activity has implications in cancer prevention [5].

 The capability of chlorophyll to protect mitochondrial membranes against gamma-radiation [6] may be beneficial during cancer treatment. The structural similarity between chlorophyll and hemoglobin, which differs by the inclusion of magnesium and iron, respectively, may explain observed hematological benefits of chlorophyll-rich substances, although a mechanism of such has not been elucidated. Wheatgrass is also rich in flavonoids, particularly apigenin, which is known for its functions in anti-oxidation [7]; in anti-inflammation [8] by inhibiting cytokine-induced leukocyte adhesion [9]; in anti-carcinogenesis [10] by antiproliferative and proapoptotic activities [11] through regulation of signaling pathways such as PI3K/Akt/mTOR [12] and MAPK [13]; and in cardiovascular protection by inducing endothelial derived hyperpolarizing factor (EDHF)- mediated vascular dilatation [14]. Vitamin C (ascorbic acid), a component of fresh wheatgrass, has been associated with the prevention and treatment of cancer through a number of mechanisms that relate to antioxidant and pro-oxidant properties, stimulation of the immune system, altering carcinogen metabolism, enhancement of collagen synthesis, and interference with cancer cell signaling [15]. The possibility that antioxidant agents may interfere with chemotherapy necessitates the specific investigation of each agent and the timing of its administration. When received simultaneously with chemotherapy, vitamins C and E were found to restore antioxidant status that had decreased due to breast cancer and its treatment, and to reduce DNA damage [16]. Choline and indoles are other components of wheatgrass that have had anti-cancer activity attributed to them [17,18].

 Phytochemical screening and gas chromatography-mass spectrometry analysis has verified, in the aqueous extract of wheatgrass: flavonoids, triterpenoids, anthraquinol, alkaloids, tannins, saponins, sterols, squalene, caryophyllene and amyrins, many of which have demonstrated anti-oxidant activity [19,20]. Oligosaccharides isolated from wheatgrass were found to stimulate the expression of inflammatory cytokines in human peripheral blood mononuclear cells [21]. The oligosaccharide, maltoheptaose, is the first immunostimulatory component of wheatgrass to be identified. Maltoheptaose was shown to activate monocytes via Toll-like receptor 2 (TLR-2) signaling. WHEAT, WHEATGRASS, WHEATGRASS DERIVATIVES The qualitative and quantitative composition of wheatgrass differs from that of other wheat products. For example, in the passage from grains to sprouts to wheatgrass, the quantity of flavonoids increases [22]. Table 1 shows the composition of a sample of fresh wheatgrass [23]. Nevertheless, the quantities of nutrients in wheatgrass products may vary according to growing conditions, such as soil composition, climate (amount of sunlight, temperature, presence of snow), duration of growth period, and height at harvest. Wheatgrass grown in soil with nutrients was found to have a higher level of ascorbic acid than wheatgrass grown in soil with tap water, or grown in tap water with nutrients [24]. Regardless of the growing medium, quantities of flavonoids and ascorbic acid were shown to increase over a 15-day growth period [24]. Table 1. Nutrient comparison of 1 oz (28.35 g) of wheatgrass juice [23].

 Nutrient Quantity Protein 860 mg Beta-carotene 120 IU Vitamin E 880 mcg Vitamin C 1 mg Vitamin B12 0.30 mcg Phosphorus 21 mg Magnesium 8 mg Calcium 7.2 mg Iron 0.66 mg Potassium 42 mg Wheatgrass can be consumed in a number of forms: fresh juice, frozen juice, tablets, and powders. Aqueous and ethanol extracts are available, following freeze drying or oven drying. The nutritional composition of the products varies according to the processes of their production, as well as the growing conditions of the wheatgrass from which they were derived. At least one commercial producer of tablets (http://www.wheatgrass.com) continues to grow wheatgrass under the same conditions implemented by Charles Schnabel during the 1930s: slow growth during the winter and harvest in the spring. The claim is that outdoor cultivation increases the chlorophyll content, while faster growth, on trays, as done by Ann Wigmore, results in more sugars and less fiber. On the other hand, the cultivation of wheatgrass without geographical or seasonal restrictions enables the widespread and continuous availability of fresh juice. Frozen wheatgrass juice (WGJ) is more convenient than fresh juice, yet was found to contain a 20% lower level of amino acids [25]. 

Ethanol extracts of wheatgrass were found to have higher phenolic and flavonoid content than aqueous extracts [24]. In a study that compared quantities of nutrients in 7-day old fresh, freeze dried and oven dried wheatgrass [26], fresh wheatgrass samples had the highest amount of ascorbic acid and chlorophyll, but the lowest amount of total flavonoids and phenolics. Ethanolic extract from freeze-dried wheatgrass had the highest level of ferric-reducing antioxidant power assay and the lowest α-tocopherol value. Freeze-dried wheatgrass samples exhibited the highest activity of 2,2-diphenyl-1-picrylhydrazyl scavenging ability. Medical Use of Wheatgrass Mini-Reviews in Medicinal Chemistry, 2015, Vol. 15, No. 0 3 Commercial wheatgrass products standardize at least some of the components. Fermented wheat germ extract (FWGE) is a concentrated extract derived from the endosperm of the wheat plant, and is sold as a dietary supplement under the trade name fwge. Fermentation with baker's yeast during preparation concentrates biologically-active benzoquinones. Inspiration for the development of FWGE came from the work of Albert SzentGyorgyi, the Nobel laureate who discovered vitamin C, and who proposed that the combination of vitamin C with methoxy-substituted benzoquinones could help treat cancer. Máté Hidvégi, the Hungarian chemist who invented FWGE in the early 1990s, aimed to develop wheat germ extracts with high and standardized levels of benzoquinone. 

The health benefits attributed to FWGE are primarily in the realm of cancer, as adjunct care against the disease and in the reduction of the side effects of its treatment. Animal experiments and clinical trials do not report any adverse events of FWGE , nor do toxicity studies in which dosages by body weight of 25-fold the recommendation for oral use were administered to rats [27]. HEALTH BENEFITS OF WHEATGRASS Early evidence of the health benefits of wheatgrass comes from anecdotal reports. Numerous in vitro studies have been conducted in recent years, particularly on the standardized FWGE, FWGE . Several animal studies have been performed, as well as a few clinical trials. Here we present central findings from studies published in the English language. We note that several studies that were presented at conferences were not published as articles, raising the possibility of publication bias. LABORATORY (IN VITRO) EXPERIMENTS Laboratory experiments have been conducted on the involvement of wheatgrass and its derivatives, particularly FWGE, in immunological, anti-oxidative, and anti-cancer activities. Both immunological and anti-oxidative activities have been investigated in the search for mechanisms of anticancer activity. Both the cancer disease and its treatment are known to impair the immunological and oxidative status of patients. 

Anti-Cancer Effect Through Immunological Activity We present two examples by which immunological effects of FWGE may impede the activity of tumor cells. In the first example, FWGE was shown to downregulate cell surface MHC class-I proteins of malignant T- and B-cells, yet not affect healthy peripheral blood mononuclear cells [28]. Overexpression of MHC-1 protects tumor cells from attack by natural killer (NK) cells. Thus, the selective downregulation by FWGE of MHC-1 proteins may reduce metastatic activity by decreasing the defense of tumor cells against natural killer (NK) cells. In the second example, FWGE was shown to induce the synthesis of ICAM-1 [29]. The decreased expression of ICAM-1 protein on the endothelial cells of solid tumor vessels inhibits the passage of leukocytes through the vessel membrane. Thus, FWGE may increase the exposure of tumor cells to leukocyte infiltration. In the same study, FWGE was shown to stimulate the immune system, by means of exerting a synergistic effect on proinflammatory cytokine production, including tumor necrosis factor-alpha, a key anticancer cytokine. High concentrations of FWGE inhibited the proliferation and survival of myeloid, though not lymphoid cells. 

Anti-Cancer Effect Through Apoptosis Several experiments have identified induction of apoptosis as a mechanism of the anti-cancer activity of wheatgrass. Wheatgrass extract was found to induce apoptosis of MCF-7 breast cancer cells [30,31], human acute promyelocytic leukemia cells [32], and HeLa cervical cancer cells [31]. Apoptosis was also determined to be a mechanism by which FWGE inhibits the growth of gastric carcinoma cells [33], human lymphoma cells [34], human colon carcinoma cells [35], human ovarian cancer cells [36], and a broad spectrum of 32 human cancer cell lines, of which the highest activity was found in neuroblastoma cell lines [37]. Induction of apoptosis by FWGE was shown to be selective, occurring in leukemic human cells, but not in healthy, peripheral blood mononuclear cells [38]. One mechanism of FWGE-induced apoptosis appears to involve the Poly (Adenosine diphosphate ribose) Polymerase (PARP) enzyme. PARP promotes DNA repair in cancer cells; thus, its cleavage results in apoptosis of these cells. FWGE was shown to induce apoptosis of leukemia and hepatocellular carcinoma cells by activating cleavage of the PARP enzyme [38,39]. FWGE stimulates the PARP pathway by regulating metabolic enzymes in the glycolysis and pentose cycles of cancer cells [38]. The application of FWGE to pancreatic adenocarcinoma cells decreased glucose uptake and increased glucose oxidation and ribose recycling in the pentose cycle, in a dose-dependent manner [40].

 Increased pentose cycle activity leads to an increase in superoxide dismutase scavenger activity, as well as increased lipid synthesis from glucose, which promotes cell differentiation and protects against oxidative stress. Wheatgrass has demonstrated effectiveness as a radical scavenger also in antioxidant assays [20]. FWGE was shown to enhance the effect of tamoxifen on MCF-7 breast cancer cells in vitro, by enhancing apoptotic activity [4]. Tamoxifen combined with FWGE significantly increased apoptosis, while tamoxifen alone showed no effect. The estrogen-receptor activity of MCF-7 cells increased with FWGE, decreased with tamoxifen, and decreased further with tamoxifen and FWGE combined [41], reflecting the synergistic effect between FWGE and tamoxifen. Other Possible Mechanisms of an Anti-Cancer Effect Other mechanisms that have been proposed for the anticancer potential of FWGE include decreasing cell motility [38] and inhibition of cyclooxygenase (COX) -2 [35], which is overexpressed in 80-85% of adenocarcinomas [42]. Gene expression data identified 2,142 genes in ovarian cancer cell lines, representing 27 biological pathways that were significantly associated with FWGE sensitivity [36]. 4 Mini-Reviews in Medicinal Chemistry, 2015, Vol. 15, No. 0 Bar-Sela et al. Wheatgrass Derivatives Combined with Cancer Drugs In vitro experiments of the combined treatment of FWGE with chemotherapy drugs show that the effects depend on the type of cancer, the specific cancer drug, and the timing of application. An aqueous wheatgrass extract was found to enhance the effect of cisplatin on MCF-7 breast and HeLa cervical cancer cells [31].