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Anticancer Effect of Spices Used in Mediterranean Diet: Preventive and Therapeutic Potentials

Cancer is one of the leading causes of death worldwide, with almost 10 million cancer-related deaths worldwide in 2020, so any investigation to prevent or cure this disease is very important. Spices have been studied widely in several countries to treat different diseases. However, studies that summarize the potential anticancer effect of spices used in Mediterranean diet are very limited. This review highlighted chemo-therapeutic and chemo-preventive effect of ginger, pepper, rosemary, turmeric, black cumin and clove. Moreover, the mechanisms of action for each one of them were figured out such as anti-angiogenesis, antioxidant, altering signaling pathways, induction of cell apoptosis, and cell cycle arrest, for several types of cancer. The most widely used spice in Mediterranean diet is black pepper (Piper nigrum L). Ginger and black cumin have the highest anticancer activity by targeting multiple cancer hallmarks. Apoptosis induction is the most common pathway activated by different spices in Mediterranean diet to inhibit cancer. Studies discussed in this review may help researchers to design and test new anticancer diets enriched with selected spices that have high activities.


INTRODUCTION
Since ancient times spices and herbs have been extensively used as a food flavoring and traditional medicines (1). Based on history and several current studies, the Mediterranean region has been recognized across generations with a rich reserve of natural medicinal plants (2). As well, the consumption of the main components of the Mediterranean diet has shown a diverse array of health benefits due to the presence of abundant natural phytochemicals (3). Besides, it is believed that using herbs and spices in the traditional Mediterranean diet is associated with emphasizing its medicinal properties and protecting against chronic diseases, including cancer (3,4). According to statistical analysis, the Mediterranean area exhibited a lower incidence rate of different types of cancer compared to other areas of the world (5). Several studies have reported the antioxidant, anti-inflammatory, and immunomodulatory impact of spices, which may be correlated with the prevention and treatment of cancer (1). Moreover, polyphenols are the main bioactive chemical compounds found in spices and culinary herbs (6). The recent research demonstrated the role of dietary polyphenols as powerful antioxidant and anticancer agents along with many medicinal properties (7)(8)(9)(10). It revealed chemo-preventive potency represented by modulation of different processes and biomarkers, such as tumor cell apoptosis, cell cycle progression, inflammation mediators, cell invasion, and metastasis (11). In literature, there are countless spice-derived secondary metabolites that exhibited potential for cancer prevention, however; they are still under research and development (12,13). This review summarized some studies about well-known spices in the Mediterranean diet demonstrating their anticancer effects and mechanisms of action. Studies discussed in this review may provide a solid base for researcher and nutritionists to develop effective anticancer nutrition.

SPICES IN THE MEDITERRANEAN DIET: FLAVOR CHARACTERISTICS AND TRADITIONAL USE
Spices are used in different Mediterranean food recipes to impart aroma, color, and taste to food preparations and sometimes mask undesirable odors (14). Spices refer to the dried part of a plant that contains volatile oils or aromatic flavors such as buds (cloves), bark (cinnamon), root (ginger), berries (black pepper), seeds (cumin, coriander) (15,16). Recently, measurements of dietary intake of spices are gaining much significance as various phytochemicals present in spices, have been recognized to have health-promoting benefits (17). Spices are used in traditional Mediterranean cuisines such as soups, cooked lamb roast, fish preparations, marinades, bouquet garni, baked fish, rice, salads, occasionally with egg preparations, dumplings, vinegar, jams, and marmalades (15).
Spices such as ginger (Zingiber officinale), that gives a refreshing pleasant aroma, biting taste, and carminative property, which make it an indispensable food ingredient in most Mediterranean food recipes (16), is used in different forms such as fresh ginger, dry ginger, ginger powder, ginger oil, and ginger paste to enhance both sweet and savory traditional Mediterranean recipes (18).
Rosemary (Rosmarinus officinalis), an aromatic herb that has been known from ancient times as a memory herb, a native to the Mediterranean from Spain to the Balkans and into North Africa (14,19). At present, rosemary is widely cultivated in Spain, Morocco, Tunisia, France, Algeria, Portugal, and China (20). The fresh and dried leaves of rosemary are used frequently in traditional Mediterranean cuisine as they have a bitter astringent taste and are aromatic, dried, and powdered leaves (21). Some spices are used in small amounts because of their intense flavor, such as clove (Syzygium aromaticum L.), clove used as a whole or ground form or in oil form that is used in a small amount, for example, curry powder uses 2 % (mild) to 3 % (sweet) by weight of ground clove buds (15). Clove oil is one of the most important essential oils used for flavoring all kinds of food products, such as sausage, baked goods (22).
Black Cumin (Nigella sativa L.) is an ancient spice with a mild odor and warm, bitter taste (23). Black cumin is used as a spice in Middle Eastern cuisines. In ancient Egypt, it was used as a preservative in mummification (24). The seeds of black cumin have a pungent bitter taste and aroma and are used as a spice in Middle Eastern cuisines. The dry-roasted nigella seeds flavor curries, vegetables, and pulses. Black cumin is used in food as a flavoring additive in bread and pickles (24,25).
The most popular and the most widely used spice in Mediterranean food is black pepper (Piper nigrum L) (15). Black pepper contributes toward flavor, taste the predominating ones being taste and flavor, and hence pepper is a multifunctional spice (26). Pepper plays an important role in the cuisines of China, South East Asia, Greece, Italy, and France such as meat dishes, fish preparations, soups, and pickles (27). Some spices such as turmeric (Curcuma longa L), is used as color agents, it is made into a yellow powder with a bitter, slightly acrid, yet sweet taste. Fresh spice is much preferred than dried spice in Spain, France, Italy, Greece, Turkey (14,28). In Egypt as early as 3000 BC. cinnamon (Cinnamomum cassia) was used in the Testament of the Bible and there indications (29). Cinnamon is used as a flavoring and coloring agent of the foods. However, it gives a sweet sensation of the food that is enhanced because of the synergetic effect between the sweet taste of sugar and the sweet aroma of cinnamon (16). Moreover, cinnamon makes a tan or brown color for food and it is used in many Mediterranean food recipes such as milk, apple pie, and cinnamon buns (30). Table 1 describes the spices classification and general characteristic.

Mediterranean Plants Used as Food Additives
There is a growing interest in the use food additives from natural sources to improve taste and appearance, preserve flavor and reduce microorganisms' growth. Because the Mediterranean area has high plant species biodiversity, many of its wild plants can be a useful source for natural food additives (31,32). In the following paragraphs selected examples of such plants are discussed. Carex distachya Desf. (Cyperaceae) is an herbaceous plant that is globally distributed in different habitats. It is a steno-mediterranean species and is known with the Italian name "carice mediterranea. Carex genus is known of the presence of high content of stilbene derivatives (32). Additionally, flavonoids, including resveratrol, flavolignans, lignans and terpenes were also isolated from the C. distachya, as well as other unusual metabolites such as feruloyl monoglyceride macrolactones and dibenzoxazepinones. The high contact of polyphenols made this plant a potential source of natural antioxidants for their food protective effect (32,33).
Teucrium chamaedrys L. (Lamiaceae) is a perennial evergreen euri-mediterranean species that is rhizomatous dwarf shrub. Teucrium species are rich in essential oils and is the most abundant source of furanic neo-clerodane diterpenes. Other phytochemicals present in this plant include phenylethanoid glycosides, iridoid glycosides and phenolic compounds (32). The medicinal use of Teucrium chamaedrys is prohibited in some countries due to its liver toxicity, however, alcoholic extracts are still permitted as flavoring agents, because they are fundamental in providing a bitter aromatic taste (32,34). Teucrium polium L. (Lamiaceae) is another plant from Teucrium genus that has medicinal properties and is used as a natural food Tumeric (Curcuma longa L) Aromatic spices Rhizome A colorant, flavoring and medium aromatic.
Black cumin (Nigella sativa L) Aromatic spices Fruits (Seeds) A strong aromatic smell and warm, bitter taste.
Clove (Syzygium aromaticum) Aromatic spices Buds A pungent, strong, and sweet with a bitter, astringent flavor (22) Cinnamon (Cinnamomum cassia) Aromatic spices Stem bark A sweet and aromatic, and less bitter. (29) preservative due to its antioxidant and antimicrobial properties (35,36). The plant contains phenylethanoid glycosides, neoclerodane diterpenes, iridoid glycosides and flavonoids (32,37). Petrorhagia velutina (Guss.) (Caryophyllaceae) is an annual sudmediterranean herbaceous plant with a characteristic densely glandular-tomentose stem. Flavonoids C-glycosides were isolated from its leaves, in addition to cinnamoyl glucose esters and phytotoxic chlorophyll derivatives (32). Due to its antioxidant properties, Petrorhagia velutina can be used as a natural food preservative, by impeding oxidation, which is a mandatory step in rotting, either by aerobic or anaerobic mechanisms (38). Arbutus unedo (Ericaceae) is a steno-mediterranean evergreen small tree that is reported to have various phytochemicals, such as flavonoids, steroids and terpenoids (32). It has antioxidant properties, and thus can also be used as a food preservative (39). Myrtus communis (Myrtaceae) is an evergreen small tree that contains important essential oils. Phytochemical investigation of this plant revealed that it contains various monoterpenoids, triterpenes, flavonoids and small amounts of phenolic acids (32). The plant was demonstrated to have antioxidant and antimicrobial properties allowing it to be used as a natural food preservative without altering the nutritional characteristics of the food products (40). In a study conducted using a number of Mediterranean spices, namely, annatto, cumin, oregano, rosemary, saffron and sweet and hot paprika, to compare the oxidative stability of refined olive oil tested by the Rancimat method with common food additives during storage at different temperatures, reported that the spice extracts have significant stabilizing effects (P < 0.05) (41).

ANTICANCER ACTIVITY OF SPICES FROM THE MEDITERRANEAN DIET: CHEMICAL CONSTITUENTS AND MECHANISMS OF ACTION Ginger
Ginger (Zingiber officinale Roscoe) rhizome is widely used as a spice and folk medicine, affiliated to the Zingiberaceae family, belonging to Southern Asia (42,43). It has various constituents which may vary as a reason of environmental factors, the place of origin and whether the rhizomes are fresh or dry. Its characteristic odor is due to the presence of volatile oil containing various monoterpenoids and sesquiterpenoids (44). The fresh rhizomes pungency is due to its gingerols content where most abundant one is 6-gingerol (1-[40-hydroxy30-methoxyphenyl]-5-hydroxy-3-decanoate). On the other hand, the pungency of dry rhizomes is due to the shogaols content, such as 6-shogaol, which are formed as a result of thermal degradation of gingerols (44). Additionally, ginger also contains terpenoids, alkanes, paradols and diarylheptanoids (45). The phenolic compounds of ginger including gingerols shogaols and paradols were found to exhibit antioxidant, anti-tumor and anti-inflammatory properties (43,46,47).
6-gingerol (Figure 1) was identified as the main active medicinal component of ginger (45). It is usually found as yellow oil and can form a low-melting crystalline solid.
Several mechanisms of action for 6-gingerol have been discussed in many studies, including its chemo-preventive and chemo-therapeutic effects.
The activation of mitogen-activated protein kinase (MAPK) signaling pathway has a role as a possible mechanism behind the chemo-preventive and chemo-therapeutic activity of ginger via the induction of cell arrest against several types of cancer as reported in scientific literature as follows: One of the studies has investigated the mechanism of the cytotoxic effect of 10-gingerol on human colon cancer cells via the activation of MAPK in a dose-dependent manner, this morphological changes lead to apoptosis that also could be obtained by way of increasing DNA in the sub-G1 phase of the cell cycle (48). The additional study stated the antiproliferation effect of 6-gingerol on human skin keratinocyte cell lines as a consequence of MAPK and AP-1 signaling pathways (49) and on mouse skin tumor cells through the activation of NF-kappa B(NF-κB), p38 MAPK, and cyclooxygenase-2 (COX-2) expression as reported in a published study (50). Interestingly; another study highlighted the suppression of oral cancer cell growth and inhibition of migration by suppressing the AKT/mTOR signaling pathway and inducing AMP-activated protein kinase (AMPK) which in turn leads to cell arrest and apoptosis (51). 6-Gingerol plays a role in fighting gastric cancer cells along with chemotherapy, particularly Cisplatin, by altering phosphatidylinositol-3-Kinase and Protein Kinase B (PI3K/AKT) signaling pathway; consequently, this will induce cell cycle arrest at the G1 phase (52). Also, 6-Gingerol leads to cell cycle arrest at the G2 phase as well, against oral and cervical carcinoma (53).
Moving to renal cells, cell-cycle G1-phase arrest could be obtained upon 6-Gingerol treatment (54). The impressive study emphasized how 6-Gingerol can induce cell arrest at the G1cell cycle phase of osteosarcoma cells, by dint of AMPK signaling activation, therefore growth abolition (55).
Furthermore, 6-Gingerol could fight human pancreatic cancer cells via the suppression and the downregulation of the ERK/NFκ B/Snail signal transduction pathway as stated in the reference (56). Reactive oxygen species (ROS) has a role as a possible mechanism behind the chemopreventive and chemotherapeutic activity of ginger via the induction of cell arrest against several types of cancer as reported in scientific literature as follows: One study approved the anti-tumor activity of 6dehydrogingerdione which is one of the active extracts of ginger against breast cancer cells in humans that causes growth suppression due to the generation of ROS (57).
Interestingly, another study figured out the inhibitory effect of 6-Gingerol against lung cancer in mice via the generation of ROS (58).
Angiogenesis could be defined as the creation of totally new blood vessels from previously existing endothelium, which is a necessary process in tumor formation (59). It's worth mentioning here the anti-angiogenesis effect of 6-Gingerol via the induction of micro-vessel normalization due to the stabilization of p-VEGFR2/VE-cadherin/β-catenin/actin complex (46). Moreover, an Invitro study showed the inhibitory effect of 6-Gingerol in the suppression of endothelial cell tube formation, therefore it prevents the tumor blood supply (60).
6-gingerol has a suppression effect on the renal cell carcinoma metastasis in vitro and in vivo, this effect was due to the upregulation of yes-associated protein (YAP) ser127 phosphorylation and the downregulation of YAP levels in cell nuclei that is responsible for cancer cell migration (61).

Peppers
Capsicum is a genera of pepper, consisting of more than 31 different species including five domesticated species, C. baccatum, C. annuum, C. pubescen, C. frutescens, and C. chinense (62). Pepper is widely used as a food spice due to its pungency and unique flavor. Pepper contains provitamin A, vitamin E vitamin C, carotenoids and phenolic compounds including capsaicinoids, luteolin, and quercetin (62). Capsicum fruits have been used in the treatment of toothache, infections, coughs, sore throat, rheumatism and for wound healing (62). The main constituent, capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) (Figure 2), which is an off-white crystalline lipophilic colorless and odorless alkaloid (63), has antioxidant, anti-inflammatory, cytotoxic and antiproliferative effects.
Capsaicin has shown a chemo-therapeutic effect against several types of cancer through the initiation of cancer cell apoptosis (64). Cellular responses upon treatment with capsaicin affect mechanisms of cell death, especially through the downregulation of β-catenin which plays an important role in β-catenin-dependent signaling that is a significant event in the development of malignancies (65). In addition, upregulation of pro-apoptotic genes in other words pro-apoptotic stimuli in tumorigenic cells (66,67). Furthermore, one study stated the anti-proliferative effect of capsicum through the suppression of FBI-1-Mediated NF-κB Pathway that led to breast cancer cell apoptosis (68).
As discussed previously the anti-angiogenesis effect plays a significant role in killing tumor cells, as it is a possible mechanism of the anti-cancer effect of capsaicin that is figured out in vivo and in vitro (69). In in vitro model, was through the inhibition of tube formation, while in vivo through the suppression of vascular endothelial growth factor (VEGF)-induced vessel formation (70,71).
Capsaicin took part in fighting metastases of cancer, by altering signaling pathways that are important in cell migration (72), Moreover, the anti-invasive effect of capsicin could be done due to the suppression of phosphoinositide 3-kinase (PI3K) signaling cascade and RASrelated c3 botulinum toxin substrate1 (RAC1), that control cancer cell migration (73).

Rosemary
Rosmarinus officinalis L., often known as rosemary, is the scientific name for a Mediterranean plant that is grown in a variety of nations (74). Recently, rosemary extract (RE) was allowed by European Union legislation, allowing food companies to use the label "antioxidant: rosemary extract" on their products (75). Rosemary has been identified as a potential anticancer medication due to its antioxidant properties. It has the ability to act on free radicals and protect DNA, proteins, and lipids from oxidative damage (76), as later discovered, rosemary derivatives are capable of producing cytotoxicity precisely through the generation of ROS in particular conditions. The main active compounds of Rosemary are summarized in Figure 3. Rosemary Extract (RE) has been shown to affect intracellular antioxidant systems by activating the activation of nuclear transcription factor (Nrf) 2 target genes (77) and increasing glutathione levels, with a reduction in its reduced form (GSH) relative to its oxidized form (GSSG) (78). However, some antioxidants, such as beta-carotene, vitamin E, and vitamin C, have shown mixed results in clinical research addressing their involvement in reducing the risk of cancer formation [the antioxidant impact and anticancer action has been questioned (79)(80)(81)(82)(83)(84)(85). Furthermore, Carnosic Acid (CA) and Carnosol (CS) inhibit endothelial cell differentiation, proliferation, migration, and differentiation capacity, as well as other angiogenic capabilities. Several data show that their effects on endothelium and cancer cell development may be related to the programmed cell death stimulation (86).
CA also inhibits cytokine-induced adhesion molecule production and monocyte adherence to endothelial cells via an (NF-kB -dependent mechanism (87,88).
Histone deacetylases (HDACs), which regulate gene expression by acting on the acetyl group of histones, have abnormal expression patterns that coincide with the beginning of malignancies (89). HDAC2 has been shown to be highly expressed in tumor cells, where it inhibits the production of p53, resulting in a decrease in programmed cell death. The effect of rosmarinic acid (RA) vs. suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor utilized as an antitumoral medication, on the survival and programmed cell death of tumor cells lines, as well as HDAC production, was recently investigated. Similar to SAHA, RA inhibited cell growth and cancer spheroid formation, as well as causing tumor cell death and blocking HDAC2 expression. RA also decreased cyclins D1 and E1 as well as proliferating cell nuclear antigens, while increasing p21. Finally, a rise in p53 generated from the HDAC2 decrease regulated the protein synthesis of intrinsic mitochondrial apoptotic pathway-related genes (90).
The antineoplastic impact of rosemary could be due to a regulatory effect on the immune system. by enhancing the innate immune response; this enhanced response is attributable to cytotoxic natural killer cells and the formation of an antiinflammatory cytokine profile, which may aid the immunological response to cancer cells (91). CS inhibited tumor growth, also resulted in a decrease in interleukin-4 (IL-4) and IL-10 (IL-10) and an increase in interferon production (92).
Along with the molecular mechanisms discussed above, additional molecular mechanisms of rosemary have been described and linked to its anticancer effects, including hormone signaling alteration (93), and the ability to interact with a broad range of molecular targets (94,95). Furthermore, rosemary has recently been shown to boost the expression of genes with known cancer-suppressing capabilities (96). Finally, rosemary phenolic compounds may play a role in a variety of metabolic pathways as well as basic cellular activities and macro-and micronutrient metabolism. These altered pathways may have a clinical impact on the initiation and course of cancer (97,98). In addition, rosemary extract has been studied in combination with antitumor agents such as 5 Fu, cisplatin, doxorubicin, paclitaxel, tamoxifen, trastuzumab, and Vinblastine. Rosemary extract has a synergistic effect and plays a role in modulating gene expression for enzymes involved in the mechanism of resistance (99)(100)(101)(102).
To summarize, while the use of rosemary and its derivatives in the treatment of neoplasms is an interesting topic of research, big and controlled studies are needed to definitively determine the substance's true influence in clinical practice. Taking into account the need to standardize the extraction procedure in order to get REs with consistent antiproliferative properties (103).

Turmeric
Turmeric (Curcuma longa) belongs to Zingiberaceae, which is extensively cultivated for its rhizomes. It is used as spice, preservative and coloring agent in addition to possessing many medicinal applications such as anti-inflammatory, antihyperlipidemic, and antimicrobial activities (104,105). Turmeric is known to contain poluphenolic compounds known as curcuminoids, including curcumin (Figure 4), demethoxycurcumin and bisdemethoxycurcumin (104,105).
Curcumin (Figure 5), the main coloring principal of Curcuma longa, is an odorless, yellowish crystalline lipophilic compound, offers a surprising number of health benefits, including  anti-inflammatory, antioxidant, chemo-preventive, and chemotherapeutic characteristics (106,107).
The intrinsic and extrinsic routes are the two primary pathways that create apoptotic signals. The intrinsic apoptotic pathway works by stimulating the mitochondrial membrane to inhibit anti-apoptotic protein expression (108), curcumin disrupts the mitochondrial membrane potential balance leading to increased suppression of antiapoptotic proteins (109). The extrinsic apoptotic pathway works by increasing death receptors (DRs) on cells and triggering tumor necrosis factor (TNF) related to apoptosis. Curcumin also plays a role in this pathway by increasing the expression of DRs (106,107,110).
In addition, findings from in vitro and in vivo investigations have indicated that curcumin has a powerful cytotoxic effect on many cancer cells by inhibiting oxidative stress and angiogenesis, as well as inducing apoptosis (111).
The PI3K/AKT signaling pathway regulates VEGF expression. Curcumin therapy decreased protein expression levels of PI3K and AKT. Curcumin therapy also dramatically reduced the levels of mRNA expression of VEGF, PI3K, and AKT (112).
Curcumin's anti-inflammatory properties would almost certainly result in its anti-tumor properties, given the close link between inflammation and cancer. Curcumin has been shown to inhibit the development of numerous types of cancer by lowering the production of inflammatory mediators (113).
Increased production of pro-inflammatory molecules such as cytokines, ROS, COX-2, and transcription factors such as NF-B, AKT, activator protein 1 (AP1), and signal transducer and activator of transcription 3 (STAT3) is induced by inflammation, leading to the initiation and progression of cancer. Curcumin's anticancer property comes from its immunomodulatory ability, which it does via interacting with a variety of immunological mediators as it inhibits the transcription of TNF-and, as a result, the expression of inflammatory genes via suppressing NF-B activity. Curcumin's immunomodulatory properties, on the other hand, are directed not only at molecular targets, but also at cellular components like macrophages, dendritic cells, and T and B lymphocytes (114)(115)(116)(117)(118).
Curcumin's anti-cancer properties are also related to its interference with the cell cycle and reduction in cyclin-dependent kinases (CDK) expression. CDKs regulate the progression of the cell cycle (119). Curcumin also inhibits STAT3, which is involved in signaling carcinogenic pathways (120).
In the early phases of cancer growth, free radicals and hazardous compounds produced by oxidative stress play a significant role. As a result, substances with antioxidant properties may be useful in avoiding cancer. Curcumin has the ability to trap free radicals, which means it can help prevent cancer from developing. Curcumin prevents DNA damage induced by oxidative causes like ionizing radiation by reducing free radicals and active oxygen species, according to several cellular and preclinical investigations (121).
Curcumin is thought to have anti-cancer properties by interfering with several cellular processes and activating or inhibiting the production of certain cytokines, enzymes, and growth factors. Curcumin's anti-cancer potential, however, has been limited, owing to its low water solubility. Curcumin compounds with improved efficacy and/or water solubility or stability have resulted through chemical modification of these moieties (107).

Black Cumin
Nigella sativa (N. sativa) is a tiny shrub with annual flowers that belongs to the Ranunculaceae family. It has white, pink, yellow, and purplish exquisite flowers with 5 to 10 petals (123). When the fruit is ripped open, it reveals a great number of black seeds known as black cumin in English, and Habbat el Baraka or Habbah Sawda in Arabic (124). Syria, Lebanon, Pakistan, India, and Afghanistan are among the Middle Eastern and Western Asian countries where the N. sativa plant is widely farmed. N. sativa are used as a spice in Indian and extensively in Middle Eastern cuisines due to its pungent bitter taste and aroma. The seeds contain many vitamins and minerals in addition to important active compounds including thymoquinone, thymohydroquinone and dithymoquinone (nigellone) (24).
The pharmacological properties of N. sativa are mainly due to its quinine constituents, primarily thymoquinone (Figure 6) because it is the most abundant monoterpene (24).
Among several therapeutic plants, N. Sativa has long been regarded as one of the most valued nutrient-rich herbs in history, and various published scientific studies are currently ongoing to confirm the traditional applications of this species' small seed (72).
Because of its low toxicity and numerous mechanisms of action, N. Sativa can be a useful tool for health improvement (125). Recent research suggests that N. Sativa oil and extracts contain anti-inflammatory and antimicrobial characteristics, as well as bronchodilator, hypoglycemic, immune booster, anticancer and antioxidant properties (126)(127)(128)(129)(130). Once the antitumor characteristics of the N. Sativa seed and extracts were established, the researchers investigated the antitumor properties of its major active components, such as thymoquinone and dithymoquinone (131). Black cumin's antitumor mechanism of action is as follows: Thymoquinone (TQ) antioxidant and cytotoxic effect has been studied in vitro and in vivo utilizing a variety of animal models and tumor cell lines.
One of the first publications pointing to N. sativa's possible anti-cancer characteristics, An aqueous extract of N. Sativa seeds were found to have considerable cytotoxic effects on tumor cell lines (HepG2, MOLT4, and LL/ 2), but not on healthy, noncancerous umbilical cord endothelial cells (132).
Both aqueous and ethanolic extracts of N. Sativa seeds were also found to exhibit significant cytotoxic effects on MCF-7 cells in the presence and lack of H 2 O 2 , apart from their antiproliferative properties (133).
A crude methanolic extract of N. Sativa also induced around 50% cytotoxicity in Sarcoma180 cells (S-180 cells), Dalton's lymphoma ascites, and Ehrlich ascites carcinoma, in an in vitro cytotoxic study (134).
Another in vivo study found that 6-month oral administration of N. Sativa seeds protected rats from methylnitrosourea-induced oxidative stress and colon carcinogenesis due to lower production of malondialdehyde (MDA), a lipid peroxidation biomarker, and nitric oxide (NO) biomarker (135).
A few researches has investigated the possibility of N. Sativa having an anti-mutagenic effect against the directly acting mutagen N-methyl-N0 -nitro-N-nitrosoguanidine (MNNG).
Due to dramatically reduced chromosomal abnormalities in primary rat hepatocytes, an ethanolic extract of N. Sativa exerted an inhibitory effect against MNNG mutagenicity. MNNG's anti-mutagenic actions were assigned to the stimulation of detoxifying enzymes that break down MNNG, chemical contact with or uptake of MNNG (or its electrophilic degradation products), increased DNA replication fidelity and enhanced DNA repair (136).
Several studies examined the impact of N. Sativa oil on the fibrinolytic capability of HT1080 human fibrosarcoma cell lines, which is a marker of malignant tumors.
In cell cultures, N. Sativa oil produced a dose-dependent downregulation of major fibrinolytic products such as urokinasetype plasminogen activator (u-PA), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor type 1. The capacity of N. Sativa to prevent local tumor invasion and metastasis is highlighted in this study (137).
In many studies, several research groups have postulated that increasing NK cytotoxic activity against cancer cells is a mechanism underlying N. sativa's anti-cancer properties (138,139).
The ability of N. Sativa to alter the activity of key enzymes has been primarily related to the key mechanisms underlying the reported anti-cancer properties of the plant (140, 141).
The inducible nitric oxide synthase (iNOS) pathway is one mechanism that has been linked to tumorigenesis. NO is an endogenous radical that is synthesized by iNOS or another NOS isoforms throughout physiological events such as inflammation and has been linked to tumor growth. In a recent study, they investigated how an ethanolic extract of N. Sativa would modify the iNOS pathway in rats with hepatocarcinogenesis induced by diethylnitrosamine (DENA). The serum levels of alpha-fetoprotein (AFP), NO, IL-6, and TNF-α factors whose production was considerably bolstered after treatment with DENA, were dramatically reduced after oral administration of N. Sativa ethanolic extract (142).
A study published recently found that a methanolic extract of N. Sativa seeds caused apoptosis in MCF7 cells in a dose-and time-dependent manner. In MCF7 cells, the methanolic extract of N. Sativa resulted in a significant increase in the expression of apoptotic factors such as caspase-3, caspase-8, caspase-9, and the p53 tumor protein, implying that N. sativa's anti-cancer activity is mediated through the p53 and caspase signaling pathways (143).
Thymoquinone, the active phytochemical of Nigella sativa, exhibited an anticancer effect toward different cancer cells. It has suppressed the expression of janus Kinase 2 (Jak2) and STAT3, as well as upregulated the ROS level, and promoted apoptosis in human melanoma cells (144). Guler et al. reported the molecular anticancer activity of TQ in glioma cells. It has mediated apoptosis via inhibiting pSTAT3, hindering matrix metalloproteinases (MMP) and GSH levels, increasing iROS generation (145). Another study revealed the cytotoxic effect of TQ in Neuro-2a cells. The caspase-3 induction, KIAP protein reduction, and uprising of BAX/Bcl2 ratio have been observed upon the treatment with TQ (146,147). Several studies demonstrated the antitumor mechanisms of action of thymoquinone, including its effect on the main cancer biomarkers and cell growth (148,149). Hence, TQ can suppress NF-Kb, IL-8, PI3K/AKT, and MAPK as well as prevent cell migration by reducing the expression of N-cadherin gene (149)(150)(151).

Clove
Cloves, Syzygium aromaticum L, dried buds, have long been used as a spice and in traditional Chinese and Indian medicine. Cloves include a diverse variety of bioactive components. Sesquiterpenes, volatile oil (eugenol), caryophyllene, tannins, and gum are among the major chemical constituents of cloves (152,153).
Clove oil is an effective antibacterial, analgesic, expectorant, antioxidant, and antispasmodic. Eugenol (Figure 7) is one of clove oil components that is responsible for its characteristic

In vitro
Produced cytotoxicity and caused a rise in the G2/M phase (166) odor, is a colorless to pale yellow oily liquid and has been found in a few anticancer formulations (154). Clove's antitumor mechanism of action as follows: The capability to inhibit oxidative stress has been defined as a protective effect against cancer formation (carcinogenesis or tumorigenesis); however, whenever cancer has formed, the antioxidant effect can contribute to cancer's development, whereas the pro-oxidant effect can evoke cancer cell death through several signaling pathways (155,156).
Notably, eugenol has been identified as an agent having a dual effect, antioxidant, and pro-oxidant, with beneficial effects in both cancer prevention and treatment (157)(158)(159).
Eugenol also exhibits ferric ion (Fe3+) reducing ability and electron donor properties, allowing it to neutralize free radicals by producing stable products (162).
Furthermore, in many studies eugenol has been shown to reduce microsomal lipid peroxidation as well as iron and OH radical-induced lipid peroxidation in rat liver mitochondria. The production of thiobarbituric acid-reactive compounds was used to evaluate the antioxidant effect (160,161).
Some inflammatory markers, such as inducible iNOS and COX-2 expression, as well as the levels of pro-inflammatory cytokines IL-6, TNF-α, and prostaglandin E2 (PGE2), were reduced in dimethylbenz[a]anthracen (DMBA)-exposed animals after treatment with eugenol, showing its anti-carcinogenic effect. Furthermore, in mouse skin with otetradecanoylphorbol-13acetate-induced inflammation, eugenol was observed to decrease the activation of NF-B (157,163).
According to certain research, eugenol can induce cytotoxicity at concentrations in the µM range. In the µM range, eugenol suppresses melanoma cell proliferation by causing cell cycle arrest in the S phase, followed by cell apoptosis (164).
In another study, Eugenol in the µM range produced cytotoxicity and caused a rise in the G2/M phase in LNCaP (androgen-responsive human prostate cancer) and PC-3 (androgen-independent human prostate carcinoma) cell lines (166) (Figure 1). Demonstrate the six spices that have mentioned in this review with their main phytochemicals ( Table 2). Summarize the anticancer activity of the main Mediterranean diet spices and their mechanisms of action.

CONCLUSION
The clue in this review suggested that spices could be part of your daily diet that may lower cancer risk and affect tumor manner of acting. This review only scratches the surface of the overall impact of spices because roughly speaking there are 180 spices widely being used for several purposes. The proof goes on those numerous processes, involving proliferation, apoptosis, angiogenesis, signaling pathways, transduction, cell cycle phases, and immunocompetence could be affected by one or more of the previously mentioned spices, which in turn is reflected on the tumor activity. The Mediterranean diet is rich source of numerous spices. Compared with other diets, it includes multiple spices instead of focusing on single one. The presence of a cocktail of spices in single diet increases the chance of possible synergistic effect that may enhance the anticancer effect of standard therapies. The most common spice in the Mediterranean diet is black pepper (Piper nigrum L). Apoptosis induction is the most common anticancer pathway activated by different spices in the Mediterranean diet. Ginger and black cumin have the highest anticancer activities by targeting multiple cancer hallmarks. Further studies are needed to design anticancer diets containing the correct combination of spices.