2.1.7. Lung Disease

Acute lung injury is caused by lung inflammation as a consequence of endotoxemia [103]. Promotion of pulmonary inflammatory cell sequestration and enhanced production of pro-inflammatory mediators affects the alveolar space resulting to lung dysfunction [104–107]. In the study of systematic endotoxin-induced acute pulmonary injury in rats [108], administered sesamol at 1–3 ppm blocks the inflammatory cells induced by LPS in infiltrating the alveolar space, suppresses the protein leakage and expression of inflammatory cytokines in bronchoalveolar lavage fluid (BALF). The NF-кB activation in alveolar macrophage is inhibited by sesamol. Sesamin at dosages 1 and 3 ppm, moreover, suppresses the generation of nitric oxide related to the alveolar macrophage.

Leukotrienes are mediators of lipid that involves the pathogenesis in asthma. The enzyme 5-lipoxygenase (5-LOX) draw in the production of inflammatory mediators through metabolism of arachidonic acid into leukotriene B4 (LTB4) and leukotriene C4 (LTC4), potent inflammatory mediators, by the presence of five lipoxygenase activating protein (FLAP). LTC4 is claimed to possess powerful inflammatory eicosanoid, which enhances vascular permeability [109]. Sesamin has been reported to reduce the levels of LTB4 more effectively than sesamol. On the contrary, sesamin and sesamol downregulates the serum level of LTC4. The cumulative effects of the combined sesamin and sesamol reduces the said inflammatory mediators, although its reducible strength is equivalent to that of the individual effects of sesamol and sesamin suggesting that the synergistic interactions are absent. For the most part, sesamin and sesamol exhibits anti-leukotriene effects, which downregulates the receptors and key enzymes of leukotriene pathway and further diminishing the pro-inflammatory leukotrienes production [110].

Non-heme iron-containing enzyme called lipoxygenases (LOX) is characterized by its catalysis activity in incorporating molecular oxygen into polyunsaturated fatty acids [111]. The conversion of arachidonic acid to hydroxyeicosatetraenoic acid (HETE) via metabolism with LOX promotes expression of leukotrienes and regulates the inflammation pathway [112].

In the study of kinetic inhibition [113], sesamol competitively inhibits LOX with IC50 value of 51.84 μM and an inhibitory constant (Ki) of 4.9 μM. The competitive inhibition is happening in either the active substrate site or the active metal ion site. Hence, in the ferric reducing ability power (FRAP) assay of the same study, 55.35 μM of sesamol reduces half of the Fe3<sup>+</sup>-LOX into Fe2<sup>+</sup>-LOX indicating the partial interaction of sesamol to the active metal ion site.


**Table 1.** In vivo models for the anti-inflammatory effects of sesame lignans.

**Table 2.** In vitro models for the anti-inflammatory effects of sesame lignans.


#### 2.1.8. Others

The extent of the effect of other sesame extracts on the inflammatory cytokines and mediators has also been investigated thoroughly. One study involves the aqueous extract of sesame oil (SOAE) [114], which has twenty-eight identified molecules that ranges from moderate to polar in nature. The SOAE-methoxyphenol derivatives (SOAE-8; i.e., vanillyl alcohol, p-hydroxyphenylacetic acid, vanillic acid, coniferyl alcohol, p-coumaric acid, ferulic acid, sinapic acid and syringic acid) are the key components for its anti-inflammatory property. The absence of the sesame lignans in SOAE paves way for the study to be engrossing. Monocyte derived macrophages (MDMs) and RAW 264.7 macrophage cells were used, and the results are positive with slight distinction. SOAE-8 successfully reduced dose-dependently the mRNA levels of the inflammatory markers (*IL-6, IL-1*β and *TNF-*α) in MDMs. Meanwhile, in RAW264.7 cells, only *TNF-*α mRNA level was not reduced dose-dependently by SOAE-8.

The ethanol extract in black sesame seeds (BSSEE) attenuates liver inflammatory response in fructose-induced NAFLD rat model [115]. Three major lignans, which are sesamin (16.33%), sesaminol (1.92%) and sesamolin (13.06%), were found in BSSEE. Three major lignans, which are sesamin (16.33%), sesaminol (1.92%) and sesamolin (13.06%), were found in BSSEE. Inflammatory cytokines are dose-dependently reduced by BSSEE when administered at 0.5–2 ppm. Infiltration of inflammatory cells is also hindered in the presence of BSSEE. Correspondingly, BSSEE with concentrations of 1 ppm and 2 ppm promotes the activation of Nrf2 and improves the levels of MAPKs and NF-кB. In another study of BSSEE, Freund's complete adjuvant (FCA)-induced arthritis rat model was used [116]. The study examined the effect of BSSEE on the inflammatory cytokines (IL-6 and TNF-α). It has been reported that at dosage 800 ppm of BSSEE, the levels of inflammatory cytokines induced by rheumatoid arthritis are reduced in the span of 28 days. Comparatively, extracts of black sesame seeds via CO2 supercritical fluid extraction (SFE) exhibits neuroprotective activity against ischemia [117]. While its composition is mainly made up of fatty acids (caprylic acid, capric acid, lauric acid, myristic acid, palmitoleic acid, margaric acid, linolenic acid, arachidic acid, behenic acid, palmitic acid, stearic acid, linoleic acid and oleic acid) and phytosterol (cholesterol, brassicasterol, stigmasterol, Δ-5, 24 stigmastadienol, Δ-7 stigmastanol, Δ-7 avenasterol, eritrodiol, campesterol + campestanol + 24 methylene cholesterol, clerosterol + Δ-5, 23 stigmastadienol, Δ-5 avenasterol and β-Sitosterol + sitostanol), its synergistic interactions play a vital role. The infiltration of leukocyte reduces when treated with CO2 SFE extracts of black sesame seeds. On the contrary, the study suggests further evaluation of its mediating-effect on the neuronal disorders.

The ethanol extract in the sesame coat (EESC) also manifests anti-inflammatory property [118]. It was reported that EESC contains sesamin, sesamolin, phenolic compounds and tetranortriterpenoids [119]. Using RAW 264.7 macrophage cell line, EESC (0.08 ppm) exhibited lowering effect on the levels of NO production as well as PGE2 production. EESC, at the same dosage, inhibits both the *iNOS* and *COX-2* protein expressions by 94% and 53%. Inhibitory effect on the LPS-induced degradation of IкB-protein has been observed when EESC was applied.


**Table 3.** In vitro/in vivo applications of different extracts from different sesame components.

#### *2.2. Anti-Cancer Activity*

During inflammation, the immune system of the body is tasked to the release of reactive oxygen and nitrogen species (RONS) to fight against pathogens and to protect the body. RONS are also responsible for tissue repair and regeneration [120]. However, these chemicals are able to obstruct DNA repair mechanisms, which can potentially lead to DNA damage. With damaged DNA, the chance of mutations significantly rises, promoting tumorigenesis. It was reported that chronic inflammation is part of the 15% precedencies of recorded cancer cases [121]. Millions of cancer cases are recorded every year and millions of people succumb to different types of cancer. In 2018, 9.6 million deaths in the world were caused by cancer [122]. Due to this, efforts in finding a cure for and attempts to understand cancer continue to rise [123]. Cancer is commonly defined as the abnormal growth of the cells, but there are indications that mark the development of cancer in the human body. It was suggested that six essential alterations in cell physiology are mainly responsible for the uncontrolled growth of the cells. Self-sufficient in growth signals, insensitive to growth-inhibitory signals, evasive of apoptosis, unlimited replication potential, maintenance of angiogenesis and invasive to tissues are the six characteristics of cells during tumor development [124,125]. However, in a study more than a decade later after these indications were identified, two other hallmarks of cancer have been discovered. It was reported that tumor cells are also able to reprogram energy metabolism and are evasive of immune destruction, with genome instability and inflammation as the primary causes [126]. Knowing these hallmarks allow the creation of defensive strategies against cancer and also understanding the fundamentals of these indications opens the opportunity for a better comprehension as to how neoplastic diseases emerge.

#### 2.2.1. Lung Cancer

Oil extracted from sesame stood out among other vegetable oils because of its exceptional nutritional characteristics [2]. Sesame lignans have demonstrated several pharmacological applications [127], including anti-proliferative activity, which made them subjects of anticancer studies. Lung cancer, which is the most diagnosed cancer in 2018 [122], has been studied by Harikumar and colleagues by treating human lung adenocarcinoma cell line H1299 with sesamin solution. Sesamin was able to significantly inhibit the proliferation of H1299 cells, with a 50% inhibitory dose of 40.1 μmol/L [128]. Sesaminol has also been proven to exhibit anticancer property against lung carcinoma A549 cells at a concentration of 50 μM and 6 h of treatment [129]. The mechanism of action of sesaminol on A549 cells is tackled in the following section (2.2.2 Breast Cancer).

Another lignan of sesame, sesamol, has also been explored for its anticancer properties [130]. It has been studied for its apoptotic effect in lung adenocarcinoma SK-LU-1 cell line. After 48 h of treatment, the lignan showed selective antiproliferation effect with an IC50 of 2.7 mM on SK-LU-1 cells and 7.6 mM on Vero cells. The apoptotic effect of sesamol was also found to be both time and dose-dependent. The higher the concentration, the higher number of cell deaths was recorded. At 2.7 mM sesamol, evident necrosis occurred in a time-dependent manner, while at 5.4 mM sesamol, early stage apoptosis was affected [131,132]. A 24-h sesamol treatment of SK-LU-1 cells showed an increase in the activities of caspase 3/7, which largely participate in the propagation of death signals. Specifically, the increase and activation of caspase 3 causes the cleavage of a DNA repair protein known as PARP. Consequently, DNA damage occurs and cell deaths are achieved. The loss of mitochondrial membrane potential (Δψm) was also investigated as one of the factors of apoptosis and a 48-h treatment revealed that longer exposure to sesamol leads to a greater decrease in Δψm. This proposes that mitochondria are part of the apoptotic pathway that occurs in SK-LU-1 cells [131,132].

Another study revealed that sesamin also has a protective action against the down-regulation of the PI3K-Akt signaling pathway in a nickel-induced apoptosis in mouse liver. PI3K-Akt pathway is responsible for the restriction of apoptosis and for the promotion of cell survival [133,134]. This action of sesamin showed that the lignan is capable of preventing DNA damage, hence, it becomes a potential anticancer agent. In line with this, it was also reported that the ability of sesamin to reduce

*COX-2* gene expressions in A549 cell line occurs through PI3K-Akt pathway [135] and inhibition of this pathway inactivates inflammatory response, which reduces restenosis [136].

#### 2.2.2. Breast Cancer

Another common type of cancer is the female breast cancer, which accounts for 11.6% of the total cancer deaths [122]. For that reason, breast cancer is also widely studied. In 2007, sesamin was examined for its effect on the proliferation of human breast cancer cell line MCF-7 for 24 h. Results showed that the inhibition is done through G1 phase growth arrest and is dose-dependent, with a cytostatic effect at 100 μM sesamin. The lignan was also used to assess the effect of down-regulation of cyclin D1 protein expression in different types of human tumor cells including the breast cancer cell line T-47D, the lung cancer cell line A549, the transformed renal cell line 293T, the immortalized keratinocyte cell line HaCaT, the melanoma cell line UACC-62 and the osteosarcoma cell line MG63, which proved that suppression of sesamin generally occurs in the tumor cells. In line with this, the down-regulation of cyclin D1 protein expression was examined as one of the main factors that cause the growth inhibitory effect of sesamin against cyclin D1-depleted MCF-7 breast cancer cells. Results showed that the inhibitory effect of sesamin is largely dependent on the presence of cyclin D1 as cyclin D1-depleted MCF-7 cells were almost insensitive to the sesamin treatment [137]. In the study of Harikumar, the same antiproliferation activity of sesamin was also observed against breast cancer cells MDA-MB-231 with an IC50 value of 51.1 μmol/L [128].

The anticancer function of sesamin also manifests even with a different mechanism of action. Sesamin was also able to impede the proangiogenic activity of MCF-7 cells. A 24-h pretreatment of MCF-7 cells and macrophages were carried out with 50 μM of sesamin, which resulted to the suppression of angiogenesis upon observation of the endothelial capillary tube assay and the network formation of the cells. Observation done on MCF-7 cells alone, however, revealed that the sesamin treatment was not significantly cytotoxic to the breast cancer cells as it did not decrease the cells' viability even at sesamin concentration of 100 μM within a 72-h treatment period. MφCM-treated MCF-7 cells were also examined for the same activity and sesamin showed a more promising inhibition performance than when MCF-7 cells were treated with the control medium. The same suppressive effect of sesamin was also exhibited against a more malignant cell line, MDA-MB-231 cells, treated with MφCM. To properly investigate the mechanism of action of sesamin on the tumor cells, vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9), which are essential factors of angiogenesis, were evaluated. MφCM treatment was found to induce *VEGF* and *MMP-9* mRNA expression in MCF-7 cells, but treatment with sesamin drastically hindered this. The same results were obtained for MDA-MB-231 cells [138].

A combined treatment with γ-tocotrienol also prevents the proliferation of MCF-7 and MDA-MB-231 cells. It was suspected that sesamin could increase the antiproliferative activity of γ-tocotrienol by inhibiting its metabolic degradation but the study revealed that the synergistic inhibiting effect of the two compounds is the result of the induction of G1 cell cycle arrest and the reduction of protein expression levels involved in the cell cycle. The synergistic inhibitory performance of sesamin and γ-tocotrienol is also effective against the murine +SA mammary epithelial cell line of a mouse [139]. An in vivo study on the proliferation of MCF-7 cells in athymic mice presented a comparison of the apoptotic activity of sesamin and of a lignan of flaxseed, secoisolariciresinol diglucoside (SDG), in which sesamin showed a more promising activity [140]. Tumor cell proliferation declined by 38% with sesamin treatment and cell apoptosis rose by 91% as opposed to the 37% reduction of proliferation with SDG treatment without exhibition apoptotic property. Their ability to down-regulate growth factor receptors known as EGFR and HER2 explains the reduced tumor growth and sesamin's ability to reduce pMAPK expression causes it to be more effective than SDG.

Just like with sesamin, sesamol was also used against the human breast cancer MCF-7 cells. Endothelial cell line EA.hy 926 was used to observe morphological changes that are caused by the exposure to sesamol. Endothelial cells were treated with sesamol for 72 h with a concentration range of

0.05-1.0 mM. It was found that lower doses of sesamol caused the cells to swell, while a concentration of 1 mM caused apparent cell death. Fragmented nuclei were present in the cells that were treated with 1 mM sesamol, indicating apoptosis. To check whether sesamol is capable of inhibiting the growth of MCF-7 cells, the cells were subjected to a 3-day treatment at 0.10 mM. This revealed that cell numbers are indeed lower than the controls. Combining PI labeling with a TUNEL assay led to the information that cell deaths occur in both S and G2/M phases [141]. Whether the inhibitory effect of sesamol on MCF-7 cells was solely caused by the apoptosis was disclosed in the study. No other pathway of inhibition was presented.

Aside from this, sesaminol has also been utilized against breast cancer cell lines MDA-MB-231 and MCF-7, along with lung carcinoma cell line A549 stated in the previous section. As mentioned, restriction of cyclin D1 expression plays a major role in some of the inhibitory pathways induced by sesame lignans. It was reported that the same statement applies to sesaminol, which was verified when a 6-h treatment at 50 μM sesaminol reduced cyclin D1 expression in MCF-7 cells. Although the same conditions did not produce the same result in MDA-MB-231 cells, it was stated that 100 μM sesaminol with 24 h to 48 h of sesaminol treatment achieved the desired result. To investigate how sesaminol manifests the same activity, sesaminol-immobilized FG beads were incubated with MCF-7 cells to identify, which proteins would bind to the beads. Among the proteins was an inner mitochondrial membrane protein known as adenine nucleotide translocase 2 or ANT2. This protein is said to be overexpressed in different malignant tumors and is recognized as an oncoprotein. To confirm whether ANT2 is involved in the anticancer activity of sesaminol, a knockdown of ANT2 in MCF-7 cells was performed. This resulted to growth inhibition and accumulation of cells in the G1 phase [129].
