Olive Leaf Extracts as a Medicinal Beverage: Origin, Physico-Chemical Properties, and Bio-Functionality

Abstract

Medicinal beverages are herbal drinks that are consumed by people in numerous countries including China, India, Brazil, Greece, Turkey, and others. These herbal drinks possess many beneficial properties for human health, such as antioxidant, antimicrobial, anti-inflammatory, anticancer, antiaging, anti-fertility, antitumor properties, and anti-diabetic effects. Flavonoids, phenols, carotenoids, sterols, glucosinolates, alkaloids, polyacetylenes, coumarins, saponins, and terpenoids are the main constituents which offer benefits to human health. In this context, this review aimed to highlight medicinal beverages’ origin, physico-chemical properties, and bio-functionality, focusing mainly on olive leaf extracts and their bioactive components that have not been extensively studied.

1. Introduction

Progress in the functional foods and food plants sector has led to the reduction in synthetic antibiotics use and the alleviation of physical disorders [1,2]. Phenols, tannins, flavonoids, stilbenes, and anthocyanins are the main constituents of plants to which the anticancer, anti-diabetic and antiaging properties are attributed [2,3,4,5]. Some representative species are rosemary, sage, and peppermint, which in addition to their contribution to the improvement and enforcement of flavors and aromas, possess anticancer, antimicrobial, and anti-diabetic properties due to their essential oils [3,6,7,8]. The high demand for the plant species is due to the easy access, lower cost in comparison to convectional drugs and demand for natural alternatives choices [9]. An important substance which is contained both in sage and rosemary is rosmarinic acid, which possesses anti-inflammatory properties [3,6,7]. The positive effects of sage in hypolipidemia, irritation and memory enhancement have also been demonstrated [7]. Other herbs and vegetables that are used for the improvement of aroma and flavor and possess antioxidants and antimicrobial properties are dill leaves, bay leaves, basil, thyme, garlic, onion, tarragon, marjoram, celery, and shallot [10]. These herbs and vegetables can also find applications in medicine. The incorporation of these herbs into meat products and several other products such as sauces, vinegar, and mustards can contribute to the improvement of product appearance and increase in nutritional and medicinal value [11,12].

All herbal drinks have physical and chemical properties. Physical properties include temperature turbidity color, solid content, odor, and taste. Mineral content is included in chemical properties [13]. The chemical composition of herbal drinks consists of phytochemical compounds such as phenols, flavonoids, sterols, carotenoids, alkaloids, glucosinolates, saponins, coumarins, terpenoids, polyacetylenes, and other sulfur-containing compounds. Flowers, roots, and leaves present proportionally different phytochemical properties compared to herbal drinks. The compounds above can inhibit product oxidation and decrease disease risk conditions. Also, these phytochemical compounds are responsible for the pharmacological action of herbal drinks, such as antibacterial, antioxidant, anticancer, anti-inflammatory, anti-fertility, and antitumor properties, and anti-diabetic effects. The pharmacological properties may differ from type to type of herbal drink. The pharmacological action of herbal drinks is in accordance with the content of bioactive constituents. The most popular herbal drinks are many species of tea and olive leaf extract. The olive leaf extract possesses a distinctive position in herbal drink category on account of its high antioxidant, anti-inflammatory, anticancer, anticholesterolemic, antihypertensive, antimicrobial and antiviral properties, which are mentioned more extensively in Section 2.

Table 1. Chemical composition of selected herb species and herbal drinks.

Herb Species and Herbal Drinks	Chemical Composition	References
Green tea	Flavonoids, terpenoids, cardiac glycosides saponins and tannins	[14]
Green tea infusions containing broccoli by-products	Catechins, hydroxycinnamic acids, flavonols and glucosinolates	[14]
Tea from Artemisia annua L.	Coumarins, terpenes, flavonoids, acetylenes and phenols	[14]
Loloh cemcem, Bali traditional drink	Tannins, terpenoids, flavonoids, alkaloids, and phenols	[15]
Olive leaf extracts	Flavonoids (luteolin, apigenin, luteolin-7-O-glucoside, etc.), ferulic acid, caffeic acid, tyrosol and hydroxytyrosol and secoiridoids (ligstroside, oleuropein dimethyloleuropein),	[16]

presents the chemical composition of some selected herb species and herbal drinks containing broccoli by-products, among others, given their high content of polyphenols.

Consumption of Herbal Drinks in Several Countries and Their Positive Effect on Human Health

Herbal drinks are used in Africa and India for medicinal purposes and health improvement [17]. Herbal drinks can be found in the market either in isolated form (roots, flowers, leaves, and bark) or as complex herbal mixtures [18]. According to the World Health Organization, more than 80% of people in Africa and Asia use herbal medicines. Herbal drinks can be prepared by several means such as ‘akar kayu’, ‘jamu’, drink, herbal tea, herbal decoction, and herbal infusion [19]. Herbal tea has gained the interest of many countries such as Turkey, China, Brazil, India and others. Herbal teas are used in traditional medicine and pharmacy in Greece and the Eastern Mediterranean countries [20]. The overall consumption of herbal teas in 152 countries increased by 25.1% from 2003 to 2013. Countries that possess the highest percent of tea consumption are China, India, and Brazil, followed by Japan, Iran, USA, Russia, Argentina, and Turkey and others. The highest number of tea drinkers was noted in Ireland, Turkey, and the United Kingdom [21]. A rapid increase in tea consumption was recorded in Japan from 1986 to 2007, and the next year, it decreased [22]. This increase was attributed to the health benefits offered by catechins. Leung cha is a tea that is consumed in China and is utilized for the cure of fever [23].

In 2016, the population of overweight and obese people increased to over 1.9 billion adults [24]. The medicinal herbs that were used for the therapy of obesity by the Brazilian Health Regulatory Agency were green tea, green tea–Camellia sinensis (L.) Kuntze; carqueja–Baccharis trimera (L.) DC; hibiscus-Hibiscus sabdariffa L. and soursop Annona muricata L. [25]. Green tea is one of the most popular consumed plant species worldwide, either isolated or intercropped. The world production of green tea is going to be increased by 7.5% per year [26]. Generally, the combination of various plant species is effective in the cure of some types of cancer and obesity [27]. As far as the effectiveness and safety of herbal mixtures are concerned, there is a need for more research. In 2013, concerns related to the need for formal recognition in the countries of origin about the commerce and consumption of products were expressed at an event in China. The recent years the health benefits of herbal teas and spices consumption, has led to a rapid expansion of the sector, concerning both organic and conventional spices and herbs, integral to herbal tea production, that is rapidly growing and is expected to reach approximately $31.95 billion by 2028 [28]. Considering the above, the main scope of the review article was to highlight the medicinal beverages’ origin, physico-chemical properties, and bio-functionality, focusing mainly on olive leaf extracts and their bioactive components that have not been extensively studied in the literature.

2. Olive Leaf Extracts

Olive leaves are the leaves of the olive tree (Olea europaea L.), which has been cultivated in Mediterranean countries for centuries [29]. For example, Egypt produces approximately 332,321 tons of olives annually and has a total cultivation area equal to 1% of the world’s total (100,000 ha) [30]. In Algeria and Tunisia, approximately 8 million hectares are cultivated with olive trees [31]. Olive leaves are a by-product that represent more than 10% of the weight of harvested olives [32]. Olive leaf extracts have been used for the cure of fever and malaria [33] and in various pharmacological applications [34]. Olive leaves contain organic matter (76.4–92.7 g/100 g dry matter), low quantities of crude protein (6.31–10.9 g/100 g dry matter), amino acids (89.9 g/100 g total nitrogen), nitrogen attached to the cell walls (49.2 and 35.4 g/100 g total nitrogen), crude fat (2.28–9.57 g/100 g dry matter), mannitol and glucose, bioactive components, and inorganic matter in different proportions, depending on: (i) terroir, (ii) cultivar, (iii) geographical position, (iv) methods of drying and extraction, (v) period of harvest, and (vi) standardized methods of phenolic compounds’ concentration, among others [35,36,37]. Bioactive components include phenols, such as flavonoids (luteolin, apigenin, luteolin-7-O-glucoside, etc.), tyrosol, hydroxytyrosol, ferulic acids and caffeic, and secoiridoids (oleuropein, dimethyloleuropein, ligstroside) [16,38,39].

The major polyphenolic compounds of olive leaves are oleuropein (24.54%), hydroxytyrosol (1.46%), tyrosol (0.71%), luteolin-7-O-glucoside (1.38%), apigenin-7-O-glucoside (1.37%), and verbascoside (1.11%) [40]. Caffeic acid, vanillic acid, and luteolin are present in lower amounts. Several factors, such as hydric deficiency, salinity, fertilization, geographical zone, sampling time, light exposition, frost stress, fungi, bacteria, genotypes, leaves age, and alternate bearing patterns of olive can change the synthesis and the number of phenols [41]. The above-mentioned polyphenols and other flavonoids, phenolic acids, phenolic alcohols, and secoiridoids are found also in different proportions in various parts of the olive tree, including the tree (branches or twigs), fruits, leaves, and virgin olive oil [12], and have antioxidant, antimicrobial, antiviral, antihypertensive, anticholesterolemic, anti-inflammatory, hypoglycemic, and anticancer activities [42,43,44,45,46,47]. Also, they decrease the risk of cardiovascular diseases and regulate blood pressure and cholesterol levels in animals [48,49]. All of the activities above are attributed mainly to the predominant phenolic compound oleuropein. Oleuropein has a diverse concentration (mg/kg) in olive leaves ranging from 5526 mg/kg to 26,471 mg/kg, whereas much lower amounts (≤2 mg/kg) are found in olive oil [49]. However, before going any further, it is mandatory to elucidate the biochemical pathway of oleuropein in olive fruits and leaves. Mostly, enzymes, including β-glucosidase, esterases and oxidoreductases, control the hydrolysis and the oxidation of oleuropein. In particular, β-glucosidase releases glucose, forming thus, the aldehydic form of oleuropein aglycon and the dialdehydic form of decarboxymethyl oleuropein aglycon, while esterases hydrolyze the ester bonds of oleuropein, producing glucosyl derivates, hydroxytyrosol and elenolic acid. In the gastrointestinal tract, oleuropein is hydrolysed by the action of enzyme β-glucosidase as a result the production of oleuropein aglycone [50]. Figure 1 shows the typical structures of oleuropein and its hydrolyzed derivatives.

2.1. Antioxidant Activity

Olive leaves exhibited the highest antioxidant activity against free radicals and reduced the oxidative stress in mice [43,44]. Oleuropein is found in the highest amounts in olive leaves and possesses antioxidant, antihypertensive, anti-inflammatory, hypoglycemic, and antiarrhythmic activities [44,51,52,53]. In a study, the intake of olive leaf extract and oleuropein by mice led to a significant reduction in the oxidative action of cyclophosphamide [54]. Oleuropein can contribute to the prevention of low-density lipoprotein oxidation (LDL). This prevention depends on its concentration [55,56]. Also, oleuropein can scavenge hypochlorous acid, which is produced in vivo by neutrophil myeloperoxidase at the site of inflammation and can oxidize the proteins and enzymes [55]. Oleuropein has also a scavenging effect against nitric oxide and increases the inducible nitric oxide synthase (iNOS) expression [57]. An experiment conducted on rabbits showed that oleuropein increases the oxidative resistance of LDL and simultaneously induces a decrease in the levels of total, free, and esterified cholesterol in plasma [58].

Manna et al. [59] investigated the protective effect of oleuropein in an isolated rat heart. Their results show that oleuropein induces a reduction in the release of a significant susceptible indicator of the exposure of the heart to oxidative stress called oxidized glutathione. A prevention was also observed in membrane lipid peroxidation. Puerta et al. [60] reported that an oleuropein concentration equal to 80 μM inhibited the leukotriene B4 generation peritoneal leukocytes of intact rats after their stimulation with calcium ionophore. Visioli et al. [61] researched the effect of oleuropein aglycone on human volunteers. The results of this study revealed that aglycone of oleuropein induced a decrease in an indicator of oxidative stress urinary production of 8-iso-PGF2α. There was no dependence between this reduction and concentration of oleuropein aglycone. According to studies, oleuropein can activate the transcription of nuclear factor erythroid 2-related factor 2, which leads to the stimulation of the intracellular antioxidant enzymes’ expression and increase in concentration of glutathione, vitamin C, β-carotene, and α-tocopherol [62,63,64,65]. In another study, oleuropein at concentrations of 50 and 100 µM caused a reduction in cell death caused by H₂O₂. The percentages of cell death were 6.4% for 50 µM and 9.2% for 100 µM [66]. Also, it was supported that the antioxidant effects of oleuropein have a relationship with the decrease in radical oxygen species in HaCaT cells. IL-1β and thioredoxin also take part in the protective action of oleuropein in HaCaT cells. Oleuropein decreases the concentration of IL-1β and inhibits the activity of caspase-1/IL-1-converting enzymes.

2.2. Anti-Inflammatory Activity

Inflammation is a crucial and defensive response that appears after an infection or damaged tissue and is a responsible factor for the promotion of many diseases such as atherosclerosis, diabetes mellitus, metabolic syndrome, cancer, and diseases of the kidney and nervous system [67]. In a previous study, oleuropein at concentrations 12 μΜ and 200 µM induced a decrease in the level of proinflammatory cytokine interleukin (IL)-6 [64]. This reduction was equal to 38.8% for 12 μM and 45.5% for 200 μM. Castejon et al. [68] found that oleuropein caused an inhibition in murine lupus nephritis NLRP3 inflammasome-related signaling pathways. Acute and chronic inflammation are the two main categories of inflammation. In chronic inflammation, the reactive oxygen (ROS) species take part and are responsible for oxidative degradation and the depletion of antioxidants [69]. IL-1, IL-6, TNF-a, and IFN-γ are the main mediators of the inflammatory response [70,71,72,73]. A damaged tissue, in addition to inflammatory cytokines or mediators, also releases monocyte chemoattractant proteins (MCP-1), cyclooxygenase (COX), inducible form of nitric oxide synthase (iNOS), metalloproteinases (MMP), and adhesion molecules. Furthermore, a significant role in the production of countless pro-inflammatory mediators plays nuclear factor Kappa β (NF-kβ) [42].

Studies have shown the reduction in serum C-reactive protein (CRP), IL-6, endothelial and monocyte adherence molecules (ICAM-1 and VCAM-1), and chemokines using the Mediterranean Diet (MD), which included extra virgin olive oil [74]. A study revealed that oleuropein inhibited the secretion of IL-1β [75]. Ryu et al. [76] reported that oleuropein downregulates the key markers iNOS, COX-2, NF-Kβ, and JNK, and of the two pro-inflammatory interleukins, IL-6 and IL-1β. In mice, the symptoms of colitis, which is caused by dextran sulfate sodium (DSS), were receded by the intake of oleuropein [77]. Oleuropein induced a decrease in COX-2, iNOS, and MMP-9, and suppressed p38 MAPK phosphorylation, which may be due to the upregulation of annexin A1 [78]. The production of TNF-α and IL-1β was attenuated by oleuropein in a mouse model of carrageenan-induced pleurisy, in a rat model of post-traumatic stress disorder, and in rats with spinal cord trauma [79,80,81]. In another study, oleuropein had a beneficial effect on ovariectomy/inflammation experimental model of bone loss in rats [82]. Larussa et al. [83] claimed that the levels of COX-2 and IL-17 and the inflammation of the colon tissue can be reduced by the provision of oleuropein on colon biopsies taken from ulcerative colitis subjects.

2.3. Hypoglycemic Activity

The symptom of diabetic patients (both type 1 and 2), chronic hyperglycemia, is responsible for damages of organs such as heart and blood vessels, nerves, kidneys and eyes [84]. Oxidative stress is responsible factor for diabetes complications and the increase in insulin resistance and diabetes mellitus [85,86,87,88,89]. Flavonoids and polyphenols can contribute to the reduction in negative effects of oxidative stress and free radicals in diabetic patients [90]. The high concentration of phenols in olive leaves is very significant for human health [91]. The most predominant phenolic compounds that possess many beneficial properties for human health are oleuropein and its hydrolysis products, elenolic acid and hydroxytyrosol [92,93]. All these beneficial properties are probably attributed to the antioxidant properties of oleuropein and its hydrolyzed products [91].

The antihyperglycemic properties of oleuropein have been supported by Gonzalez et al. [92] and Romani et al. [93]. Also, according to Al-Azzawie et al. [94], oleuropein may cause inhibition in hyperglycemia and oxidative stress caused by diabetes, and the supply of oleuropein may suppress diabetic complications that come from oxidative stress. In a study, the supply of oleuropein in diabetic type 1 rats induced a significant inhibition in the increase in glucose compared to the untreated rats with oleuropein rats [95]. The provision, for 4 weeks, of oleuropein (8 mg/kg body weight) and hydroxytyrosol (16 mg/kg body weight) rich extracts caused a significant reduction in the serum glucose levels [63]. Oleuropein on physiological concentrations activated the adenosine monophosphate-activated protein kinase on cultured mouse byoplast, which has been connected with the increased cellular internalization of glucose [96]. Oleuropein caused a decrease in glucose transporter GLUT-2 (glucose transporter-2) of cultured cells [64]. GLUT-2 regulates the absorption of intestinal glucose and insulin secretion.

According to Wu et al. [97], the secretion of insulin by pancreatic β-cells is facilitated by oleuropein, which is connected with the activation of the MAPK pathway. Also, the cytotoxicity of β-cell-caused amyloids is prevented by oleuropein. A recent study showed that the pancreatic β-cell cytotoxicity induced by human islet amyloid polypeptide aggregates was inhibited by oleuropein aglycone at a concentration of 10 μΜ, as a result the protection of the plasma membrane from permeabilization and the prevention of cell death [98]. A reduction was achieved in the concentration of blood glucose, plasma insulin, and hepatic glycogen of mice after the intake of oleuropein at concentrations of 5–10 ppm per day [99]. Also, another study showed a significant decline in blood glucose levels, an increase in glucose tolerance, a reduction in the homeostasis model assessment insulin-resistance index, and a promotion of protein kinase B activation after the intake of oleuropein at concentrations of 200 ppm for 15 days in diabetic mice [100]. All the histopathological changes in mice are restored by the oleuropein. Also, important changes were recorded in the composition of the gut microflora of diabetic mice after oleuropein consumption. A study demonstrated that polyphenols of extra virgin olive oil (EVOO) had important beneficial effects on intestinal bacteria [101]. Generally, the dysregulation of gut microbiota can induce a modification in intestinal permeability and affect several pathophysiological mechanisms that can have a negative effect on glucose metabolism. Oleuropein participates beneficially in glucose metabolism and can cause changes in the glucose transport and intracellular metabolism, increase in insulin susceptibility and glucose-stimulated insulin production by pancreatic β-cells.

The consumption of polyphenol-rich EVOO (25 mL per day, 577 mg of phenolic compounds per kg of body weight) for 4 weeks induced an important decrease in body weight, fasting plasma glucose, and glycated hemoglobin [102]. These changes were connected with an important mitigation in serum visfatin, a pro-inflammatory adipocytokine. Another study showed that the intake of EVOO decreased the risk of developing type 2 diabetes by 16% [103]. Also, the levels of plasma glucose and glycated hemoglobin in type 2 diabetics were significantly reduced by the supplementation of EVOO, showing that the consumption of EVOO could be useful for the treatment of type 2 diabetes. The intake of oleuropein in capsules (51 mg/day) or a placebo for 12 weeks significantly improved the insulin sensitivity and pancreatic β-cells secretory capacity of 46 middle-aged, overweight men [104]. In a study, the intake of oleuropein (35 to 200 mg/day) reduced the post-prandial blood glucose response after the intake of sucrose but did not affect post-prandial glucose after the consumption of bread or glucose [105]. Oleuropein inhibited the sucrase and GLUT-2-mediated transport, but α-amylase was not affected significantly. A study showed a fast reduction in blood glucose after the oral supplementation of two capsules of Tensiofytol per day (100 mg of oleuropein and 20 mg of hydroxytyrosol) by hypertensive patients suffering from obesity and/or diabetes [106].

Blood glucose and dipeptidyl-peptidase 4 (DPP-4) protein concentration and activity were lower after the supplementation of 20 mg oleuropein in human patients subjected to the Mediterranean diet compared to those people who were subjected to the Mediterranean diet without the addition of oleuropein [98]. Also, higher levels of serum insulin and glucagon-like peptide-1 (GLP1) were monitored. The supplementation of oleuropein at 20 mg before the lunch on blood glucose and DPP-4 was reported, and an important decline in markers of oxidative stress, such as soluble NADPH oxidase-derived peptide activity and 8-iso-prostaglandin-2α was reported [107]. Mediterranean diet meals containing the amount of oleuropein that is contained in 10 g of EVOO reduced blood glucose levels and DPP-4 activity and increased insulin and GLP-1 levels in comparison to a conventional Mediterranean diet meal without oleuropein [108]. In another study, blood glucose levels exhibited a lower increase in type 2 diabetic patients after the consumption of 40 g of oleuropein-enriched chocolate in comparison to the intake of conventional chocolate [109]. These findings prove that oleuropein might induce a decline in post-prandial blood glucose by its involvement in a mechanism that counteracts oxidative stress-mediated incretin downregulation.

2.4. Anticholesterolemic Activity

The supply of oleuropein to diabetic rats inhibited significantly (24.80%) the increase in cholesterol compared to the no-fed-oleuropein diabetic rats (

Table 2. Effect of oleuropein on triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very low-density lipoprotein (VLDL) in diabetic rats [95].

Parameter	Control	Diabetic	Diabetic + Oleuropein
TG (mg/dL)	82.33 ± 14.75	126.57 ± 18.59	97.69 ± 13.91
TC (mg/dL)	72.01 ± 16.35	110.88 ± 28.48	83.38 ± 20.75
HDL (mg/dL)	39.00 ± 13.29	25.01 ± 9.19	38.13 ± 10.67
LDL (mg/dL)	26.52 ± 21.58	60.57 ± 28.18	25.73 ± 21.06
VLDL (mg/dL)	16.47 ± 2.49	25.30 ± 3.44	19.55 ± 2.78

) [95]. The provision of oleuropein and hydroxytyrosol-rich extracts for 28 days significantly decreased the cholesterol levels of rats [63].

A study conducted in mice treated with oleuropein (100 mg/kg/day) for 42 days showed that oleuropein acts as a ligand of the peroxisome proliferator-activated-receptor alpha (PPARα), which is responsible for the activation of gene encoding enzymes that tale part in fatty acid cell uptake, mitochondrial β-oxidation, microsomal ω-oxidation, genes that encode many apolipoproteins and a family of genes that critically participate in many metabolic pathways [110]. Consequently, PPARα controls the circulating fatty acids and biochemical processes associated with insulin sensitivity and glucose metabolism. The oral intake of oleuropein at a concentration of 50 mg/kg by cholesterol-fed rats induced mitigation in body weight and adipose tissue mass [111]. This reduction in body weight is related to the significant inhibition of peroxisome proliferator-activated-receptor gamma (PPARγ) and an increase in serum adiponectin levels. The hypocholesterolemic activity of oleuropein was reported by Romani et al. [93].

2.5. Antihypertensive Activity

The hypotensive effect of olive leaves has been well studied [112,113,114]. The most popular phenolic constituents that are contained in olive leaves are oleuropein and oleacein; however, the latter is active only in good quality extra virgin olive oil [115]. According to Petkov and Manolov [116] oleuropein had a hypotensive, coronary dilating, and antiarrhythmic action. Another study demonstrated that hydroxytyrosol exhibited a calcium-antagonistic action [117]. Hansen et al. [118] reported that oleacein can inhibit the distinct angiotensin-converting enzyme (ACE). Bioactive peptides that act as inhibitors of ACE have been obtained from various sources of animal and vegetable origin. The majority of ACE inhibitory peptides are isolated by the action of specific proteases on different sources of dietary proteins. These include dairy and fermented milks, eggs, soybeans, chickpeas, peanuts, tuna, sardines, shrimp, chicken, and squid, among others. A representative example of ACE inhibitor peptides is found in hydrolysates of milk proteins.

The development of severe hypertension and atherosclerosis in insulin-resistant rats was prevented after the supply of isolated triterpenoids from African wild olive leaves, Greek olive leaves, and olive leaves from Cape Town [119]. The isolate of the African wild olive leaves contained 0.27% iso-molecular mixture of oleanolic acid and ursolic acid. The isolate of Greek olive leaves consisted of 0.71% oleanolic acid, and the Cape Town olive leaves contained 2.47% oleanolic acid. The provision of Arbequina table olives (3.85 g/kg) in rats that suffered from hypertension caused a transient reduction in blood pressure, which was observed on the second week after the provision and lasted until the seventh week [120]. Generally, some studies have demonstrated the antihypertensive effect of olive oil polyphenols in rats that suffered from hypertension [121], people who suffered from high cholesterol levels [122], pre-hypertensive [123,124] and people who suffered from hypertension [125], and coronary heart disease patients [126]. A study has shown that the consumption of olive oils with at least 150 mg/L (ppm) of polyphenols may have positive effects on systolic blood pressure [127]. A significant blood pressure reduction has been observed by the consumption of triterpenes for many years [128,129]. Recently, a study revealed that olive oils containing high amounts of triterpenes (86–389 ppm) did not affect blood pressure [130]. A study showed that OLE exhibited a protective action against the rise in blood pressure caused by nitro-L-arginine methyl ester at concentrations of 50 and 100 ppm [131]. A decrease in systolic left ventricular pressure and heart rate of rabbit hearts was achieved after the intake of oleuropein [132]. The possible mechanisms of the cardioprotective effects of olive leaf extract are the blockade of beta receptors [133], angiotensin-converting enzyme (ACE) inhibition, Ca² + antagonism [134], and vasodilatory NO pathways [93].

In a study conducted in 20 monozygotic adult twin pairs suffering from mild hypertension in Germany, the intake of olive leaf extract at a dose of 0.5 or 1.0 g daily for 56 days led to a significant decline in systolic and diastolic blood pressure [135]. The consumption of olive (Olea europaea L.) leaf extract (EFLA^®943) by patients at the dose of 0.5 g twice daily in a flat-dose manner for 56 days caused a mitigation in systolic and diastolic blood pressures of patients who were taking Captopril at a dose of 12.5–25 mg twice daily [136]. An important decrease in blood pressure in patients was observed after the intake of oleuropein in a dose of 1.0 g four times daily for 84 days [137]. A reduction in the digital volume pulse stiffness index was also found after the consumption of 51 mg of oleuropein once by 18 male and female volunteers [135]. Consuming olive leaf extract containing high amounts of polyphenols (136 mg) for 42 days reduced systolic and diastolic blood pressure throughout the day, and the 24 h measure in comparison to the control group [137]. Another study demonstrated that the consumption of olive leaf extract tablets containing 16% oleuropein and at least 0.62 mg of luteolin induced a decrease in inflammatory factors associated with hypertension, while no changes were exhibited in renal and liver functions [138].

The provision of olive leaf extract containing oleuropein at concentrations of 20–60 ppm on type 2 diabetic animals accompanied by renal hypertension, caused a decline in systolic blood pressure and a rise in nitric oxide concentration, which is connected with beneficial vasodilator effect [126]. Researchers have attributed the protective action of oleuropein against the cardiovascular system in type 2 diabetes to the inhibition of the angiotensin-converting enzyme action, the stenosis of calcium channels, the swelling of blood vessels, the restoration of endothelium function, and the scavenging of oxygen free radicals [64,139,140]. Oxidative stress plays an important role in the pathogenesis of cardiovascular disorders, and the increase in reactive oxygen species in human carotid arteries of older people, which results in the excessive accumulation of superoxide ions by NADPH oxidase [141]. The increase in oxygen-reacted species in the vascular tissue of hypertensive rats has been connected with the generation of hypertension [142]. A significant beneficial effects of different polyphenol-rich plant products on hypertension-induced alterations of the carotid have been reported [143,144]. This beneficial effect leads to a significant reduction in blood pressure and systemic oxidative stress.

2.6. Anticancer Activity

Generally, cancer is treated by surgery in the early stage or by specific chemotherapies and immunotherapies when it has spread. The disadvantage of chemotherapies and immunotherapies is the resistance to drugs and the generation of host side effects. One way for the reduction in drug resistance and increase in treatment efficacy is the combination of conventional treatment with biological agents (complementary therapy).

Bouallagui et al. [145] observed an inhibition of accumulation of luminal breast cancer cells (MCF-7 cells) by the supply of extract rich in oleuropein and its derivative hydroxytyrosol. Similar results were reported by Han et al. [146]. Oleuropein can also negatively affect the NF-kB expression, which is responsible for the control of many genes driving cancer development and progression [147,148]. Oleuropein exhibited a pro-oxidant and anti-proliferative effect in androgen-insensitive DU145 prostate cancer cells [146]. Oleuropein also prevents azoxymethane, which causes pre-neoplastic lesions of the colon and decreases dysplasia and DNA damage [147]. A previous study reported that HIF-1a was inhibited significantly by oleuropein and hydroxytyrosol, and the expression of p53 in HT-29 human colon adenocarcinoma cells was promoted. The latter led to apoptosis [148]. The oleuropein inhibitory effect on human colon carcinoma cells may be facilitated by long-chain fatty acids and their esters, such as hydroxytyrosol oleate [149]. A high anti-proliferative activity on pancreatic cancer cells (MiaPaCa-2 cells) by oleuropein has also been reported for Corregiola leaf extracts.

In a previous study, 51 analogs of oleuropein were studied on several human cancer cells and it was reported that analog 24 expressed the highest inhibitory effect in vitro (human colon cancer cells HCT-116, human cervical carcinoma cells HeLa, MCF-7 cells) as well as in vivo (B16-F10 mouse melanoma cells) [150]. It was found that oleuropein promotes apoptosis in cancer cells, like HeLa cells [151], HepG2 human hepatoma cells [152], SH-SY5Y human neuroblastoma cells [153], and HCT116 cells [154]. Also, oleuropein exhibited pro-apoptotic activity against HL60 human promyelocytic leukemia cells [155]. Studies have proven the effect of oleuropein on the most aggressive skin cancer, melanoma [156]. A study revealed that oleuropein altered the dynamics of intracellular Ca²⁺ in mesothelioma cancer cells and changed the T-type Ca²⁺ channels [157]. Another study showed that oleuropein can affect the modulation of onco-miRNAs (miRNA-21 and miR-155) genes, which suppress tumor development [158]. Tezcan et al. [159] claimed that oleuropein may affect the expression of miRNAs, miR-137, -145, and -153, in glioblastoma multiforme (GBM) cancer stem cells. A significant role in cancer treatment, play the histone deacetylases (HDAC2 and HDAC3) inhibitors. In a previous study, it was supported that oleuropein may inhibit histone deacetylases inhibitors (HDAC2 and HDAC3) [160].

Obesity is a reason for the development of many cancers and metastatic dissemination. In mice melanoma cells, oleuropein inhibited tumor growth and lymph node metastasis and abrogated angiogenesis and lymphangiogenesis through the decrease in the peroxisome proliferator-activated receptor γ and the infiltration of M2 macrophages [161]. Anter et al. [155] reported that oleuropein displayed anticancer/pro-apoptotic activity on leukemic cells, like HL60 human promyelocytic leukemia cells. Oleuropein showed anticancer activity in glioma cells by reducing the ability of MMP-2 and -9 to invade extracellular tissues [162]. The promotion of a decline in the metastatic ability of breast cancer cells by oleuropein was demonstrated by Hassan et al. [163]. This reduction was attributed to MMP-2 and MMP -9 decrease, strengthened by the promotion of tissue inhibitors of MMP (TIMP) 1,3,4. The authors also reported that the cancer cell invasion and propagation were regulated by the balance of MMP and TIMP.

3. Additional Anticancer, Antiviral, and Antimicrobial Properties of Oleuropein and Olive Leaf Extracts

Several studies have been carried out on the use of oleuropein in complementary cancer therapy on account of its anticancer properties, anti-proliferative, and pro-apoptosis activity. The side effects and toxicity in tumor-bearing patients caused by anticancer drugs could be treated by oleuropein. The cell accumulation of PC3 prostate cancer cells was affected by the application of co-treatment with very low amounts of doxorubicin and Ole [164]. This co-treatment could cause a significant promotion of autophagy. The relationship of cancer cells by autophagy reveals a new therapeutic treatment for Ole. The protection of cardiotoxicity of doxorubicin by oleuropein could be a good reason for the application of oleuropein in cancer co-treatments [165]. Oleuropein synergistically increases bevacizumab’s effect, which is used in the treatment of the most malignant human cancer GMB by preventing VEGFA, MMP-2, and -9 activities [166]. A study has shown the participation of PI3K/Akt/mTOR in the promotion of protein synthesis and suppression of autophagy [167].

The mTOR inhibition and autophagy induction caused by oleuropein could be used in the development of new therapeutic methods against GBM. Oleuropein can affect cell proliferation in BRAF melanoma cells by downregulation of the pAKT/pS6 pathway [168]. Also, oleuropein enhances the cytotoxic effect of dacarbazine against BRAF melanoma cells. The exposure of melanoma cells to oleuropein leads to reverse trastuzumab resistance in HER2-overexpressing breast cancer cells [169]. Oleuropein at a concentration of 200 μΜ can enhance the decrease in MMP-7 gene expression in HepG2 cells caused by cisplatin [170]. This was attributed to the reduction in the cancer-promoting ability of nerve growth factor (NGF) on HepG2 cells. The mature NGF is produced from its precursor form, pro-NGF, through MMP-7 proteolytic cleavage and exhibits a pro-survival effect on HCC cells. The stimulation of oleuropein with cisplatin simultaneously potentiates caspase-3 gene expression, which leads to the enhancement of apoptotic rate. A previous study supported the idea that oleuropein potentiates the sensitivity of nasopharyngeal carcinoma cells to radiation [171]. Generally, oleuropein can discriminate the cancer from normal cells, prevent accumulation, and promote apoptosis in several tumors such as mesothelioma, GBM, and melanoma. These abilities above have gained the researchers’ interest in the discovery of new cancer methods of treatment.

3.1. Antiviral Activity

The antiviral activities of O. europea against viral hemorrhagic septicemia virus (VHSV), herpes virus, hepatitis virus, rotavirus, bovine rhinovirus, canine parvovirus, and feline leukemia virus have been proven [172,173]. In Vero cells, olive leaf hydroalcoholic extract displayed antiviral activity against the herpes simplex virus type at concentrations higher of 1 mg/mL [174]. Leaf extracts from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) showed significant antiviral activity against the herpes simplex type 1 (HSV-1) were assessed on Vero cells at a concentration of 200 ppm and 820 ppm, respectively [175]. Also, Lee-Huang et al. discovered that olive leaf extracts-displayed antiviral action against HIV-1 infection and replication at a concentration of 200 ppb (μg/L) [176]. The replication of HSV-1 in HeLa cancer cells was inhibited by the half-maximal effective concentration of oleuropein 0.241 mg/mL [177]. A study showed that oleuropein extracted from Jasminum officinale L. var. grandiflorum inhibited HBV action in HepG2 2.2.15 cells test in vitro and DHBV activity in ducklings test in vivo [178]. Oleuropein effectively blocks HBsAg production in HepG2 2.2.15 cells. The effectiveness of the block production of HBsAg was analogous to the oleuropein dose with IC50 equal to 23.2 μg/mL. The reduction in DHBV replication by oleuropein in infected ducklings by DHBV in vivo was achieved at a concentration of 80 mg/kg of oleuropein.

According to a recent study, olive leaf phytoconstituents induced a significant inhibition in SARS-CoV-2 [179]. The standardized olive leaf extract (SOLE) containing 20% oleuropein exhibited moderate antiviral activity against SARS-CoV-2 with an IC50 of 118.3 μg/mL. Also, olive leaves can significantly inhibit respiratory viruses, such as the syncytial virus and type 3 parainfluenza virus [180]. Oleuropein and other phytochemical constituents of olive leaves (3,4-hydroxyphenylethanol, 3β-Hydroxyolean-12-en-28-oic acid, 2α,3β-Dihydroxyolean-12-en-28-oic acid, 3′,4′,5,7-Tetrahydroxyflavone, 7-O-beta-D-Glucosyl-5,7,3′,4′-tetrahydroxyflavone, verbascoside, apigenin-7-O-glucoside and 3,4′,5,7-Tetrahydroxyflavone) have shown in silico antiviral activities against SARS-CoV-2 viral proteases (Mpro/3CLpro, PLpro), TLRs, ACE2, RBD, NSP15, HSPA5, SBDβ, TMPRSS2, S protein and Furin [181,182,183,184,185,186,187,188]. Hydroxytyrosol, luteolin, and kaempferol have shown antiviral action against SARS-CoV-2 in vitro [181,188,189,190,191]. Non-structural protein 12 (nsp12), 3C-like protease (3CLpro), and papain-like protease (PLPro) are essential keys for the preparation of targeted antiviral drugs (DAAs) against COVID-19. The 3C-like protease (3CLpro) and papain-like protease (PLPro) take part in the SARS-CoV-2 replication cycle by controlling the polyprotein produced during transcription into functional subunits [192,193,194].

3.2. Antimicrobial Activity

Oleuropein and other phytochemicals found in olive leaf extracts can inhibit or delay the growth rate of a plethora of bacteria and fungi. Olive extract’s oleuropein has shown high antimicrobial activity on a wide range of Gram-positive and Gram-negative bacteria and fungi, such as Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Candida albicans, and Cryptococcus neoformans in vitro [195,196]. Oleuropein can suppress the growth of both ATTC bacterial strains and clinical bacterial strains (e.g., Haemophilus influenzae, Moraxella catarrhalis, Salmonella typhi, Vibrio parahaemolyticus, Staphylococcus aureus) [197,198]. The dialdehydic form of decarboxymethyl oleuropein and ligstroside aglycons, hydroxytyrosol, and tyrosol exhibited bactericidal activity against harmful bacteria of the microflora of gut Clostridium perfringens and Escherichia coli and against beneficial bacteria such as Lactobacillus acidophilus and Bifidobacterium bifidum [198]. Foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, Yersinia sp., and Shigella sonnei are killed after contact with the phenols above for 1 h. In a study, the growth of Staphylococcus aureus and Bacillus subtilis were inhibited at minimum inhibitory concentrations 0.6 to 1.6 mg/mL and 1.2 to 1.8 mg/mL, respectively [199]. According to Bisignano et al. [200], Staphylococcus aureus exhibited minimum inhibitory concentration for oleuropein from 0.0625 to 0.5 mg/mL for certified strains of the American Type Culture Collection (ATCC) and from 0.0312 to 0.250 mg/mL for clinical isolates. In a study, it was found that oleuropein displayed high inhibitory action against Campylobacter jejuni, Helicobacter pylori, and Staphylococcus aureus with minimum inhibitory concentration as low as 0.31% v/v, 0.62% v/v, and 0.78% v/v, respectively [200]. Previous studies have demonstrated that Campylobacter jejuni isolates were more sensitive to various natural compounds compared to other microorganisms such as Escherichia coli, Salmonella, and Listeria [201].

A previous study reported that Staphylococcus aureus exhibited the highest sensitivity to oleuropein extracts, while Escherichia coli O1577:H7 displayed the highest resistance [202]. Leaf extracts of Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) exhibited antimicrobial action against Gram-positive bacteria at minimum inhibitory concentrations of 7.81 and 15.61 μg/mL and Staphylococcus aureus ATCC 6538 at minimum inhibitory concentrations of 15.61 and 31.25 μg/mL [174]. Moreover, oleuropein may enhance the bactericidal action of peracetic acid against L. monocytogenes biofilms [203]. The combination of oleuropein with commercial sanitizers increased their bactericidal action, especially for Chlorhexidine digluconate, which resulted in an approximately 60-fold decrease in the minimum inhibitory concentrations values for Staphylococcus aureus and Listeria monocytogenes (2.5 mg/mL) [204]. Oleuropein can interact with the phosphate group and modify the phospholipid/water interface properties [205]. In another study, it was reported that oleuropein interacted with phosphatidylglycerol and phosphatidylethanolamine of Staphylococcus aureus and Escherichia coli, and the antimicrobial activity was exhibited only at the cell surface because oleuropein is not able to form stable mixed monolayers with the lipids in the interface [206]. This phenomenon may be attributed to the prevention of the glycosidic group of oleuropein diffusion through the cell membrane [197]. Juven et al. [207] reported that oleuropein affected a significant leakage of glutamate, potassium, and inorganic phosphate from Lactobacillus plantarum. Oleuropein did not change the rate of glycolysis when incorporated into resting cells of L. plantarum, but it decreased the ATP content of the cells, whereas other studies [208,209,210,211] have reported a considerable antimicrobial activity and sanitizing effects of oleuropein.

4. Conclusions

Olive leaf extracts give a great and useful opportunity for the discovery of new treatment methods due to their high antioxidant, anti-inflammatory, anticancer, anticholesterolemic, antihypertensive, antiviral, and antimicrobial activities. These properties above are mainly associated with the phenolic constituents of olives, olive oil, and herein in olive leaf extracts, such as oleuropein and its hydrolyzed derivatives elenolic acid, and hydroxytyrosol. Therefore, the food and beverage industry should consider olive leaf extracts as an excellent ingredient in the development of new fortified beverages that strengthen the immune system and longevity. In this context, more extensive research studies are required in the near future to optimize the extraction methods of oleuropein and its hydrolyzed derivatives and convey more exhaustive clinical trials to fix and standardize the effective concentration of these polyphenols against chronic or other pathophysiological disorders.