Rosmarinus officinalis L. (rosemary) as therapeutic and prophylactic agent

Phytocompounds and pharmacological activities

R. officinalis L., popularly known as rosemary, is a plant belonging to the family Lamiaceae and originated from the Mediterranean region. However, it could be found all over the world. It is a perennial and aromatic plant, shrub-shaped with branches full of leaves, having a height of up to two meters and green leaves that exude a characteristic fragrance. R. officinalis may be used as a spice in cooking, as a natural preservative in the food industry, and as ornamental and medicinal plant [1–4].

Several phytocompounds presenting pharmacological activities may be isolated from essential oils and extracts of R. officinalis L. (Fig. 1), varying the concentration of these molecules in each specimen of the plant. The phytocompounds most reported include caffeic acid, carnosic acid, chlorogenic acid, monomeric acid, oleanolic acid, rosmarinic acid, ursolic acid, alpha-pinene, camphor, carnosol, eucalyptol, rosmadial, rosmanol, rosmaquinones A and B, secohinokio, and derivatives of eugenol and luteolin [5–8]. Pharmacological effects of phytocompounds from R. officinalis L. were showed in Table 1.

Phytocompound	Pharmacological effect	Reference
Caffeic acid	a. Antibacterial	[168]
	b. Antioxidant	[169]
	c. Inhibitory effect of tumor cell immigration	[170]
	d. Inhibitory effect of tumor cell proliferation	[171]
	e. Protective effect of transplanted livers	[172]
	f. Apoptotic effect on tumor cells	[173]
Carnosic acid	a. Antiproliferative	[174]
	b. Protective effect of photoreceptor cells	[175]
	c. Antitumor	[176]
	d. Anti-inflammatory	[177]
	e. Inhibitory effect of digestive enzymes (lipase, α-amylase, and α-glucosidase)	[178]
	f. Suppressive effect of lipogenesis	[179]
Chlorogenic acid	a. Antioxidant	[180]
	b. Protective effect against lead-induced renal damage	[181]
	c. Protective effect against colitis	[182]
	d. Anti-infective	[183]
Oleanolic acid	a. Antiviral	[184]
	b. Protective effect against oxidative stress-induced apoptosis	[185]
	c. Antiproliferative	[186]
	d. Antitumor	[187]
	e. Antioxidant	[188]
Rosmarinic acid	a. Neuroprotective	[189]
	b. Protective effect against nicotine-induced atherosclerosis	[190]
	c. Complementary agent to the anticancer chemotherapy	[191]
	d. Anxiety control	[192]
	e. Antiproliferative	[193]
	f. Antiviral	[194]
Ursolic acid	a. Cytotoxic for tumor cells	[195]
	b. Anticancer	[196]
	c. Inducer of osteoblastic activity and reducer of osteoclastic activity	[197]
	d. Hypouricemic	[198]
	e. Proapoptotic	[199]
	f. Inductor of insulin sensitivity	[200]
	g. Protective effect against diabetic nephropathy	[201]
	h. Reducer of weight gain and atherosclerosis	[202]
Alpha-pinene	a. Antibacterial	[203]
	b. Antimicrobial	[204]
	c. Protective effect against aspirin-induced oxidative stress	[205]
	d. Protective effect against peptic ulcer	[206]
Camphor	a. Immunomodulatory	[207]
	b. Antiproliferative	[208]
	c. Hypoglycemic	[209]
	d. Antimicrobial	[210]
	e. Antifungal, antihyphal, and antibiofilm	[211]
Carnosol	a. Antiproliferative	[212]
	b. Protective effect against renal ischemia-reperfusion injury	[213]
	c. Antifungal	[214]
	d. Proapoptotic and proautophagic	[215]
	e. Anti-inflammatory	[216]
	f. Anti-atopic dermatitis	[217]
	g. Antidiabetic	[218]
Eucalyptol	a. Proapoptotic	[219]
	b. Antibiofilm	[220]
	c. Control of infection and inflammation	[221]
	d. Anti-inflammatory	[222]
	e. Antinociceptive	[223]
	f. Antiviral	[224]
Rosmanol	a. Antinociceptive, antidepressant, and anxiolytic	[82]
	b. Anticancer	[225]
Eugenol	a. Acaricidal	[226]
	b. Antifungal	[227]
	c. Chemotherapeutic on cervical cancer cells	[228]
	d. Antiproliferative	[229]
	e. Anti-inflammatory and antioxidative	[230]
Luteolin	a. Anti-inflammatory	[231]
	b. Anti-atopic dermatitis	[232]
	c. Proapoptotic and proautophagic	[233]
	d. Antimicrobial	[234]
	e. Antiproliferative	[235]
	f. Protection of microglia against rotenone-induced toxicity	[236]
	g. Inhibitory effect of glucocorticoid-induced osteoporosis	[237]

R. officinalis L. can promote several pharmacological effects due to the interaction between the molecules of the plant and the organic systems. The effects demonstrated by this plant include (1) ability to attenuate asthma, atherosclerosis, cataract, renal colic, hepatotoxicity, peptic ulcer, inflammatory diseases, ischemic heart disease [9, 10]; (2) antioxidant and anti-inflammatory actions of rosmarinic acid [11, 12]; (3) control of hypercholesterolemia and oxidative stress and relief of physical and mental fatigue [13]; (4) myocardial blood pressure reduction with rosmarinic acid [12]; (5) antiulcer action [14]; (6) lipid peroxidation reduction in heart and brain [15]; (7) antiangiogenic and neuroprotective effects of carnosic acid and carnosol [16]; (8) prevention of problems related to atherosclerosis [17]; (9) anticancer and antiproliferative effects [18–21]; (10) antiviral [22]; and antimicrobial actions [23]; (11) hepatoprotective [24], nephroprotective [25] and radioprotective-antimutagenic capacities [26]; (12) glycemia reduction [27]; (13) muscle relaxant and treatment for cutaneous allergy [28]; (14) ability to treat depressive behavior [29].

Extraction methods

The extract of plant can be obtained from roots, stems, leaves, flowers, fruits, seeds, and bark. Therefore, fresh or dried samples can be used. However, according to Vongsak et al. [30], a higher level of flavonoids was detected in dried samples of Moringa oleifera leaves, as compared to fresh samples.

Another relevant aspect is the size of the particles that can interfere in the extraction process. Since, the smaller the particle size, the higher the interaction between the plant sample and the solvent to obtain the extract. Thus, powder samples have better contact with the solvent than crushed samples. Nanoparticles containing Centella asiatica presented higher yields than microparticles, when in contact with methanol [32].

The solvent to extract active compounds may interfere with the final yield of these molecules. In Psidium guajava L. leaves extracts, the concentration of alkaloids, carbohydrates, flavonoids, saponins, and tannins was higher in ethanolic and hydroalcoholic solvents than petroleum ether, chloroform or water [33]. The presence or absence of some chemical constituents in the solvent can interfere in the pharmacological activities; e.g., the antioxidant activity was more prominent in Garcinia atroviridis methanolic extract than in aqueous extract. On the other hand, the aqueous extract of this plant presented better antihyperlipidemic effect [34].

The chosen method for extraction of compounds may interfere the yield of the sample. The use of high temperatures (90 °C) for C. asiatica extraction provided an increased yield of phenolic compounds which provided better antioxidant activity [35]. However, in microwave assisted extraction, a duplicate increase in the yield of C. asiatica triterpene was detected, compared to the Soxhlet extraction [36].

The purpose of the study

Background

Myocardial infarction is a condition characterized by the cardiac muscle necrosis, due to cell death caused by inflammation, which may be initiated by oxidative stress that produces cytokines synthesis, such as tumor necrosis factor-α (TNF-α) and interleukins (IL-1β and − 6); reabsorption of necrotic tissue; exacerbated collagen deposition; and hypertrophy. Both reactive oxygen species (ROS) and cytokines may induce the action of metalloproteinases (MMP), as well as the collagen accumulation, which is responsible for causing changes in the size, weight and function of the heart. Besides, the continued presence of metabolites from affected cells may also provide these changes. Thus, a forced adaptation of the organ to a new reality may occur, providing a cardiac remodeling that could lead to heart failure [37, 38]. In this manner, the use of antioxidants has been evaluated in these cases. Also, other types of medications have been used, such as the vasodilators prazosin, diltiazem, and felodipine, showing no satisfactory outcome regarding mortality reduction or hospitalization [39–41]. Positive inotropic drugs, which have a good hemodynamic effect, can also present significant side effects, about patient survival due to the neurohormonal activation and ventricular arrhythmias. Milrinone, flosequinan, pimobendan, ibopamine, and vesnarinone have caused an increase in mortality in chronic heart failure [42–45]. To control the oxidizing agents, the effectiveness of some natural products, including R. officinalis L., has been investigated, mainly by the presence of bioactive molecules with antioxidant capacities, such as rosmarinic acid, carnosic acid, and carnosol [46].

Methodology

The effect of the supplementation with R. officinalis L. leaves was evaluated on cardiac remodeling after myocardial infarction in male Wistar rats [47]. For this, healthy animals and infarcted animals were fed with standard chow or chow containing 0.02% or 0.2% of R. officinalis L. leaves for 90 days. The animals were evaluated by transthoracic echocardiographic exam. Other analyses were performed on the left ventricle from the sacrificed animals, such as: (i) checking of the infarct extension in size and length; (ii) muscular viability by endocardial and epicardial circumferences; (iii) chemical mediators levels, such as TNF-α, IFN-γ, IL-10, MMP-2 and TIMP-1; (iv) total protein and lipid hydroperoxide levels; (v) enzymatic activity of glutathione peroxidase, superoxide dismutase, catalase; and (vi) cardiac metabolism checked by the activity of β-hydroxyacyl coenzyme-A dehydrogenase, lactate dehydrogenase, citrate synthase, Complex I (NADH:ubiquinone oxidoreductase), Complex II (succinate dehydrogenase), and ATP synthase.

Findings

No deaths were observed in healthy animals; however, among infarcted animals, two deaths were verified in the groups that received standard chow and supplementation with 0.02% of R. officinalis L. leaves, besides one death in the group supplemented with 0.2%. Nevertheless, the infarction size was similar among infarcted animals and was not found any difference between the weight gain and systolic arterial pressure in all groups. The infarction generated an adverse cardiac remodeling, demonstrated by increased of left ventricular diameter; high collagen percentage; alterations in the diastolic and systolic functions; intensification of oxidative stress; metabolic changes evidenced by modification of enzymatic activity; increased of MMP-2 activity and decreased of IL-10, TNF-α, IFN-γ levels. However, the supplementation with R. officinalis L. leaves improved diastolic function, reduced muscle hypertrophy, provided morphological and functional changes in the heart of infarcted animals, verified by increased β-oxidation of fatty acids and reduced lactate oxidation, besides improved respiratory chain performance. R. officinalis L. significantly decreased oxidative stress, even though the used concentrations provided different scenarios regarding the diastolic function and hypertrophy, since the supplementation with 0.02% presented lower left atrium and supplementation with 0.2% demonstrated higher Complex II activity. Additionally, collagen percentage, cytokines levels, and MMP-2 activity were not altered with any of the supplementations.

Background

Lipid metabolism may be altered and lead to increased levels of total cholesterol, low-density lipoprotein cholesterol and triacylglycerols, in the blood, causing cardio- and cerebrovascular disorders [55]. The use of some medications may induce to dyslipidemia, including antirheumatics [56], second-generation antipsychotics [57], antiretrovirals [58], and antibiotics (gentamicin) [59]. In this way, plant products to control dyslipidemia has been investigated.

Methodology

The toxicity caused by gentamicin was attenuated in Sprague-Dawley rats with administration of R. officinalis L. extract [60]. In this study, the animals received gentamicin by intraperitoneal injection and 8% R. officinalis L. aqueous extract orally (10 mL/kg), the control groups were treated with saline solution (0.9% NaCl) or gentamicin (60 mg/kg). Doses were daily given over 10 days.

Findings

The body weight of the animals increased significantly in the group treated with gentamicin compared to the control group (saline), demonstrating that the antibiotic could change the body mass. On the other hand, there was an inhibition of the body weight gain using the co-administration of the extract. Besides, the plant product also significantly reduced the liver weight of the animals compared to the group treated with antibiotic. The liver injuries caused by gentamicin were reversed with the administration of the extract. The harmful effects of this antibiotic were attenuated with plant extract at the liver level, providing a significant decrease in alanine aminotransferase and aspartate aminotransferase activity and total bilirubin levels. R. officinalis L. extract presented hypolipidemic effect, as evidenced by significant reductions in total cholesterol, phospholipids, triacylglycerols and atherogenic index. Additionally, the co-administration of the extract also protected against DNA damage, demonstrated by the absence of genetic material fragmentation in treated animals.

Background

The localized blood flow reduction in the brain is known as cerebral ischemia, caused by arteries obstruction or systematic hyperfusion, causing irreversible damage. Inflammation and oxidative stress may be related to this physiological disorder [65]. The unexpected decrease of vital supplies (oxygen and nutrients), due to ischemia, may lead to stroke [66], which is caused by the edema from the rupture of the blood-brain barrier [67]. This liquid accumulation contributes to increase the brain mass [68] and consequently promote cell death [69]. Thus, recombinant tissue plasminogen activator (TPA) has been used for the treatment of this problem; however, this product has caused a worsening of the lesions as a result of the cerebral ischemia, contributing to the increase in the infarct size, cerebral edema, and hemorrhage intracranial [70]. Thus, alternative products for the treatment has been studied, including the plant products.

Methodology

Hydroethanolic extract obtained from the R. officinalis L. leaves has been demonstrated in providing brain tolerance to artificially induced ischemia [71]. For this analysis, the authors promoted occlusion of the middle cerebral artery with intraluminal nylon filament implantation for 60 min in adult male Wistar rats, restoring blood flow after this period. Moreover, the animals were previously treated with the extract at 50, 75 or 100 mg/kg/day or with the vehicle (control) for 30 days. Ischemia induction occurred 2 hours before the last treatment. Non-ischemic animals were also included in the study for comparative purposes. The reperfusion period was 24 h and then the analyses were performed on the animals, including: (i) total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-c) and high density lipoprotein (HDL-c) levels, quantified on the 30th day of treatment before surgery; (ii) neurological functions assessed by means of scores, such as 0 (“no neurological dysfunction”), 1 (“failure to extend opposite forepaw”), 2 (“circling to the contralateral side, when held by tail with feet on floor”), 3 (“falling to the left”), 4 (“unable to bear weight on affected side”/“no spontaneous walking and a depressed level of consciousness”), and 5 (“death”); and (iii) neurological behavior, including volume of the infarct and edema and permeability of the blood-brain barrier.

Findings

After the 30th day, all animals gained weight; however, it was lower in the group treated with 100 mg/kg. R. officinalis L. extract decreased TC, TG, and LDL-c and increased HDL-c levels. In the treated groups were observed low levels of LDL/HDL and TG/HDL after 30 days. These data demonstrated that R. officinalis L. extract had no dyslipidemic effect. Regarding the neurological functions, the plant extract contributed to reducing the neurological deficit, since the untreated group presented score 3 (“falling to the left”), and after using doses of 50, 100 and 75 mg/kg the score was 1 (“failure to extend opposite forepaw”). The extract also provided a reduction in the infarction volume, presenting an excellent protection in the groups treated with 75 and 100 mg/kg. On the other hand, in untreated groups, the induced ischemia caused severe infarction in the subcortex and cerebral cortex regions. The edema formation was controlled in the animals treated with R. officinalis L. extract since protection against rupture of the blood-brain barrier and non-extravasation of liquid were observed.

Background

Cyclooxygenase inhibitor medicines have been used to treat pain, such as non-steroidal anti-inflammatory drugs (NSAIDs), although the prolonged use of these medications may lead to cardiovascular, renal, and gastric complications [75, 76]. In contrast, products obtained from medicinal plants can operate synergistically with these medicines to control pain.

Methodology

The synergistic and antinociceptive activities of R. officinalis L. ethanol extract was reported in a study conducted in Wistar rats [77]. The animals were previously treated with plant extract, phytocompounds (ursolic acid and oleanolic acid), ketorolac, an NSAID, and ketorolac associated with plant products. Nociception was induced by subcutaneous injection of 1% formalin in the right paw dorsum.

Findings

Plant extract (0.58 μg/paw) and ketorolac (0.88 μg/paw) provided antinociception of 66.5%. Higher doses of these products (10 μg) showed values of 38.5 and 42.6%, respectively. Thus, the drug interaction presented more effective antinociceptive action using lower doses. The administration of ursolic acid and oleanolic acid provided antinociception of 48.7 and 47.5%, respectively. Additionally, the association of extract or ursolic acid with ketorolac presented a nociception reduction of 61.1 and 71%, respectively. The phytocompound may be one of the responsible for the synergistic and antinociceptive effects of R. officinalis extract.

Background

Biofilms are formed by microbial communities of different species adhered to the biotic or abiotic substrate, being surrounded by polysaccharide extracellular matrix produced by the microorganisms. This structure offers protection to the microorganisms against the external environment, actions of the host’s defense system and antimicrobial agents [87, 88]. The proportion of microbial cells and extracellular matrix may range between 10 and 25% of cells and 75–90% of polymeric substances [89]. The microorganism arrangement in these three-dimensional structures gives them about a thousand times more antimicrobial resistance than in planktonic cells, being directly related to cases of infectious diseases [90]. Therefore, the development of new products or strategies to combat microorganisms in biofilms is important. Another concern of the scientific community is the constant emergence of antimicrobial-resistant strains, which has been stimulating the search for alternative methods to control pathogenic microorganisms.

Methodology

Phytotherapy is a wide field that can use plant products as an antimicrobial. The results of some studies have been increasingly promising and motivating. R. officinalis L. glycolic extract is an example of this, since its ability to control mono- and polymicrobial biofilms were cited [91]. In this study, the authors proposed to evaluate the effect of this plant extract on microorganisms that cause oral infections, such as Candida albicans, responsible for pseudomembranous/erythematous candidiasis and angular cheilitis [92]; Staphylococcus aureus, related to periodontitis due to its presence in supra- and subgingival biofilms [93]; Enterococcus faecalis, associated with asymptomatic endodontic infections characterized by formation of periapical lesions [94]; Streptococcus mutans, one of the agents that promote the development of dental caries [95]; and Pseudomonas aeruginosa linked to more aggressive periodontitis [96]. According to de Oliveira et al. [91], the action of R. officinalis L. extract was analyzed both on monomicrobial biofilms of each species and polymicrobial biofilms formed by C. albicans associated with S. aureus, E. faecalis, S. mutans or P. aeruginosa. These microbial associations were carried out once these species cause important clinical manifestations or present peculiar behavior when they are together. It has been reported that C. albicans may favor the development of S. aureus [87] and, besides, this bacterium was found in 27% of candidemia in nosocomial infections [97]. The association of C. albicans with E. faecalis both are benefited, and their pathogenicity may decrease, unlike when they are alone [98]. The interaction of C. albicans with S. mutans results in an extremely virulent biofilm on the teeth [99]. The development of C. albicans may be regulated by enzymes secreted by P. aeruginosa; these proteins affect the process of cellular respiration and hyphal formation [100]. Therefore, mono- and polymicrobial biofilms were formed in microplate for 48 h. After, planktonic cells were discarded by washes with saline solution (0.9% NaCl) and the biofilms were treated with R. officinalis L glycolic extract (200 mg/mL) for 5 min, considering its use as dentifrice or mouthwash. The affected cells were removed by other washes with saline solution, and the biofilms were disaggregated by ultrasonic homogenizer using a potency that caused no damage to the structure of the microorganisms (25%/30 s). Subsequently, the generated microbial suspension was diluted in saline solution and added in solid medium to form colonies. For polymicrobial biofilms, the suspensions were added in selective medium to determine how much each specie was affected in the mixed biofilm, both by the microbial interaction and by the extract action. This analysis was performed by counting of colony-forming units, being presented in concentration per milliliter (CFU/mL).

Findings

R. officinalis L. extract provided a significant monomicrobial biofilms reduction after 5 min treatment, with rates of 99.96 ± 0.07% for C. albicans; 67.84 ± 12.05% for S. aureus; 77.64 ± 15.67% for E. faecalis; 79.32 ± 7.34% for S. mutans; and 98.23 ± 2.17% for P. aeruginosa. Regarding the polymicrobial biofilms, the plant extract was also effective due to a decreased CFU/mL concentration observed in the treated groups. In the association of C. albicans with S. aureus, the yeast was more affected (89 ± 13.89%) compared to the bacterium (56.75 ± 22.58%). In the biofilm of C. albicans with S. mutans was also observed reductions of 92.04 ± 5.24% and 64.55 ± 15.12%, respectively. On the other hand, the associations of C. albicans (85.87 ± 17.48%) with E. faecalis (93.03 ± 2.44%) and C. albicans (85.19 ± 10.48%) with P. aeruginosa (83.33 ± 17.79%) significant differences were not found. These results demonstrated the potential antibiofilm effect of R. officinalis L. extract on microorganisms that may cause oral infections, as well as the possibility of its insertion in oral hygiene materials to control biofilms adhered to surfaces, such as teeth, oral mucosal, prostheses, and orthodontic appliances.

Background

Lead (Pb) is a toxic heavy metal and harmful to living things when mainly carried by food, water, and air. It may present accentuated toxicity to the liver and kidneys [112] as evidenced by a post-mortem analysis in individuals intoxicated by Pb [113]. The contact with Pb is initiated by its entry in the organism from drinking water contaminated with the metal from the pipelines, canned foods due to solder used in the cans, and ceramic enamels [114]. Lead may provide a variety of disorders, including hematological [115] and immunological changes [116], cardiac [117], nervous [118], metabolic and reproductive problems [119], and cancers [120]. However, ethylenediaminetetraacetic acid (EDTA) calcium disodium salt, magnesium dimercaptosuccinic acid (DMSA), D-penicillamine (PCA), and dimercaprol (BAL) have been used to treated Pb poisoning cases. These substances are chelating and provide Pb reduction in the body [121]. However, these medications can cause intoxication due to high dosage or allergic reaction regards to the penicillin. The common side effects are: (i - EDTA) nephrotoxicity, headache, fatigue, myalgia, thirst, fever, nausea and vomiting, sneezing, nasal congestion, lacrimation, rashes, anemia, and hypotension; (ii - DMSA) nausea, diarrhea, rashes, transient elevation of the serum aminotransferase; (iii - PCA) rheumatoid arthritis, urticaria, maculopapular reactions, lupus, pemphigoid, myasthenia gravis, renal toxicity progressing to nephrotic syndrome, leukopenia, thrombocytopenia, and aplastic anemia; and (iv - BAL) rise in the blood pressure, tachycardia, vomiting, abdominal pain, headache, burning sensation in mouth and throat, lacrimation, blepharospasm, rhinorrhea, sweating, anxiety, fever, hemolytic anemia [122].

Methodology

Alternatively, R. officinalis L. ethanolic extract has been evaluated as a protective option against the hepato-nephrotoxic effect caused by the Pb [123]. In this study, male albino rabbits received distilled water (control group), R. officinalis L. extract or lead acetate (PbA) for 30 days at 30 mg/kg. Also, another group of animals received plant extract for 30 days and then PbA for the same period. Blood of sacrificed animals was collected for analysis of total erythrocyte, packed cell volume, hemoglobin, mean cell volume, mean corpuscular hemoglobin concentration, and total leukocyte, granulocyte, lymphocyte, and monocyte. A biochemical analysis was performed from the serum of the animals, verifying the presence of markers related to damage in the liver and kidneys, as well as activities of aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), from liver, and levels of urea (ERU) and creatinine (CRE), from kidneys. The activity of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) were quantified. Besides, lipid peroxidation (LIP), glycogen (GLY), and tissues protein (TSP) levels were checked. Histopathological and histochemical analyses were performed by hematoxylin/eosin staining and mercury bromophenol blue method, respectively.

Findings

Animals exposed to the PbA showed significantly reduce of activity and body weight. Absolute weight of liver and kidneys also decreased to 42 and 62%, respectively. On the other hand, the treatment with R. officinalis L. extract previously promoted normalization of the absolute weight of liver (66%) and kidneys (80%). These data indicated the protective effect of the plant extract against the damages caused by Pb, regarding the changes in the mass of the organs. The animals exposed for 30 days to PbA presented a decreased in total erythrocyte, packed cell volume, hemoglobin, mean cell volume and mean corpuscular hemoglobin concentration. The number of total leukocytes (neutrophils and monocytes) was increased; however, the levels of lymphocyte and eosinophil were dramatically decreased. In rabbits pretreated with plant extract, the cell concentration was similar to the control group, demonstrating that the R. officinalis L. extract had a protective effect, even with prolonged exposure to the Pb. The production of markers related to hepatic and renal damages was higher in animals exposed to the PbA. The activity of AST (173%), ALT (259%), ALP (162%) and the levels of ERU (161%) and CRE (153%) were significantly increased. However, in animals pretreated with extract, lower concentrations of AST (168%), ALT (129%), ALP (136%), ERU (121%) and CRE (112%) were observed. Thus, the potential of R. officinalis L. extract to protect the organisms against the harmful effects of the Pb was verified by biochemical tests. In animals exposed to PbA, the activity of antioxidant enzymes was significantly reduced, including CAT (52%) and SOD (47%), whereas MDA level (181%) was increased. In samples obtained from kidneys suspension, higher rates of CAT (57%), SOD (62%), and MDA (375%) were found. CAT (26%), SOD (45%) and MDA (63%) levels from the liver, and CAT (16%), SOD (33%) and MDA (87%) levels from kidneys were controlled only in animals pretreated with plant extract. A significant glycogen reduction was observed in the groups treated by PbA, both in the liver (41%) and in the kidneys (21%). In contrast, the animals treated with R. officinalis L. extract presented indexes of 20 and 7%, respectively. The tissue protein levels were statistically similar between the group exposed to PbA and pretreated with extract, presenting reductions in both cases. Histopathological analyses showed no alterations in the liver of rabbits treated with R. officinalis L. extract or distilled water (control), as well as in pretreated animals. Histopathological changes were observed instead in animals exposed to the PbA, demonstrating necrotic areas. Regarding the kidneys, abnormalities were not observed in the control, pretreated and treated groups. In contrast, severe tissue and pathological disorders were observed in groups exposed to the PbA. High concentrations of neutral mucopolysaccharides of hepatocytes and renal tubules were demonstrated by histochemical analysis in rabbits from control and treated groups. A carbohydrates reduction was observed in animals treated by the PbA, both in their liver and kidneys; however, a higher concentration was found in the animals preliminarily treated with R. officinalis L. extract. As for proteins, a reduction was observed in the exposed groups to the PbA, both in the liver and kidneys. Despite this, the protein content was not affected in the group pretreated with the plant extract. Hence, the protective effect of R. officinalis L. extract was also histologically proven against hepato-nephrotoxicity of the Pb.

Background

In the face of imminent or fanciful danger, the organism precedes these events and intensifies some chemical reactions, generating a series of physiological signals that may affect the senses and some systems and cause symptoms, such as tachycardia, intense phobia, excessive perspiration, abdominal pain, and autonomic nervous system dysfunction [127]. Thus, the anxiety is installed. For the treatment, anxiolytics and antidepressants medications have been used, including the benzodiazepines, which are highly addictive and therefore should be consciously consumed [128]. However, many of these medicines may cause side effects such as hypotension, arrhythmias, and anticholinergic effects [129]. Selective serotonin reuptake inhibitors (SSRIs) are medications used to treat anxiety, being the most commonly prescribed antidepressants. Nevertheless, this drug may present side effects, including nausea, vomiting, insomnia, restlessness and dysfunction [130]. Due to these undesirable results, the use of plant medicines as adjuvant or primary treatment has been considered to control psychiatric, and neurological disorders [131].

Methodology

The antianxiety effect of R. officinalis L. hydroethanolic extract was evaluated in rats submitted to a stressful situation [132]. In this study, the animals received doses of plant extract (100, 200 or 400 mg/kg) by intraperitoneal injection. Rats from the control groups received saline or diazepam (1 mg/kg). The effect of the products on anxiety in rats was evaluated by the elevated plus maze device, which is used to generate and measure the anxiety, as well as to check the effect of anxiolytic medicines. It is a device composed of two platforms (width: 10 cm; length: 40 cm) that cross each other. One of the platforms is walled (high: 40 cm) while the other has no wall. The center of the labyrinth has an area of 10 cm², where the animal is placed with its head facing the non-walled region. These platforms are 50 cm above the ground. An illustration of this device can be seen in Fig. 2. This test lasts for 5 min, and the animals are often stressed due to device height and unprotected regions; thus, they tend to seek shelter in walled areas. Hence, the anxiety may be measured by the entries and permanence of the animal in the protection-free area. Consequently, the absence of anxiety is related to the ability to cope with these challenges. The permanence time and the entries number in each maze area are quantified separately. The time spent and the entries percentage in unprotected regions, as well as the locomotor activity, should also be evaluated to measure the anxiety of the animal. The entries number in any maze may measure locomotor activity. This device was used in the study by Abadi et al. [132], which rats were previously injected with the products according to their experimental group and the test was conducted after 45 min.

Findings

The permanence of the animals treated with R. officinalis L. extract in the regions with no walls was increased, indicating that these rats were less anxious and stressed. In this evaluation, higher concentrations of the extract were more effective and the dose of 400 mg/kg provided a similar effect to the diazepam. Additionally, the permanence time in protected areas was reduced using the extract at 400 mg/kg, similar to the standard medicine. Regarding the entries number in protected areas, a significant reduction was observed according to the dose, while in areas with no walls the entries number was increased. By locomotor activity analysis, it was found that the animals of all the groups presented similar behavior regarding the exploration of the maze. Based on these results, the antianxiety potential of R. officinalis L. extract was effectively demonstrated. The capacity this plant product to provide stress relief in situations of real danger could be an alternative to the conventional medicines, which may lead to various side effects and addiction.

The nature of the tumors

Solid tumors consist of tumor cells and a characteristic tumor vascularization, which is different from the vascularization of healthy tissues. Thus, this microenvironment is physiologically formed by high interstitial fluid pressure (IFP), low oxygen tension, and low extracellular pH. The regular balance of growth factors present in healthy tissues is totally unregulated in a tumor tissue. This fact contributes to the development of an abnormal vascularization that compromises tissue structure and function. Therefore, the nutrition and excretion of tumor tissue products are compromised [137].

The high IFP in the tumor tissue is due to the vascular content accumulation caused by poor tumor vascularization [138]. Thereby, factors such as decreased blood vessel activity and lymphatic, osmotic pressure, and contractility of tumor stroma, cooperate for increased IFP [138, 139]. As a consequence, the flow of cells with antitumor activity and therapeutic substances are greatly impaired in tumor tissues [140]. It was verified a heterogeneous distribution of oxygenation in the tumor tissue since some portions receive low concentrations of oxygen and others are not attended, due to the insufficient vascularization [141]. As for the extracellular pH, it was observed that tumor tissues have acidic pH [142], due to the accumulation of lactate, produced by the glucose metabolism, that inside the tumor cell is found in high levels [143, 144].

Therapeutic barriers to treat the cancer

Anticancer therapy can be compromised precisely by the three factors cited above: high IFP, low oxygen tension and low extracellular pH.

The high IFP in tumors compromises the delivery of the antitumor agent, mainly antibodies and other proteins, by decreasing the vascular flow, as well as its transport from the circulation to the tumor. Patients with lymphoma or melanoma have shown better results with chemotherapy when decreased IFP occurs during treatment [145].

Deficiencies in tissue oxygenation can inhibit the therapeutic effect of radiation, since oxygen is a potent radiosensitizer that contributes to tumor cell death [146]. Also, hypoxia has also been reported as a problem in the treatment with chemotherapeutic agents requiring oxygen for maximum efficiency, such as mephalan, bleomycin, and etoposide [147]. Besides, lack of oxygen compromises the cell division, thus, antiproliferative drugs lose their effectiveness on tumor cells [148].

Acid extracellular pH can impair the delivery of many chemotherapeutic agents [149]. The acidic condition in the tumor tissue can affect many drugs at the molecular level, preventing these agents from crossing the cell membrane [150]. Additionally, some therapeutic molecules are sequestered by acidic endosomes located inside the tumor cell [151].

Breast adenocarcinoma (MCF-7) and cervical adenocarcinoma (HeLa) [91]

Epithelial colorectal adenocarcinoma (CaCo-2) and histiocytic lymphoma cell line (U-937) [152]

Esophageal squamous cell carcinoma (KYSE30) and gastric adenocarcinoma (AGS) [153]

Lung carcinoma (A549) [154]

Action mechanisms

Cancer cells can survive and develop tumors higher than non-tumor cells, even under chemo- and radiotherapy conditions. Among the strategies used by cells, the capacity to form new colonies is an important ability they present [154]. On the other hand, these authors proved that R. officinalis L. extract could inhibit the formation of new colonies of lung cancer cells (A549).

The tumor cells proliferation occurs on the oxidative stress influence, which exerts on the cells a force for them to survive to this adversity. Therefore, this situation activates the redox signaling and, consequently, activator protein (AP-1) and NF-κB are also activated. Subsequently, tumor suppressor genes inhibition can also be observed [155]. In contrast, the antitumor activity of R. officinalis L. has been attributed to the antioxidant effect that the plant presents, such as free radicals elimination and lipid peroxidation control [156, 157]. This fact has been proven on cancerous lineages such as MCF-7 and colorectal adenocarcinoma cells (HT-29) [158, 159].

The cytotoxicity of R. officinalis L. observed on tumor cells can be related to the interference in the cell cycle and also to apoptosis induction. R. officinalis L. methanolic and ethyl acetate extracts impaired distribution of U937 cell cycle in S phase, and provided a decrease in the G1 and G2/M phases. In addition, the methanolic fraction of the extract inhibited the CaCo-2 growth, with a decrease in the G2/M phase [152].

In response to the action of a therapeutic agent, the tumor cell can release ROS to provide the necessary oxidative stress for its proliferation, using the mitochondrial pathway [160, 161]. However, rosmanol, a phytocompound from R. officinalis L., caused apoptosis in colorectal adenocarcinoma cells (COLO 205), increasing the levels of apoptosis-inducing factor (AIF) and cytochrome c [162].

Other phytocompounds from R. officinalis L. have been cited as responsible for antiproliferative activity against cancer cells, as the ursolic acid that can act on the NF-κB pathway and provide NF-κB phosphorylation repressors inhibition. Thus, this phytocompound can attenuate the action of agents involved in oncogenesis, such as COX-2, MMP-9, cyclin D1, C-Jun, and C-fos. The antioxidant potential of ursolic acid has also been observed [153]. Besides, carnosol has shown a blocking effect on NF-κB [163], and carsonic acid has neutralized ROS and, consequently, protecting cell membranes against lipid peroxidation [153].

There is a class of proteins that after activation by cleavage acts on the DNA repair or even lead the cell to apoptosis, in case of impossible repair of the genetic material. These enzymes are called poly ADP ribose polymerase (PARP), and were activated by DNA breaking caused by ROS or other reactive species [164, 165]. In the study conducted by Moore et al. [154] increased PARP cleavage was observed in A549 cells by exposure to the R. officinalis L. extract, indicating an induction to the apoptosis of these cancer cells. These authors also proved that the plant extract can control the Akt phosphorylation, an important enzyme responsible for the regulation of metabolism, apoptosis, and cell proliferation. Blocking of this pathway (P13K/Akt) can result in improvements in the cancer treatment by chemo- or radiotherapeutic agents [166]. Besides, Moore et al. [154] have also shown that the extract can inhibit the activation of mTOR and p70S6K, mammalian targets of rapamycin that are cancer signaling proteins. Probably, this occurs due to the ability of R. officinalis L. extract in interfering with protein synthesis by DDIT4 gene induction, which is capable of inhibiting the synthesis of mTOR and p70S6k [167].