Globally, therapeutic plants have been used by various communities, having a relevant role in the treatment of human and animal diseases. Today, they have been investigated increasingly often because of their benefits and fewer side effects when compared to pharmacological drugs. They can also be used as a complementary treatment to boost therapeutic progress [1].

Rosmarinus officinalis L., which belongs to the Lamiaceae family, is an aromatic evergreen plant with upright stems, whitish-blue flowers, and dark green leaves. It is commonly known as rosemary and is native in countries of the Mediterranean region. Fresh and dried leaves represent the most relevant part of the plant and can be used as a spice or to make herbal tea ^[2][3][4][2,3,4].

Rosemary’s chemical composition varies in different extracts, but its analysis shows that phenolic diterpenes, triterpenes, and phenolic acids are the most relevant active constituents. Regarding phenolic compounds, carnosic acid, carnosol, and rosmarinic acid, have been declared to have the main therapeutic effects, such as antioxidant, anti-inflammatory, antiviral, and antibacterial activities ^[2][3][4][2,3,4]. Plant extracts can be obtained from roots, stems, leaves, flowers, fruits, seeds, and bark, using selective solvents and standard procedures. Qualitative and quantitative studies on bioactive compounds isolated from plants depend on the proper selection of extraction method, which is a vital choice for obtaining satisfactory results ^[1][4][1,4].

The aerial parts of Rosmarinus officinalis have been widely used in different cultures as a food preservative and also as a flavoring agent in foods, beverages, and in cosmetics [2]. They are reported to have a variety of specific therapeutic properties, such as being hypoglycemic, antiatherogenic, antihypertensive, hypocholesterolemic, antioxidant, anti-inflammatory, hepatoprotective, antidepressant, antiproliferative, and antibacterial. It may also improve asthma, cataract, renal colic, peptic ulcer, and physical and mental fatigue ^[2][3][4][2,3,4].

Inflammatory diseases are widely known to be the main cause of morbidity across the global population. If inflammation is not controlled, it may result in numerous diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, immune-inflammatory illnesses, and neoplastic transformations. Moreover, chronic inflammation is also associated with stages of tumorigenesis, presenting a risk factor for the occurrence of certain types of cancers. Chronic diseases tend to manifest as a sustained low-grade inflammation. In some of those diseases, treatment still represents a challenge, given the lack of safe and effective medications. As a response to the difficulties in finding safe and effective treatment options to control inflammation, many animal models have been developed to study and evaluate drug anti-inflammatory activities. To carry out these studies, the choice of the appropriate animal model for the preclinical experiment represents a challenge; in order to, afterward, establish the efficacy and translation of the drugs therapeutic properties in humans. Even though there are numerous in vivo models of inflammation, developed to access the potential of anti-inflammatory drugs, the proper selection of an animal model is always crucial. Unsuitable selection of animal models may lead to a false positive or false negative result and, therefore, prevent the identification of a possibly promising drug [5].

Regarding the plant, Rosmarinus officinalis, it can be used in its entirety or in the form some of its isolated compounds.

The plant extract was widely investigated in several clinical diseases by researchers. In some cases, specific compounds of the plant were isolated and then tested to evaluate their activity. Phytotherapy consists of plant-derived treatments where the whole plant is used to produce an extract, and its activity results from a synergic effect between the various compounds. The difference from pharmacotherapy is exactly in this synergy, because this consists of benefits from a single active substance of a drug; the same happens to an isolated compound [1]. A limitation of phytotherapeutic drugs is the natural variability of extracts. In a plant extract, the level of compounds varies, and this causes these drugs to lose biochemical consistency. Ultimately, this results in a reduction of the optimization of safety and efficacy. The natural variability can lead to inconsistent results, and this may impair the extract in being accepted as a phytotherapeutic medicine by the scientific community [1]. The reproducibility of a beneficial effect from a plant extract is greatly reduced, given the fact that multiple factors influence an extract’s activity. Some factors, such as the harvesting of the plants at different times and locations, and different extraction and quantification methods, are perhaps the reason for that limitation. The compounds isolation, purification, and structural characterization should be more profoundly developed, and for that reason, methods must be improved. Another limitation in the development of drugs from plants is that the isolation of compounds with therapeutic activity is only done in small quantities, and therefore, is not sufficient for the production of a new drug [1]. Plant extracts have been widely investigated in various clinical diseases by researchers. In some cases, specific compounds of the plant are isolated and then tested to evaluate its activity. Not all studies reported how extracts or plant compounds were extracted because, in most of these cases, they were purchased or donated. When it was possible to access this information, many different ways of extraction were mentioned, with maceration being the most prominent. Afterward, the most widely used extraction methods were hydrodistillation, followed by Soxhlet extraction and steam distillation. Regarding maceration, the most used solvents were ethanol, followed by water, and both of them mixed. In the case of Soxhlet extraction, the most used solvent was ethanol. In studies that used maceration as the extraction method for Rosmarinus officinalis, mostly dried and ground leaves were used ^{[15][16][17][18][19]}[19,23,24,37,62]. Powder extraction was performed in a mixer with shaking ^[18][37], or slowly ^[19][62], with distilled water ^[15][19], ethanol ^[16][17][23,24], or both ^[18][19][37,62]. The extraction time varied according to the study. The temperature used was not mentioned in all studies, but the studies that noted this used room temperature ^[15][16][19][19,23,62]. The extract was filtered, and the solvent was evaporated ^{[16][17][18][19]}[23,24,37,62] on a rotary evaporator ^[17][18][24,37]. Rosmarinus officinalis extracts can be obtained from several parts of the plant, such as the roots, leaves, stems, or flowers. The size of the particles influences the extraction. Thus, smaller particles are preferable, because they have more contact with the solution, which improves the extraction. Consequently, particles in the form of powder provide better extracts, because there is a much higher contact between the plant’s particles and the solvent [4]. The solvent’s temperature and pressure, as well as the extraction time, also affect the efficiency of the extraction process. 

The solvent used for extraction influences which compounds are extracted, and individual extracts have a different activity depending on their compounds. The extraction method chosen will influence the final compounds in the extract, so the choice of method should take into account the properties of the plant [4].

2.3. Dose and Route of Administration

2.3.1. Gavage

Gavage (esophageal or gastric) is frequently used in research investigations to guarantee a well-defined and accurate dosing of animals, preferably combining substances with food or water. In one of the studies, by Faria et al. (2011), the effective dose of Rosmarinus officinalis was evaluated and determined to be 300 mg/kg. Furthermore, according to Takaki et al. (2008), 3000 mg/kg was determined to be the maximum dose that did not show any cases of lethality or signs of toxicity. The rosmarinic acid doses ranged between 10 and 300 mg/kg and provided an average dose of approximately 70 mg/kg, significantly higher than the other compound’s average. The carnosic acid average dose was roughly 25 mg/kg, varying within 5 and 60 mg/kg. Comparing these data, itwe can verify that the doses used of isolated compounds of the plant were significantly lower.

There is a higher number of studies were conducted to analyze the effects of Rosmarinus officinalis in comparison with those to evaluate an isolated compound of the plant. Even though rosmarinic acid was more common overall, having significantly more studies than carnosic acid and carnosol. Through analyzing the data, it seems that the use of the whole plant corresponds with a necessity of higher doses, compared to studies of a concentrated substance.

Gavage presents some limitations, such as a delayed onset of the effect when compared with parenteral administration, decrease of absorption of substances, and substance degradation by digestive enzymes and acid. Furthermore, a potentially significant first-pass effect by the liver, may reduce the drug’s efficacy for the substances metabolized via this route. In this sense, the dosage through oral gavage tends to be higher ^[20][84].

2.3.2. Intraperitoneal

The intraperitoneal route consists of injecting substances into the peritoneal cavity, and this is a widespread method in laboratory rodents ^[20][84]. This route was the only one used to verify the effects of the plant and all the isolated compounds mentioned earlier. Intraperitoneal delivery is recognized as a parenteral route of administration. Parenteral administration methods usually provide the largest bioavailability. Those methods tend to evade the first-pass effect, which occurs commonly with oral administration. Therefore, in cases of intraperitoneal administration, the dosage tended to be lower in comparison with oral delivery ^[20][84].

2.3.3. Oral

The administration of substances directly into the oral cavity, such as inclusion in diet (food or water), is well-established in laboratory animal experimentation. Oral administration is more economical, convenient, and moderately safe. Doses of the plant or an isolated compound given through the oral route were included in the diet, and the animals had ad libitum access to food. Consequently, this makes it difficult to truly evaluate the real results, because not all animals ingested the same amount of the plant or the compound under analysis.

As verified for every route of administration, the use of the whole plant, as an extract, always represents a higher dosage than the administration of an isolated compound. Previous reports established the use of higher doses when studies are conducted with extracts, with lower doses of isolated constituents, when studying anti-inflammatory properties [5].

2.4. Frequency and Duration

There are numerous factors to take into consideration when establishing the frequency of administration in a treatment. The specific model of inflammation, route, and dose used must be considered in the decision-making process.

The duration of treatment depends directly on the animal model, adapting to the conditions under analysis.

2.5. Biomarkers Evaluated

TNF-α, IL-1β, IL-6, and myeloperoxidase (MPO), were by far the most evaluated. In all cases, an increase in these inflammatory biomarkers confirmed the onset of the inflammation. As expected, the level of those biomarkers of inflammation suffered a reduction following treatment. Interleukin 10 (IL-10), an anti-inflammatory cytokine, was also evaluated in a variety of studies, and treatment also caused the increase of this cytokine; therefore, contributing to diminishing the inflammation. These effects are evidence of the anti-inflammatory activity of Rosmarinus officinalis, as well as the isolated compounds analyzed. Inflammation and oxidative stress are intertwined in the numerous pathophysiological events of various diseases [5]. Carnosol and carnosic acid, the main phenolic diterpenoid compounds of rosemary, have been noted to inhibit NO production. The inhibitory effects of carnosic acid in NO and TNF-α production are the result of the suppression of iNOS and COX-2 expression. Moreover, this inhibits the nuclear translocation of NF-κB. Carnosol attenuates the levels of iNOS and also downregulates NF-κB ^[21][87]. Regarding inflammatory pathways, the literature reports that the transcription factors NF-κB and signal transducer and activator of transcription 3 (STAT3); inflammatory enzymes, particularly COX-2 and matrix metalloproteinase-9 (MMP-9); and last, inflammatory cytokines such as TNF-α, IL-1, IL-6, and IL-8 are the main molecular mediators of an inflammatory response. Among these mediators, transcription factor NF-κB is the principal regulator of the immune system and the inflammatory response and controls several genes encoding the cytokines, cytokine receptors, and cell adhesion molecules associated with inflammation triggering ^[5][22][5,89].

3. Conclusions

Rosmarinus officinalis was mostly used in its entirety or as an extract of rosmarinic acid. Rosmarinus officinalis was used at a dose of 400 mg/kg via gavage and rosmarinic acid at a dose of 10 mg/kg via IP. Overall, the treatments were scheduled as daily administrations for 28 weeks. Rosmarinus officinalis showed anti-inflammatory activity, before and after induction treatments, with a decrease in the levels of inflammatory biomarkers and an increase of oxidative stress biomarkers.

Although the potent anti-inflammatory properties of rosemary extract have been well recognized in this review, more reliable trials are required in the future. Further evaluation of Rosmarinus officinalis and its main active compounds’ safety and efficacy in managing different pathological conditions is crucial.