First phytochemical study and biological activity of the leaves ethanolic extract from Cissus spinosa

Aims: The objective of this study was to identify the phytochemical profile and to evaluate the biological effects of the crude ethanolic extract (EE) and the ethanolic fraction (EF) of leaves of the species Cissus spinosa Cambess, after oxidative stress induced by cyclophosphamide (CP) in mice. Methods: Phytochemical profile was performed detecting functional groups and, analysis of total flavonoids and phenols concentration, as well as the antiradical activity in EE and EF. The phytochemical characterization was done for the identification of flavonoids present in the leaves of the plant. In the biochemical tests, hematological parameters, glucose, total cholesterol, creatinine, alkaline phosphatase and aminotransferases dosages in plasma, enzymatic and non-enzymatic antioxidants and lipid damage marker were evaluated in different tissues (liver, kidney and heart), besides genotoxic and immunological analyzes. The animals received 15 days of treatment, via gavage, with EE (50 mg kg-1) or EF (50 mg kg-1) and on the 15th day, an intraperitoneal injection of CP (100 mg kg-1) or saline (0.9%). After 24 h the last treatment, the animals were anesthetized for blood withdrawal, sacrificed and removal of the organs. Results: In the phytochemical analyzes, the presence of alkaloids, flavonoids and phenols was identified, the latter presented a higher concentration for EF. Eight flavonoids were identified Rutin, Quercetin-3-β-D-glucoside, Quercitrin, Taxifolin, Quercetin, Canferol, Luteolin and Apigenin. In the biochemical analyzes, in general, EE showed a better antioxidant action against oxidative damages, hypoglycemic and antitilipemic action when comparing with EF, probably due to the synergism caused by flavonoids. It was observed the reduction and an increase of micronucleated polychromatic erythrocytes, due to the action of antioxidant compounds and alkaloids present in the plant, also considering the question of the seasonal period that directly interferes in the production of these compounds. In the immunological analysis, the extracts did not stimulate the spontaneous production of oxygen peroxide (H2O2) and nitric oxide (NO•). Conclusions: Other studies, such as the variation of the chemical composition of the plant by local seasonality, hypoglycemic and antilipemic action, should be carried out to better delineate the biological action present in this plant.


Introdução
Plants are used for thousands of years for ornamental, aesthetic, culinary and, mainly, medicinal purposes. In several regions of the world, medicinal plants are used as a resource for various diseases [1,2]. This knowledge has been passed from one generation to another for many years, and it is from this ethnopharmacological information that most scientific research has been carried out to confirm the biological action on the individual's organism and its supposed toxicity [1,3]. The biological effect promoted by these plants originates from the secondary metabolism, which is coming from the pathways of shikimic acid and acetyl coenzyme A, producing compounds that are important in protection, development and reproduction, as well as possessing actions beneficial to the organism [4,5].
One of these actions is the antioxidant effect, which is able to inhibit or retard cell degradation, such as damage to lipids and membrane proteins and mutations in Deoxyribonucleic Acid (DNA), caused by molecules such as reactive oxygen species and reactive nitrogen species [6]. These substances are produced through physiological processes of the cell such as respiration and can also be produced after exposure to xenobiotics [7][8][9].
The genus Cissus is considered the most abundant within the Vitaceae family with approximately 350 species worldwide distributed in the tropical regions [10]. The extracts of plants belonging to this genus are used in the treatment of various pathologies [11][12][13][14][15]. Some examples are the anti-inflammatory action present in leaves of C. pteroclada due to the presence of bergenin derivatives, which inhibit the production of cytokines, modulating the expression of pro-inflammatory substances [16]. Bharti et al. showed that the high concentration of tannins in the leaves of C. quadrangularis, has an astringent action in which it can prevent bleeding and skin diseases [17]. The presence of substances derived from kaempferol and quercetin in the leaves extract of C. sicyoides have an antidiabetic, antilipemic, antiinflammatory and gastroprotective activities [18]. The hypoglycemic and antilipemic effect of C. verticillata leaves extract may be due to the presence of the flavonoids kaempferol, quercetin and rutin [19].
The species Cissus spinosa Cambess (C. spinosa) is known as cipó-de-arraia, due to its popular use as a plaster for stinging stingrays. It belongs to the group of lianas, which is between 2 and 8 meters in length or extension, with a zigzag stem, prickly and producing flowers almost all year round. Its distribution in the Brazilian territory is concentrated in the central-western region, but also in the states of Bahia, Minas Gerais Cyclophosphamide is a chemotherapeutic drug, belonging to the group of the nitrogen mustards, considered an alkylating agent. It is widely used in the treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and cancer, such as myeloma, lymphomas and leukemias [21,22]. It is metabolized in the liver by cytochrome P-450 and converted to acrolein and phosphoramide mustard, which are its active metabolites [23][24][25]. These can also bind to DNA causing cross-linking and formation of micronucleus, cytotoxicity in important organs such as the heart, liver and kidneys, and reactive oxigen species production. The increase of these substances modifies the levels of molecules and important enzymes in the pro-oxidant-antioxidant balance, such as reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) [23,[26][27][28].
The lack of scientific study and the pharmacological activity of this species, of the genus Cissus, stimulated to identify the phytochemical profile of the C. spinosa Cambess species and to evaluate the biological, genotoxic and immunological effects on oxidative stress induced by the drug cyclophosphamide in mice.

Animals and In Vivo Treatment
Male Swiss mice, with an average weight of

Hematological and Biochemical Analysis
The analysis of whole blood for red blood

Micronucleus Analysis
The micronucleus test was performed

Immunological Analysis
The animals were divided into three groups

Statistical Analysis
Statistical data (mean ± standard deviation) of the immunological, hematological and biochemical analyzes were analyzed by parametric and non-parametric tests, if necessary, as one-way analysis of variance (ANOVA), followed by Tukey test or Kruskal -Wallis followed by Dunn's test, respectively, to verify the differences between the experimental groups. The results were considered significant when p<0.05. For the micronucleus frequency test, the chi-square test was used according to Pereira [47].

Phytochemical profile
The dried leaves yielded of approximately 261 g.
After extraction by maceration with ethanol, the crude ethanolic was submeted extract chlorophyll, yielded The concentration of total phenols and total flavonoids were higher in EF when compared to EE ( Table 1).  (Figures 1 and 2) ( Table 2).

Hematological Analysis
In the analysis of whole blood, the EE was able to increase the amount of red cells -red blood

cells, and CP reduced the white cells -leukocytes.
The other parameters, hemoglobin, hematocrit and platelets did not have significant differences between the study groups ( Table 3). spinosa leaves on Cyclophosphamide-induced oxidative stress (100 mg kg -1 ) in hematological parameters in whole blood of the mice: hemoglobin, hematocrit, red blood cell count, platelet count, and white blood cell count. In plasma analysis, EE decreased glucose and cholesterol levels ( Figure 3A and 3B), and EF only cholesterol. For the markers of renal and hepatic damage, CP increased the concentration of both creatinine (renal damage) ( Figure 3C) and ALP activity (liver damage) ( Figure 4A), but EE for creatinine and EF, for creatinine and ALP, were able to prevent the alterations caused by CP. EE was also able to reduce ALT and AST activities ( Figure 4B and 4C) when comparing with the respective control groups.

Markers
In the hepatic tissue, the SOD and glutathione-S-transferase enzymes had their activities decreased in the CP group when compared to the control group and EE and EF didn't alter these antioxidants (Figure 5A and 5B). The EE increased the enzymatic activity of CAT in the liver, but also prevented its decrease before CP administration, in the heart. On the other hand, EF reduced the activity of the enzyme in the same tissue. There was no significant difference in renal tissue ( Table 4). EE contributed to the increase in GSH levels in the hepatic and renal tissues.
However, EF and CP reduced GSH in the liver and heart, when compared to the control group. In the hepatic tissue there was an increase of TBARS in the groups pre-treated with EF and also with the inducer of the damage. In the cardiac tissue, both the extract and the fraction increased this biomarker. In renal tissue, there was no significant difference between groups ( Table 4).

Immunological Analysis
In the immunological analysis, both EE and EF did not stimulate the spontaneous production of H 2 O 2 and NO in treated animals when compared to the control ( Table 6).  In the phytochemical screening the presence of flavonoids, tannins/phenols and alkaloids was verified, which is in accordance with the work on C. quadrangularis described by [48]. In Another plant that also has such effects is Cissus sicyoides, popularly known in the northeastern region of Brazil as "vegetal insulin" [11,72]. It is also used as antilipemic, anti-inflammatory, antirheumatic, in the treatment of gastric ulcers, epilepsy and in the cerebrovascular accident [18,73,74].  such as NO • [78,79]. Inflammation is an important defense mechanism used by the body, whose main function is to restore the structure and normal function of the affected tissue, either by injury or by the presence of microorganisms