1. Introduction
Cyclophosphamide (CPA) is a drug extensively used as an alkylating agent in the treatment of several malignant neoplasms, including breast cancer, multiple myeloma, kidney disease, rheumatoid arthritis, juvenile dermatomyositis, systemic sclerosis, interstitial lung disease, lupus vasculopathy, systemic vasculitis, and refractory treatment of thrombocytopenic purpura [
1,
2]. CPA can be administered orally or intravenously, with oral dosing administered daily (every 24 h) and in pulses, adjusting the dose according to hematologic and renal toxicity. Adverse reactions to CPA include bone marrow suppression, susceptibility to infections, sterility, amenorrhea, nephrotoxicity, and cystitis, as well as cardiovascular complications such as sinus bradycardia, pericarditis, myocarditis, and heart failure [
1,
2,
3,
4].
The search for new drugs that help treat pathologies while exerting fewer side effects is essential to improve patients’ quality of life and to advance medicine. The wealth of substances derived from plants has aroused great interest in this field among scientific community as a promising source for the development of new drugs. The search for innovative medicinal agents, especially those derived from natural sources, is driven by the presence of several molecules in the secondary metabolism of plants [
5,
6,
7]. Many drugs originate directly or indirectly in plant compounds, and plants harbor a variety of phytoconstituents, each with unique and distinct properties [
8].
Protium heptaphyllum (
P. heptaphyllum) is a plant commonly known as almacega, almíscar or breu branco, and it is typically found in the Amazon [
9]. Its leaves and resin are used in folk medicine due to their stimulating, anti-inflammatory, and healing properties [
10,
11]. It derives from the Burceraceae family, comprising 18 genera and more than 700 species divided into three tribes, with the genus Protium (tribe Protieae) being the main member of the family [
12,
13].
The resins, oils, and leaves of the genus Protium are extremely rich in terpenes and polyphenols, such as flavonoids [
5,
12,
14]. Phenolic compounds, including flavonoids, are known as free radical scavengers and are highly efficient in preventing autoxidation. Studies indicate that flavonoids can reduce cellular stress, which includes neuroinflammation, oxidative stress, proteotoxicity, and endoplasmic reticulum stress [
15]. Antioxidants, in addition to providing several beneficial effects, can inhibit or delay the emergence of tumor cells, delay aging, and prevent other cellular damage resulting from redox imbalance [
16,
17,
18].
Liposome nanotechnology is one of the new, innovative therapeutic approaches based on plants. This approach improves the solubility, stability, and specific targeting of active substances, overcoming absorption and bioavailability challenges [
19]. By encapsulating an active substance, liposomes offer controlled release, minimize side effects, and maximize therapeutic efficacy, presenting a promising frontier for the optimization of phytotherapeutic treatments in contemporary medicine [
19,
20]. Thus, herbal medicines conjugated using new technologies, such as using liposomes, can aid in treatment by offering effective, accessible solutions, with fewer side effects, for various pathologies due to the variety of chemical compounds, such as saponins, flavonoids, and catechins, present in plant species, highlighting the need for studies on the biological activity and toxicity of these plants to encourage investments from the pharmaceutical industry [
16,
17,
21]. Therefore, this research sought to investigate the preventive chemoprotective effects of the ethyl acetate fraction of
P. heptaphyllum extract inserted in liposomes against the oxidative processes caused by CPA.
3. Results
The measurements of initial body weight, final body weight, and feed and water consumption did not show statistically significant differences between the groups analyzed, as shown in
Table 1. However, there were changes in the weights of the organs investigated, such as the liver and heart. In the liver, the weight increased in the CPA group when compared to that in the C group, and the LP + CPA group showed a reduction in weight when compared to that of the LP and CPA groups. For the heart, the CPA group showed an increase in weight compared to that of the C group, and in the LP + CPA group, there was a decrease when compared to the results for the CPA group, as indicated in
Table 1. The other organs investigated did not show statistically significant changes.
Plasma glucose, cholesterol and aspartate aminotransferase (AST) enzyme activity levels did not show statistically significant changes in the groups analyzed. There was a decrease in alkaline phosphatase (ALP) activity in the LP + CPA group compared to that in group C. In addition, creatinine levels were reduced in the CPA, LP, and LP + CPA groups compared to those noted in group C. As for triglycerides, a decrease was observed in the CPA group compared to the levels in group C, as shown in
Table 2.
Table 3 shows the frequency of micronucleated polychromatic erythrocytes (PCEMN) after the pretreatment of mice with liposomes containing
P. heptaphyllum extract on chemically induced damage by CPA. The group treated with LP + CPA showed a significant reduction (
p ≤ 0.05) in the frequency of micronuclei when compared with that of the positive control group, showing a chemoprotective effect of the extract and thus, the possibility of producing benefits related to the prevention of DNA damage as an antimutagenic agent. On the other hand, the group treated only with the liposome containing the extract did not show a mutagenic effect when compared with the negative control group.
We did not observe statistical changes between the groups studied for cytokine levels in the liver and kidney tissue (
Table 4).
In the liver, a decrease in the activity of the catalase enzyme was observed in the LP + CPA group compared to that in the C groups and the CPA group (
Figure 1B). On the other hand, the GST enzyme showed an increase in activity in the LP + CPA group compared to the LP group (
Figure 1C). As for the SOD and GPx enzymes, no statistically significant changes were observed (
Figure 1A and
Figure 1D, respectively). In the renal tissue, an increase in the activity of the SOD enzyme was observed in the CPA group compared to that in the C group (
Figure 1E). No significant differences were observed in the activity of the catalase enzyme between the groups studied (
Figure 1F), while the GST enzyme showed decreased activity in all treated groups, as illustrated in
Figure 1G. The GPx enzyme increased its activity in the LP + CPA group when compared to that in the CPA group (
Figure 1H).
GSH levels in the liver tissue decreased in the CPA, LP, and LP + CPA groups compared to those found in group C. In addition, carbonyl protein levels (carbonyl) increased in the CPA group compared to those in group C (
Table 5). The other parameters analyzed in the liver tissue (ASA and TBARS) did not show significant changes in their results when compared to those of the control group (C) (
Table 5). No statistical differences were observed in redox state markers such as GSH, ASA, and carbonyl in the renal tissue of the animals analyzed. TBARS showed a significant difference, with a decrease in the LP group compared to the levels in group C (
Table 4).
We did not observe any significant changes in the histological analyses.
The activity of the SOD enzyme in brain tissue increased in the LP group compared to that observed in the control group. However, in the LP + CPA group, a reduction in this activity was observed in relation to the LP group (
Figure 2A). Regarding the activity of the CAT and GST enzymes, no statistically significant changes were detected (
Figure 2B,C).
In cardiac tissue, a significant increase in the activity of the SOD enzyme was observed in the group treated with LP + CPA compared to the group that received only CPA (
Figure 2D). Regarding the analysis of the activities of catalase and GST, no changes were identified between the groups analyzed (
Figure 2E,F).
We observed a reduction in GSH levels in the brain tissue in the LP + CPA groups compared to the levels in the CPA group. Regarding the levels of ASA, TBARS, and carbonyl, no statistically significant differences were identified between the groups investigated (
Table 6).
Regarding the markers GSH, TBARS, and carbonyl, in the cardiac tissue, no statistically significant changes were identified between the groups investigated. However, regarding the dosage of ASA, a decrease in its levels was observed in the group treated with CPA compared to those in the Control group (
Table 6).
4. Discussion
It is known that polyphenols, especially flavonoids, exhibit several pharmacological attributes, such as antioxidant, anti-inflammatory, and anticancer properties, but their usage is still minor compared to their immense therapeutic potential [
38]. Although many investigations have been carried out by several authors to study the protective effects of polyphenols, especially flavonoids, obtained from different plant extracts against various types of chemical residues in animal experiments, it is suggested that flavonoids are directly effective for antioxidant activities via the neutralization of free radicals [
39]. Many extracts exhibit low bioavailability and rapid degradation, which limits their clinical efficacy. Encapsulating them in liposomes can significantly improve their stability, bioavailability, and specific targeting to tissues [
38]. This synergy between phytotherapy and nanosystems allows for the creation of more effective treatments, with less toxicity and fewer side effects, marking a promising advancement in the area [
38,
40]. In this work, we sought to improve the use of the ethyl acetate fraction from the crude extract of
P. heptaphyllum in the form of liposomes, and the results obtained revealed several interesting aspects related to the impact of liposomes containing the plant extract during 14 days of preventive treatment against mutagenesis subsequently induced for 24 h with CPA.
The results showed no statistically significant difference in initial or final body weight, as well as feed and water consumption, between the groups analyzed here, and similar data were found in the study by Patias et al. [
18], where Wistar rats were treated with the same plant formulation, but at a dose of 1 mg/mL. In a study using mice over a period of 21 days, treatment with CPA (100 mg/kg) and metformin (3 mg/mL) significantly decreased body weight, in addition to reducing the survival rate of the animals [
41]. On the other hand, it was observed that the flavonoid quercetin has a significant protective capacity against changes induced by CPA (150 mg/kg) in rats, reversing the decrease in body weight and food intake [
42], which suggests that in our study, LP, administered for 14 days, and CPA for 24 h, were not toxic to the point of altering these parameters.
Among our findings regarding organ weight, an important observation was the statistical increase in liver and heart weight in the CPA group compared to the levels in the control group (C). This increase in weight could suggest that CPA may have a direct impact on the morphology and function of these organs. When we refer to liver weight, this may be linked to a possible hepatotoxic effect of CPA, since the liver is often the target of toxicity caused by chemotherapeutic agents or may even be indicative of inflammation or liver damage, often associated with CPA treatment [
43]. Another observation was that pretreatment with LP in animals exposed to CPA (LP + CPA) caused a reduction in liver and heart weights compared to those observed in the groups that received only CPA. This finding could indicate that LP may have the ability to reduce the adverse effects caused by CPA in these organs, even if they were only exposed to the mutagenic agent for 24 h. Similarly, Dolgava et al. demonstrated that CPA treatment can affect heart and liver weight in mice [
44].
Plasma analysis showed no change in AST in the treated groups, but indicated a decrease in ALP activity, a hepatobiliary marker, in the LP + CPA group, and in creatinine levels, a marker of renal function, for all groups treated with LP and CPA. In contrast, plasma triglyceride levels were elevated in the group treated with CPA alone, possibly due to changes in lipid metabolism caused by CPA. Studies by our group using
P. heptaphyllum liposomes in the treatment of obesity caused by a high-calorie diet in Wistar rats showed a reduction in AST activity and creatinine levels in both the groups with liposome-containing extract and in the obese group. A similar reduction in ALP was also noted in the obese group and in the group treated with liposomes [
18]. On the other hand, in the study by Ref. [
14], the administration of EAF from this same plant at a dose of 100 mg/kg in mice for 7 days did not modify the activities of AST and ALP, triglycerides, body weight, and anthropometric parameters. Quercetin was effective in reversing the decrease in weight and the imbalances in hepatic transaminases, urea, and creatinine in rats subjected to oxidative stress by cyclophosphamide (150 mg/kg) [
40], which leads us to suggest that the response pattern of LP and CPA significantly depends on the experimental model used, the form of extract administration, the dose, and the treatment time.
Cyclophosphamide is oxidized by P450 enzymes in the liver to become pharmacologically active, where it is converted to highly toxic metabolites—acrolein and phosphoramide mustard [
45]—which induce oxidative stress and mutagenesis.
Regarding the micronucleus test, CPA induced a significant increase in PCEMN compared to that in the control, as observed in other studies employing mouse bone marrow cells [
46,
47,
48]. CPA causes chromosomal damage by covalently binding to DNA and interfering with the cell cycle [
49]. The liposome containing the
P. heptaphyllum extract showed antimutagenic activity in the LP + CPA group. Furthermore, LP per se was not mutagenic. The chemoprotective effect attributed to medicinal plants is largely due to the bioactive compounds present, such as flavonoids [
50].
Recent studies report similar results with extracts of plants rich in flavonoids. For example, nanoparticles from
Rhaphidophora pinnata (50, 100, and 200 mg/kg) demonstrated antimutagenic activity against cyclophosphamide (50 mg/kg) [
51]. The methanolic extract of
Dalbergia latifolia revealed antimutagenic potential against cyclophosphamide (100 mg/kg) [
52], and the flavonoid from
Kigelia africana demonstrated antimutagenic activity against oxidative stress induced by cyclophosphamide (100 mg/kg) [
53]. Considering the anti-inflammatory activity of other compounds present in
P. heptaphyllum, the essential oil present in the resin of this plant showed, through studies of its chemical composition, cytotoxic action in breast cancer cells (MCF-7), antimicrobial activity, and antimutagenicity in vivo [
54]. The possible chemopreventive activity of
P. heptaphyllum resin essential oil is attributed to monoterpenes, in addition to the absence of cytotoxic and pro-apoptotic effects. Thus, the antioxidant activity of
P. heptaphyllum [
55,
56] could potentially explain the antimutagenic activity observed in the present study, since the generation of reactive oxygen species (ROS) and oxidative stress plays a critical role in DNA and chromosome damage [
57,
58,
59].
Animal research conducted in recent decades has demonstrated that CPA-induced hepatotoxicity is related to oxidative stress [
39,
43], as it triggers many liver deficiencies due to the generation of ROS [
60,
61]. The administration of CPA into liver tissue resulted in increased protein carbonylation in the CPA group, which is consistent with the metabolite acrolein that is incorporated into the proteins, generating carbonyl derivatives [
62]. On the other hand, there was a decrease in CAT activity in the LP + CPA group compared to that in group C, an increase in GST in the LP + CPA group compared to that in the LP group, and a decrease in GSH in all treated groups (CPA, LP, and LP + CPA), and although not significant, there was a trend towards an increase in GPx for all treatments. In this sense, the GSH depletion caused by CPA is due to the production of the metabolite acrolein, which is able to form conjugations with GSH, reducing its cellular level [
63]; however, GSH plays an important role in protecting cells against oxidative damage [
64]. Furthermore, studies carried out by Kaushik and Kaur [
65] showed the modulation of GST and GPx enzymes in relation to their coenzyme GSH during cold-induced oxidative stress for 21 days and found that enzyme activity increased, even in the presence of low GSH. It is also noteworthy that CPA demonstrated a tendency to increase CAT activity in this organ, and LP seemed to normalize this action, possibly due to the presence of bioactive compounds in the liposomes. Research using EAF from
P. heptaphyllum in mice also showed the normalization of enzyme activity and GSH levels after paracetamol-induced oxidative stress [
14]. Furthermore, a study using triterpenes from
P. heptaphyllum restored hepatic GSH levels depleted by paracetamol in mice [
5].
It is known that GST is involved in cellular detoxification, and its increase in the LP + CPA group may be related to an adaptive response of the liver to the stress caused by CPA. A study with
Carica papaya Linn extract triggered an increase in GST and CAT activity in the livers of mice exposed to a single dose of CPA (75 mg/kg) [
46]. Although there were changes in the redox state of the liver, no changes were observed in the immunological and histological parameters in the tissues of the groups analyzed. Similar to the results observed in this study, Patias et al. [
18] did not see changes per se in LP for histological analyses and for TNF-α, IL-6, IL-17, but observed a positive effect for IL-10, and in obese animals, for IL-1β, which suggests that the animal species interferes with the cytokine response.
In renal tissue, markers of lipid and protein damage were not modified by the treatments, except in the case of LP, which showed a physiological reduction in TBARS. Furthermore, it revealed a significant increase in GPx activity in the LP + CPA group and a reduction in GST for all treatments. Contrary to our findings, studies with
Solanum scabrum and
Cola verticillata extracts, plants rich in flavonoids, demonstrated a decrease in renal GPx activity in rats exposed to CPA (100 mg/kg) [
66]. It appears that the
P. heptaphyllum liposome induced a response that requires greater GPx activity, possibly signaled by an additional production of hydrogen peroxide and also due to the reduction in GST. In the work of Patias et al. [
14], EAF did not promote changes in GST activity per se, such as those noted in this study. On the other hand, in this study, the form of delivery of the extract in the liposomes in the group treated only with extract resulted in a decrease in TBARS. Although we observed changes in some biomarkers, the mutagenesis inducer was not effective in causing damage within 24 h of administration in the renal tissue, and consequently, the LP did not cause the activation of the immune response or the immunomodulation of the cytokines.
In brain tissue, we observed an increase per se in SOD enzyme activity in the LP group, and its activity returned to control levels in the presence of CPA compared to the levels in the LP group, which suggests that the presence of CPA may interfere with the activity of this enzyme in this tissue. The brain is a tissue composed mostly of lipids; thus, it is highly vulnerable to oxidation, and it is noteworthy that CPA did not induce changes to the point of causing damage in this tissue, which can be observed by the absence of statistical differences between the tissues of the groups studied. There are reports that curcumin improves redox balance and shows protection against oxidative damage induced by cyclophosphamide (150 mg/kg) in the brains of rats [
67], and quercetin showed promising neuroprotective effects against brain oxidative damage induced by cyclophosphamide in several studies [
42,
68]. Furthermore, flavonoids such as quercetin, apigenin, and genistein have demonstrated the ability to reverse dysfunction caused by oxidative stress in brain endothelial cells [
69]. These findings collectively highlight the neuroprotective potential of flavonoids in combating CPA-induced brain stress. In addition, we can suggest that the short time (24 h) of CPA treatment in this study was insufficient to generate brain damage, but the 14-day treatment with LP was sufficient to positively modulate SOD enzyme activity.
In cardiac tissue, there was a tendency for reduced SOD activity in the CPA group, but pretreatment with LP promoted a significant increase in this activity, suggesting that the liposome promoted an improvement in enzymatic activity. On the other hand, there was a statistical decrease in ASA levels in the CPA group, indicating a general reduction in the antioxidant capacity of this marker in cardiac tissue due to treatment with CPA, and the
P. heptaphyllum liposome was unable to modify this parameter. Unlike the results of our investigation, the work of Ye et al. [
70], in which animals were orally treated with the flavonoid chrysin (25 and 50 mg/kg/day) for 35 days and exposed to cyclophosphamide (100 mg/kg) once a week for four weeks, showed that the activities of cardiac antioxidant enzymes, such as SOD and CAT, as well as the GSH levels, were suppressed. Studies by Luiz et al. [
45] also did not observe major changes in oxidative stress parameters in the hearts of mice treated with CPA (75 mg/kg) for 24 h, as was the case in our study. It is likely that this short time of exposure to CPA was not enough to cause many changes in the parameters investigated in this study for the brain and heart, but considering the results for the liver, which is the organ that metabolizes this drug, and the kidney, which is the site of excretion, these tissues did demonstrate that they are more affected by the treatment, as observed in our findings.