Chemical composition, antitumor activity, and toxicity of essential oil from the leaves of Lippia microphylla

1 Introduction

According to the World Health Organization (WHO), the overall impact of cancer has more than doubled in the last 30 years. It is estimated that in 2008, there were roughly 12 million new cancer cases and seven million deaths due to cancer worldwide. Future projections indicate that cancer mortality will continue to rise, reaching 13.2 million in 2030 [1].

Natural products still serve as the major source of new therapeutic agents [2, 3]. More than two thirds of the drugs used in cancer treatment come directly from natural products or are developed using knowledge gained from the activity of their ingredients [4]. A variety of such natural products are, for example, vincristine, vinblastine, paclitaxel, docetaxel, etoposide, doxorubicin, among others [5]. Many mono- and sesquiterpenes, widely found in essential oils isolated from different species, are also known in the literature for their potent antitumor effects [6].

The family Verbenaceae contains several species that are used in therapy; this is primarily due to the presence of essential oils [7]. Most of them are traditionally utilized as gastrointestinal and respiratory remedies [8]. Several studies have demonstrated antitumor activity of isolated constituents of species of this family [9, 10]. Constituents isolated from species of the genus Lippia, the major genus of the family, have been proven to be cytotoxic against various tumor cell lines [11, 12]. Lippia microphylla Cham., popularly known as alecrim-de-tabuleiro, is rarely mentioned in either the phytochemical or pharmacological literature; there are reports of the popular use of its leaves in the treatment of respiratory diseases or as an antiseptic [13]. From the roots of the plant, tecomaquinone I and naphthoquinones have been isolated [14]. These substances displayed significant cytotoxic activity against various cell lines. Antifungal and antioxidant properties have also been reported [15, 16]. Volatile compounds from this oil have been shown to enhance the antibiotic activities of both gentamicin and norfloxacin [17]. Herewith, we report on an investigation into the essential oil from the leaves of Lippia microphylla (OEL), its chemical composition, its antitumor activity, and its toxicity in vitro and in vivo.

2 Materials and methods

3 Results and discussion

Twenty-six compounds, representing 99.9% of the OEL were identified. The main chemical components identified were thymol (46.5%), carvacrol (31.7%), p-cymene (9%), and γ-terpinene (2.9%) (Table 1). The chemical composition of essential oil from the leaves of Lippia microphylla differs from that published by [25]. This difference is expected, since the plants were collected at different times and in different regions; these are factors that can change the chemical composition of volatile components. However, the chemical composition of Lippia microphylla presented here is consistent with that of other Lippia species in which thymol is always a major component [26–28].

To evaluate the toxicity of OEL in non-tumor cells, a hemolytic assay with Swiss mice erythrocytes was performed. The percentage of hemolysis increased in an OEL concentration-dependent manner. The concentration producing 50% hemolysis (HC₅₀ value) was in the range of 300 (283.4–317.9) μg/mL, (Figure 1), suggesting that OEL has low toxicity against cells which are commonly affected in anticancer treatments [5].

Figure 1:

Percentage of hemolysis in red blood cells of Swiss mice upon treatment with OEL (μg/mL). Each dot represents the average±SEM of three experiments with three replicates, with a 95% confidence interval.

Mice transplanted with sarcoma 180 were treated i.p. with OEL at (50 and 100 mg/kg) for 7 days. In comparison to the tumor control group, a significant reduction in tumor weight was observed in both groups. Eight days after implantation, the average tumor weight was 2.12±0.20 g in the tumor control group, and 1.31±0.17 g, and 0. 85±0.09 g in OEL treated groups at 50 and 100 mg/kg, respectively. In mice treated with 5-FU (50 mg/kg) tumor weight was also significantly reduced (0.29±0.06 g). Inhibition was 38, 60 and 86% for treatment with OEL at 50 and 100 mg/kg, and 5-FU, respectively (Figure 2). Thus, OEL displayed significant in vivo antitumor activity for both tested doses.

Figure 2:

Effect of OEL and 5-FU on tumor weight and inhibition of tumor growth in mice transplanted with sarcoma 180. Data are expressed as average±SEM of six animals analyzed by ANOVA followed by Tukey test. ^ap<0.05 compared to tumor control. ^bp<0.05 compared to OEL (50 mg/kg). ^cp<0.05 compared to OEL (100 mg/kg).

Considering the various toxic side effects of anticancer agents on normal cells, we proceeded to investigate possible OEL toxicity. Virtually all cancer drugs promote gastrointestinal disorders, including anorexia. Anorexia is directly linked to malnutrition and weight loss [29]. Table 2 provides data on the animals’ water and feed consumption for 7 days of treatment with OEL, and their change in weight. A significant decrease in water and feed consumption of animals treated with the highest dose of OEL and 5-FU was observed, which corresponded to a significant decrease in weight. These results indicate toxicity of OEL to the gastrointestinal system at 100 mg/kg, however, such effects are observed with most anticancer drugs currently in use, as seen here for 5-FU. As such, this effect is not a principally limiting factor for pre-clinical pharmacological studies.

Concerning biochemical and hematological parameters, no significant changes were observed in urea and creatinine levels in all the groups (Table 3). Urea is a product of amino acid metabolism, and creatinine is a breakdown product of muscle creatine; both substances are excreted by the kidneys [30]. Upon kidney failure, degradation products of metabolism, particularly nitrogenous substances such as urea and creatinine, accumulate in the body, resulting in increased serum levels. Several anticancer drugs currently in clinical use trigger some form of renal toxicity [21]. However, renal toxicity was not observed for OEL.

With regard to liver function, significant increases in the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were observed in the serum of OEL-treated, but not FU-treated, mice (Table 3). Tumor implantation alone increased AST activity in the tumor control group as compared to the healthy group (Table 3). Like kidneys, the liver is susceptible to the effects of anti-neoplastic agents. In cases of liver injury, AST and ALT spill into the blood thus allowing diagnosis and monitoring of liver injury [31]. However, while AST is found in liver cells, it is also found in skeletal and cardiac muscle, kidneys, pancreas, and erythrocytes. When any of these tissues is damaged, AST is released into the blood. Since there are no routine laboratory methods available to determine the origin of AST in blood, diagnosis of the cause of its increase should take into account the possibility of injury to any of the organs where it resides [32]. The significant increase in transaminase activity suggests oil-induced liver toxicity for the higher dose employed here. We noted an increase in AST activity in all transplanted animals, regardless of the treatment, which may be related to tumor-caused damage to one or more of the tissues harboring AST. Skeletal muscle is one of the likely sources, due to mechanical trauma caused by a tumor invading the sub-axillar region. The liver is the other likely source, because it is a complex organ involved in many functions, among them, participation in the mononuclear phagocyte system, and it is the primary site of the removal of antigen-antibody complexes from circulation.

Regarding hematologic parameters, unlike most antineoplastic agents, OEL did not significantly affect the major hematological parameters of the animals (Table 4). There was, however, an increase in the value of the mean corpuscular hemoglobin concentration (MCHC). The erythrocyte indices MCV, MCH and MCHC are used in the diagnosis of anemias, but this particular result may be related to some degree of hemolysis of the samples since no significant changes were observed in RBC, MCV and MCH [31].

In the tumor control group, the percentage of monocytes was significantly increased above that of the healthy group. The primary function of a monocyte is phagocytosis and digestion of large particulate matter such as senescent cells and necrotic cellular debris [33]. This can explain the increase of this cell type in the tumor-bearing mice. A comparable increase of this cell type was not observed in animals treated with 5-FU or OEL, probably due to the inhibitory effect on tumor growth and hence reduced need for phagocytosis.

Additionally, it should be noted that the percentage of neutrophiles was increased in all transplant groups, regardless of treatment. This phenomenon is observed in cases of tissue necrosis and in the presence of tumors [34]. Likewise, the percentage of lymphocytes was decreased in all transplant groups, which is likely related to the reduced lymphocyte counts observed in moribund animals [34].

A marked leukopenia (decreased white blood cell count) with an increase in the lymphocyte percentage was evident in animals treated with 5-FU. Such changes are expected in antineoplastic treatment [35].

To evaluate possible toxic effects of OEL on the animals’ organs (liver, kidneys, spleen and thymus), these were excised, weighed and analyzed macroscopically for the presence of necrosis or bleeding. No changes were observed in any of the treated animals, with the exception that the spleen index was reduced significantly in animals treated with 100 mg/kg OEL, thereby the increase of this parameter caused by tumor inoculation was reversed (Table 5). 5-FU, a drug currently used in clinical medicine, which reduces spleen and thymus indices, exerts a potent immunosuppressive effect [36]. There is a close relationship between the occurrence, growth, and decline of a tumor, and the general state of the immune system. Immune function in an organism may respond not only during the generation and development of the tumor, but it may also be an important factor in preventing the patient’s tumor from returning [37]. Thus, one of the major drawbacks of current anticancer therapies, such as chemotherapy and radiotherapy, is the suppression of the immune system [38], which was not observed in the treatment with OEL.

5-FU also reduced the liver index, revealing the hepatotoxic effect of this drug, which corroborates literature data [36].

For a more detailed evaluation of possible toxic effects on the organs of the treated animals, a histological analysis was carried out. The kidneys of the mice from all groups did not differ histologically (Figure 3), indicating low OEL toxicity in kidneys, thereby corroborating the results of the biochemical analysis of renal function (Table 3). On the other hand, while the livers of animals treated with 5-FU or OEL at a dose of 50 mg/kg displayed preserved lobular architecture, lobular and portal venous congestion was observed (Figure 4A), as well as small and sparse foci of hepatocellular necrosis with deposition of thin collagen fibers (fibrosis) (Figure 4B), and Kupffer cell hyperplasia (Figure 4C). The same changes, albeit more frequent, were observed in animals treated with a dose of 100 mg/kg. In addition, in two animals treated with an OEL dose of 50 mg/kg a hepatocellular proliferative effect was observed (Figure 4D). These findings suggest a toxic effect on this organ, corroborating the finding of increased AST and ALT activities as a consequence of OEL treatment (Table 3). These changes, common to both groups treated with OEL, have been reported in the literature as evidence of weak hepatotoxicity during which withdrawal or reduction of the dose of the drug usually leads to rapid recovery from the damage [39]. The liver is an organ with great regenerative and adaptive ability, even when hepatocellular necrosis is present, as observed in the livers of the OEL-treated animals. If the conjunctive tissue is preserved, regeneration can be almost complete [40].

Figure 3:

Histopathology of kidneys of experimental groups: (A) Glomerulus preserved with thin Bowman’s capsule, capillary tuft supported by delicate mesangium, proximal convoluted tubule – Control; (B) Proximal convoluted tubule – 5-FU (50 mg/kg); (C) Loop of Henle – OEL (100 mg/kg) and (D) Distal convoluted tubule – OEL (100 mg/kg). Hematoxylin-eosin.

Figure 4:

Histopathology of liver of experimental groups: (A) lobular and portal venous congestion – 5-FU (50 mg/kg); (B) reticulin collapse – OEL (50 mg/kg); (C) Kupffer cell hyperplasia – OEL (100 mg/kg) and (D) isolated mitoses – OEL (100 mg/kg). Hematoxylin-eosin (A, C and D) and Gordon and Sweet method for reticulin (B).

The medical use of essential oils is not regulated in many countries, even though little is known about their toxicity. Few publications on essential oils contain data on their “latent” toxicities such as teratogenesis, carcinogenesis, or mutagenesis [41]. The genotoxic effects of anticancer drugs in non-tumor cells are of special significance for the possible induction of secondary tumors in patients undergoing therapy [42]. To evaluate possible in vivo genotoxic effects of OEL, we checked the frequency of micronucleated erythrocytes in peripheral blood, but found no such effect of OEL (Table 6). This suggests that OEL does not have in vivo genotoxic (clastogenic and/or aneugenic) effects, an important aspect of its therapeutic applicability.

4 Conclusions

OEL has significant in vivo antitumor activity, along with low toxicity. Our results may assist in developing novel OEL based chemotherapeutic agents.