Preclinical Evaluation of the Encelia canescens Lam Extract: Medicinal Properties useful for Cancer Treatment

Ipomoea reniformis Chaos is claimed in Indian traditional medical practice to be useful in the treatment of epilepsy and neurological disorders. In the present study, pretreatment effect of methanolic extract of Ipomoea reniformis on epilepsy and psychosis was evaluated in rodents using standard procedures. Besides evaluating epileptic and behavioral parameters, neurotransmitters such as Gamma-Amino Butyric Acid (GABA) in epilepsy and in psychosis dopamine, noradrenaline and serotonin contents in the rodent brain were estimated. The extract pretreatment reduced maximal electro shock; Isoniazid (INH) and Pentylenetetrazole (PTZ) induced seizures and also significantly inhibited the attenuation of brain GABA levels by INH and PTZ in mice. These results suggested that the observed beneficial effect in epilepsy may be by enhancing the GABAergic system. The test drug also inhibited the apomorphine induced climbing and stereotyped behavior and showed significantly reduced levels of brain dopamine, noradrenaline and serotonin which may be due to blocking of central dopaminergic, noradrenergic and serotonergic pathways or by enhancing the GABAergic system. The results obtained in present study suggest that the title plant possesses antiepileptic and antipsychotic activities in rodents.


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].
The plant contains various neuroprotective chemical constituents such as caffeic, p-coumaric, ferulic and sinapic acid esters. Petroleum ether extract contains fats and fixed oil while aqueous extract contains amino acids, tannins (condensed and pseudo tannins) and starch [5]. IR has been reported to possess various pharmacological actions, mainly antidiabetic [6], antiinflammatory [7], nephroprotective [8], antibacterial [9], antioxidant and antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological

Introduction
Through the years, humans have used agents from natural sources to cure their pathological conditions 1 . In this sense, plants have formed the basis of traditional systems of medicine that have been in use for hundreds of years. Currently, these systems are still developing roles in healthcare. The World Health Organization (WHO) has estimated that approximately 80% of people worldwide have used traditional medicine for health care 2 . Moreover, many secondary metabolites of plants, including alkaloids, glycosides, polyphenols, coumarins, saponins, terpenes and terpenoids, which have interesting medicinal properties, including antineoplastic activity, are known to be potentially useful for protecting the human body from diverse pathological conditions 3,4 .
Currently, phytopharmacology and ethnomedical ancestral knowledge is being combined with the modern basic and clinical pharmacological knowledge to fight pathological conditions. The present tendency retains the use of the whole medicinal plant in the form of standardized extracts combined with the use of numerous pharmaceutical technologies to create products that do not differ in appearance or quality from traditional allopathic medicines.
The WHO defines a medicinal plant as any plant species that contains substances that can be used for therapeutic purposes or whose active ingredients can act as catalysts for the synthesis of new drugs. Folk knowledge about medicinal plants is based on efficiency or is accepted and adopted based on observations of previously used plants; however, a problem in popular phytotherapy is the difficulty of controlling the dose and the product quality, and this problem can elicit risks and damage to health. Despite the low toxicity of the active ingredients of some plant species, they can elicit health problems due to factors such as microbiological contamination and the presence of pesticide residues, heavy metals or herbicides.
In contrast, cancer is a complex disease and the leading cause of death worldwide, and its treatment, control and prevention requires a multidisciplinary approach. Therefore, researchers are constantly searching for new compounds that can fight this disease and improve patient quality of life. In this search, plants are the main potential source of anticancer agents 5 .
Although the development of cancer treatments has been spectacular and there are many potential drugs for fighting the proliferation of tumour cells [5][6][7][8][9] , these drugs that are created by chemical synthesis and/or derived from plants do not have the expected clinical effects. Therefore, more effective and less toxic antineoplastic agents are needed, and their modes of action require further active research.
Among the natural products with anticancer activities, the best-known examples are the vinca alkaloids (vinblastine and vincristine) isolated from Madagascar periwinkle, Catharantas roseus which is used in many cultures for the treatment of diabetes. During an investigation of this plant, these compounds were discovered to exhibit strong potential as hypoglycaemic agents, but their discovery was indirectly attributed to the observations based on an unrelated use.
Other good examples include the epipodophyllotoxins (etoposide, etoposide phosphate, and teniposide), the taxanes (paclitaxel and docetaxel), and the camptothecin derivatives (irinotecan and topotecan). Several other plant-derived compounds are currently in preclinical and clinical trials 3 . Thus, research that is conducted on a natural product and progresses until the agents are obtained may be considered to be a complete cycle for a plant that was originally used for the treatment of cancer.
The new interesting and potential antitumouralplant reported here is Encelia canescens Lam (nv: mancapaqi, mataloba, matalobo, mucle, or "coronilla de fraile"), which is a 30-80 cm high shrub by perennial plant that grows in the Atacama and Coquimbo regions of Chile up to 1,700 m above sea level. The plant infusion has been used locally for at least 100 years to treat cancer symptoms and other pathologies in northern Chile. Until now there is not literature evidence for its anti-cancer properties. Based on this observation, the present study was conducted to gather information about the properties of Encelia canescens Lam extracts (ECEs) to examine their potentialclinical applicationsfor cancer treatment.

Plant Material
The aerial parts of Encelia canescens Lam.

Extraction and Identification
Encelia canescens Lam leaves were pulverized, boiled in distilled water or ethanol at 100 g/L for 10 min, and clarified by filtration through Whatman #1 paper.

Cell Line and Culture Media
The SaOS-2 cell line, which is a non-transformed cell line derived from a human osteogenic sarcoma (American Type Culture Collection ATCC ® : HTB-85 German Collection of Microorganisms and Cell Cultures DSMZ: ACC 243), was used for the antiproliferative studies. The cells were cultured in bovine foetal serum supplemented with McCoy's 5A media with 100 U/mL of penicillin, streptomycin (0.07 M), amphotericin (0.5 µM) and L-glutamine (2mM; Gibco ® ). Incubation was performed at 37 °C for 48 hours in 95% air and 5% CO 2 . A protocol for assessing the growth profiles of individual cell groups under study was applied to perform cell counts from the three plates for each group at regular intervals of 24 hours over a total of 5 days (i.e., the cell count times were 0, 24, 48, 72, 96 and 120 hours). The medium for each cell group was replaced every 48 hours (i.e., at 48 and 96 hours).

Antimicrobial Activity (Bioautography)
The following clinically isolated microorganisms were used in this study: Staphylococcus aureus, Bacillus  10,11 was employed to evaluate the antimicrobial activities of the extracts. 20 milligrams samples of the plants were dissolved in 1 mL of methanol and applied in volumes of 10 μL to two glass plates coated with silica gel 60 (5 x 7 cm) that had previously been UV sterilized. The solvent solution used for elution was hexane:ethyl acetate (6:4). A Thin Layer Chromatography (TLC) plate was placed into a sterile petri dish and incubated at 37°C for 24 h for the bacteria or at 28°C for 48 for the fungi. The TLC plate was then stained with a solution of tetrazolium bromide dye at 5 mg/mL for 1 h. Purple or colourless halos indicated growth inhibition.

Determination of the Total Phenolic Compounds
Using a modified Folin-Ciocalteu method 12 , the total phenol contents of the extracts were determined. Onemillilitre aliquots of the extracts were mixed with 5 mL of water-diluted (1:10) Folin-Ciocalteu reagent and 4 ml (75 g/l) of sodium carbonate. The tubes were vortexed for 10 sec and allowed to stand for 30 min at 40°C for colour development. Absorbance was then measured at 765 nm using a UNICAM UV-VS spectrophotometer. The samples of the extracts were evaluated at a final concentration of 0.1 mg/ml. The total phenolic contents are expressed as mg/g tannic acid equivalents.

Determination of Total Flavonoids
The estimations of the total flavonoids in the plant extracts were performed using the method of 13 . One-half millilitre of 2% AlCl 3 ethanol solution was added to 0.5 ml of the sample. After one hour at room temperature, the absorbance was measured at 420 nm. A yellow colour indicated the presence of flavonoids. The extract samples were evaluated at a final concentration of 0.1 mg/ml. The total flavonoid contents were calculated as quercetin equivalents (mg/g) using the following equation, which was based on the calibration curve: y = 0.0255 (X), R 2 = 0.99812, where X is the absorbance in quercetin equivalents (mg/g).

DPPH (2,2-diphenyl-1-picrylhydrazyl) Test
For the proper measurement of the antioxidant potentials of the tested extracts, the DPPH free radical scavenging method was utilized 14 . The scans were run against pure ethanol or distilled water in a UNICAM spectrophotometer at 517 nm. The measurements in the measuring cuvettes were performed 30 min after the addition of DPPH in darkness to allow sufficient time for the reactions of the antioxidants with DPPH. All measurements were performed in triplicate. The results are presented as trolox equivalent antioxidant capacities (µmol trolox/100 g dry extract).

ABTS (2, 2'-azino-bis-3ethylbenzothiazoline-6-sulphonic acid) Radical Scavenging Assay
The method of 15 was adopted for the ABTS radical scavenging assay. The stock solutions included 7 mM ABTS solution and 2.4 mM potassium persulfate solution. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted by mixing 1 ml of the ABTS solution with 60 ml methanol to obtain an absorbance of 0.706 ± 0.001 units at 734 nm on the spectrophotometer. Fresh ABTS solution was prepared for each assay. Plant extracts (1 ml) were allowed to react with 1 ml of the ABTS solution, and the absorbance was taken at 734 nm after 7 min using the spectrophotometer. The results are presented as trolox equivalent antioxidant capacities (µmol trolox/100 g dry extract).

Oral Toxicity
Albino CF-1 mice were used to evaluate the oral toxicities 16 of the ECEs extracts following the administration of 10 mg to 2 g/kg of body weight of the extracts suspended in gum arabicin 5% saline. For this study, we generated 12 groups of 5 animals per dose plus a control group (1 ml of tap water). Parameters including convulsions, sedation, hyperactivity, grooming and accelerated breathing were observed for ten days following treatment while food and water temperature were controlled. Upon the completion of testing, the animals were sacrificed by cervical dislocation. Necropsies were performed on the main organs of the animals following the observation period to determine whether changes occurred.

Acute Dermal Toxicity
Sprague-Dawley (200-250 g) rats were used to evaluate dermal toxicity according to the guidelines of the Environmental Protection Agency 17 . Eight groups of five rats of both sexes were assayed. The administered ECE doses were 10mg/kg, 30mg/kg and 50mg/kg. A non-treated group was used as a control. Upon the completion of testing, the animals were sacrificed by cervical dislocation, and necropsies were performed to evaluate potential changes in the animals' internal organs.

Topical Analgesia
Individual male CF-1 mice were placed in acrylate boxes (30 x 30 x 30 cm) with mirrors on three sides to aid observation for approximately 15 minutes prior to the initiation of the test for habituation. The animals were pre-treated with the ECEs or ibuprofen as a standard analgesic for three minutes via the immersion of the tails of the animals in 5% w/v of the test sample in Dimethyl Sulfoxide (DMSO). Subsequently, 10% formalin (saline solution) was intradermally injected in the first third of the tail using a 29G needle, and the animals were immediately placed in the boxes. Pain-like behaviours (i.e., licking/biting and flinching) were recorded in 5-minute time bins using the Pocket Observer (Noldus) for 45 minutes 18 . The animals were euthanized immediately at the end of the study. All experiments were recorded using a video camera to create back-up information. The control mice were submitted to the same procedure, but the tails were immersed in a DMSO solution that lacked ECEs and ibuprofen. Topical analgesic activity (AA) was calculated according to the following formula: %AA = 100-(median licking time for the test/ median licking time for the control) x 100.

Oral Analgesia
The peripheral analgesic activities of the ECEs were measured with the acetic acid-induced writhing test as described earlier 19 . Briefly, the inhibitions of writhing produced by ECEs that were administered via gastric feeding tubes (80 mg/kg mouse weight) were determined by comparison with the inhibition exhibited by the control group. Sodium naproxen at an oral dose of 50 mg/kg was used as the standard analgesic agent. Intraperitoneal injections of acetic acid (0.6%) at 1 ml/100 g of body weight were used to create pain sensations (algesic agent). The animals were individually placed in a glass chamber with mirrors on three sides. The numbers of writhings were calculated over 5 min following the application of acetic acid. Oral analgesic activity (AA) was calculated according to the following formula: %AA = 100-(median writhing time for the test/ median writhing time for the control) x 100.

Inhibition of the Growth of Tumour Cells in Culture
For the cell proliferation study, we generated five study groups of SaOS-2 cell line cultures according to the following treatments, 1. Control group, 2. 1% A-ECE, 3. Water, 4. 1% E-ECE, and 5. 0.5% ethanol. A protocol for assessing the growth profiles of the individual cell groups under study was applied. This protocol involved the performance of cell counts from three plates for each groupat regular intervals of 24 hours over a total of 5 days (i.e., the cell counts occurred at 0, 24, 48, 72, 96 and 120 hours). After every 48 hours elapse of cultivation (i.e., at 48 and 96 hours in the protocol), the culture medium of each cell group was replaced with fresh media.

In vivo Inhibition of Tumour Growth in the Mice
A trial was conducted on the A/J strain mice according to the recommendations of the National Health Institute 20 for assessing the antineoplastic effects and safety and toxicity of chemical substances following acute exposure. This trial was based on the recommendations of the OECD provided in regulation No. 451 21 . Subcutaneous tumours were generated via the injection of TA3 oncogenic cells (10 6 cells/0.1 ml) mixed with 0.9% NaCl solution at a 1:1 ratio into the backs of the mice. The tumours were allowed to grow for seven days. The extracts were orally administered daily for 15 days beginning at day 7 of tumour cell injection. Fourteen

Statistical Analyses
The experimental results are expressed as the means ± the standard errors (SEs) of three replicates. When applicable, the data were subjected to one-way Analyses of Variance (ANOVA), and the differences between samples were determined with Kruskal-Wallis tests using STATA 10.0. P values < 0.05 were regarded as significant.

Qualitative Screening
As a first approach, the qualitative physicochemical characterization for several extracts from Encelia canescens Lam was assessed with Thin Layer Chromatography (TLC). The results revealed the presence of several potentially relevant compounds, specifically, saponins, sterols, terpenes, flavonoids, coumarins and tannins (Table 1.), and some specific compounds representative of these families were detected and quantified with High-Performance Liquid Chromatography (HPLC) and nuclear magnetic resonance spectroscopy (H 1 RMN) (Figures 1 and 2).

Antimicrobial Activity
The antimicrobial activities of the extracts were examined with bioautography assays.

Determination of the Total Phenolic Compounds and Total Flavonoids
The total phenolic compounds, expressed as mg (mean ± SD)per g of tannic acid, were 19 ± 1.14 for the A-ECE and 23 ± 1.60 for the E-ECE. The total flavonoids, expressed   as mg (mean ± SD) per g of quercetin, were 0.15 ± 0.02 for the A-ECE and 0.13 ± 0.06 for the E-ECE (Table 2).

Antioxidant Activity
The antioxidant capacities of the extracts were evaluated as Trolox Equivalent Antioxidant Capacities (TEACs) using DPPH and ABTS assays. The results revealed both extracts exhibited good antioxidant activity, and the E-ECE was the slightly more powerful antioxidant extract; i.e., for DPPH and ABTS, the activities of E-ECE were 354±23 µmol trolox/100 g dry extract and 2,354±568 µmol trolox/100 g dry extract, respectively, compared with the DPPH and ABTS activities of 303±15 µmol trolox/100 g dry extract and 1,856±453 µmol trolox/100 g dry extract, respectively, for the A-ECE (Table 3).

Toxicity Evaluation
Assays of the acute dermal and oral toxicities were performed (data not shown). No dermal effects (oedema and/or erythaema) were observed. Furthermore, no changes in weight relative to the control group or alterations in the internal organs were observed at any of the tested doses. Similarly, no evidence of toxicity was observed in the oral toxicity assay; i.e., there were no signs of toxicity during or at the end of the treatments. Thus, the movements of animals were normal, and no hind drop, drowsiness, wheezing, cyanosis, diarrhoea, emesis, salivation or piloerection were observed. The  necropsies revealed no differences in the colours, sizesor textures of the internal organs of the extract-treated animals compared with the animals in the control group.

Analgesic Activity (%AA)
The results displayed in

Antiproliferative Activity in Cell Culture
The comparative analysis of the cell counts for the groups of cells treated with the two extracts (aqueous and ethanolic) in relation to the control cells and the cells treated with the aqueous or ethanolic phases (vehicles) revealed statistically significant differences in the reductions in the potentials for the proliferative growth of the SaOS-2 cells (p < 0.001) (Figure 3). The E-ECE extract was slightly more efficient in terms of this antiproliferative effect.

In vivo Antitumour Activity
The tumour incidences were 100% in the control and treated groups of mice prior to extract treatment. There Journal of Natural Remedies | ISSN: 2320-3358 www.informaticsjournals.org/index.php/jnr | Vol 15 (2) | Dec 2015 were no significant changes in the weights of the mice during the course of treatment, which corroborated the lack of toxicity of the ECEs that was previously observed (data not shown). Analyses of the changes in the tumour sizes of the control and ECE treatment groups revealed a lower tumour growth rate and a significant (p<0.05) decrease in tumour size in the mice treated with the lyophilized extract at the dose of 8 mg/kg x day (Table 5).
In contrast, a between-group evaluation of survival performed with a Kruskal-Wallis analysis of variance test for independent samples revealed significant differences between the control mice and the mice treated with the lyophilized extract at the doses of 4 and 8 mg/kg x day and mice treated with A-ECE at the dose of 16 mg/kg x day (Table 6).

Discussion
The search for plants and active compounds with anticancer properties began in the 1950s following the discovery of vinca alkaloids and podophyllotoxins. Subsequently, plants became a prime source of highly effective conventional drugs for many cancer treatments 22 , and this process has created a list of many anticancer phytochemicals that includes saponins, sterols, terpenes, flavonoids, and alkaloids among others 8,23 . However, the question presently posed is why some of these compounds are more toxic and occasionally less active than whole-plant extracts. Therefore, the study of extracts seems to be a promising area of research that has recently become a major concernacross the world [5][6][7]9 .
Considering that there are probably many anticancer plants in nature that have yet to be explored in terms of their antitumoural activities, there is an urgent need to develop physicochemical, preclinical and clinical studies to explore these activities. Therefore, in the present study, we examined the toxicological, antibacterial, antioxidant, analgesic, antiproliferative and antitumoural properties of extracts of E. canescens Lam, all of them useful in cancer patients. The families of active compounds identified by TLC screening and HPLC and NMR analyses in the present workwere saponins, terpenes, flavonoids, coumarins and tannins. Evaluations of the antioxidant capacities of the E-ECE and A-ECE extracts revealed that both extracts exhibited strong antireactive oxygen species properties. Moreover, neither extract exhibited toxicity in the acute topical or oral toxicity tests.
It is important highlight that at this stage we decided not to investigate antineoplastic effects of the individual components of the extracts to instead evaluate the potential utilities of the whole extracts. In this respect, there is growing evidence that whole extracts can be effective anticancer reagents that are occasionally superior to the individual components. In 24 reported that whole cell extracts from Urtica membranacea, Artemisia monosperma and Origanum dayi post exhibit particularly strong anticancer capabilities. Similar findings have been reported in relation to other pathologies; for example, it is quite clear that mixed herbal components and plant extracts exhibited superior efficacies in the treatment of malaria compared with single compounds 25 .
In contrast, patients with cancer are at a significant risk for infection due to chemotherapy, radiation or surgery treatments. Additionally, many researchers have proposed that bacterial infections can cause cancer 26,27 . Therefore, we examined the antibacterial properties of Encelia canescens Lam extracts to analyse their usefulness for addressing this collateral factor. Unfortunately, we did not observe any antibacterial activity for the water or ethanol extracts.
The evidence for the anticancer effects reported here is mainly based on results related to the antiproliferative effects in tumour cell cultures and the antitumouractivities in mice. The antiproliferative study clearly demonstrated substantial inhibitory effects of both the E-ECE and A-ECE extracts ( Figure  1). Moreover, the in vivo antitumour assays revealed that the A-ECE extract at the dose of 16 mg/kg x day and the lyophilized aqueous extract at 4 and 8 mg/kg x day efficiently increased the survival of the mice with tumours and that the lyophilized extract was able to significantly reduce tumour size at the dose of 8 mg/kg x day.
As has previously been reported by several authors, antioxidant activity seems to be directly related to anticancer potential 23,28 ; thus, the potent antioxidant activities observed for both the E-ECE and A-ECE extracts might reinforce this idea.  Finally, the observed analgesic effects are also interesting because analgesic use is an important issue for cancer patients (NCI, 2015).

Conclusion
The results presented here establish that the extracts of Encelia canescens Lam have specific antiproliferative and antitumour activities. Additionally, the observed antibacterial and analgesic effects of both extracts are positive findings of the present research that demonstrate additional features of this potentially antineoplastic plant. Moreover, considering the innocuous natures of the extracts and their interesting antioxidant activities, clinical and pharmaceutical studies should be approved by health authorities and performed to support the effectiveness and safety of the use of extracts of this plant in cancer patients.