PHYTOCHEMICAL SCREENING, TOXICITY EVALUATION OF POLYCARPAEA CORYMBOSA LAMK AND ITS EFFECT ON CANCER BIOMARKERS OF EHRLICH ASCITES CARCINOMA-INDUCED MICE COMPARED WITH THE REFERENCE STANDARD DRUG 5- FLUOROURACIL

Objective: The current investigation focuses on the study of efficacy of whole plant of Polycarpaea corymbosa Lamk in Ehrlich ascites carcinoma (EAC) inoculated Swiss albino mice.
Methods: The whole plant of P. corymbosa Lamk (WPC) was extracted with solvents of increasing polarity and their percentage yields were calculated. The major phytoconstituents present in the plant extracts were determined by standard chemical tests. Tumor was induced in mice by intraperitoneal injection of EAC cells (1×106 cells/mouse). The in vivo antitumor effect of extracts was assessed by monitoring the mean survival time, tumor volume, effect on hematological parameters, determination of lysosome specific cancer markers (cathepsin-D), β-D glucuronidase and acid phosphatase, liver marker enzymes (5’-nuclotidase and lactate dehydrogenase), membrane bound ATPase (Na+/K+ ATPase and Mg2+ ATPase), DNA, and RNA content.
Results: The percentage yield obtained were 9.87%w/w, 7.88%w/w, and 16.56% w/w for petroleum ether, ethyl acetate, and ethanol extract, respectively. The phytochemical screenings of those extracts were performed. The order of activity of extracts was ethanol extract > ethyl acetate > petroleum ether. Among the extracts, Ethanol extract of P. corymbosa Lamk. showed a significant increase in life span and decrease in viable cancer cell number and tumor volume. The protective effect of the extract on the hemopoietic system at the dose 200mg∕kg was noted. The alterations in the hematological profile, lysosome-specific cancer markers, liver-specific cancer markers, and membrane-bound ATPases DNA and RNA were restored.
Conclusion: The ethanolic extract of P. corymbosa Lamk. possesses in vivo anticancer activity when compared to the tumor control group.


INTRODUCTION
Cancer is the second most cause of the death worldwide which is characterized by abnormal growth of cells due to the insensitivity of genes. It affects people at all ages [1] where damage to the genetic material of the malignant cells in progeny was observed in common. The imbalance in pro-oxidant and antioxidant is an important cause of cancer. The oxidative stress build up in turn generates reactive oxygen species inside the cell which further progresses to cellular transformation, genomic instability, hyperproliferation, immortalization, angiogenesis, and metastasis [2]. Understanding the key enzymes involved in the disease progression is important to identify the new drug targets. The screening of new molecules or natural agents with potential antitumor activity was vital to spot the right drug for treating the disease with ease. Hence, the requirement of substitutes for the conservative chemotherapeutic drugs which is economical, easy accessible, and therapeutically active at nontoxic doses and combats chemoresistance to control the disease progress is required [1].
The plants which belong to the Caryophyllaceae family have potential source of secondary metabolites and have many medicinal properties [3]. The whole plant Polycarpaea corymbosa Lamk belongs to the family Caryophyllaceae (Synonyms-Achyranthus corymbosa). P. corymbosa Lamk was an annual or perennial herb consisted leaves which were opposite or appear in whorls, linear up to 3.5 cm long with a brittle at the tip. The flowering season of this plant was from August to September. Flowers were borne in compact heads at the end of stems. Sepals were silvery white, turning rich brown with age. Petals were small, pink to orange in color. Fruit was a minute ellipsoid capsule. It was distributed in India, Burma, Ceylon, Western Peninsula and ascending the western Himalayas to 7000 feet [4]. It was used both internally and externally as remedy for venomous bites from reptiles and animals [5]. In traditional Chinese medicine, it was used to treat cough and in Indian traditional medicine; it was used in the treatment of jaundice and inflammatory swellings [6]. This plant has proven antioxidant [7][8][9][10], anti-inflammatory [11,12], antiulcer [13][14][15], antidiarrheal [16], antimicrobial [17], analgesic [18], hepatoprotective [8,19,20], and androgenic [21] properties. The cytotoxic potential against colorectal adenocarcinoma, breast cancer, leukemia, and prostate carcinoma of human cell lines were evaluated, in which potential activity against breast cancer cell line [22] was observed. Hence, the current study was investigated to determine its efficacy in Ehlrich ascites carcinoma (EAC)-induced mice, an in vivo model.

Collection and identification of plant
The whole plant of P. corymbosa Lamk (WPC) was collected in the month of August and September from Palayamkottai, Tirunelveli District, Tamil Nadu, India. The taxonomic identification of the material was authenticated by Dr. G.V.S. Murthy, Ph.D., Scientist F and Head, Botanical Survey of India, Southern Regional Centre, Coimbatore, India. The plant material was dried under shade, segregated, pulverized by a mechanical grinder, and passed through a 40 mesh sieve. The powdered plant material was stored in airtight containers and used for further research.

Preparation of extracts and preliminary phytochemical screening
The air-dried powdered material of about 2 kg from the whole plant was successively extracted by hot continuous percolation method in Soxhlet apparatus by increasing the polarity of the solvents such as petroleum ether, ethyl acetate, and ethanol. Solvents used for this entire work were of analytical grade (Merck, Mumbai). The extracts were collected and filtered through Whatmann filter paper No. 40, and the solvents were recovered from the extracts under reduced pressure using rotary evaporator. The concentrated filtrate was then evaporated to dryness in vacuum at 35-40°C. Then, the extracts were stored in screw capped vials at 4°C until further use. The percentage yield was calculated with reference to the crude material was calculated, and the preliminary phytochemical screening for alkaloids, carbohydrates, proteins and amino acids, glycosides, saponins, flavonoids, tannins and phenolic compounds, phytosterols, fixed oils, and fats was carried out [23,24].

Animals
Swiss Albino mice (25-30 g) were maintained under standard laboratory conditions temperature (27±2°C) with relative humidity of 55±5%, in an experimental room under a 12 h light/12 h dark cycle as per CPCSEA. Before the experiment, mice were kept under quarantine for a week for acclimatization to the laboratory conditions. They were fed with commercial pellet diet and normal drinking water was provided ad libitum. The approval of the Institutional Animal Ethical Committee was obtained whose reference number is 1/243/CPCSEA dated 22 November 2013.

Acute toxicity study
As per guideline of Organization of Economic Co-operation and Development 423, the acute toxicity studies of petroleum ether, ethyl acetate, and ethanol extract of WPC were carried out [25].

Tumor cells
EAC cells were obtained from the Amala cancer Research Centre, Kerala (Thrissur), India. The EAC cells were maintained in vivo in Swiss albino mice, by intraperitoneal (i.p) injection of 1×10 6 cells/mouse after every 10 days [12].

Grouping of animals
Male Swiss albino mice were divided into 6 groups (n=6) and Group I served as normal control; Group II served as disease control (Inoculated with 1×10 6 EAC cells/mouse (i.p); Group III treated with pet ether extract (200 mg/kg b.w p.o) during induction; Group IV inoculated with 1×10 6 EAC cells/mouse and treated with ethyl acetate extract (200 mg/kg b.w p.o) and Group V inoculated with 1×10 6 EAC cells/mouse and treated with ethanol extract (200 mg/kg b.w p.o.); Group VI Inoculated with 1×10 6 EAC cells/mouse and treated with standard drug 5-fluorouracil (20 mg/kg b.w) for 14 days.

Evaluation of body weight:
Body weight of the animals was recorded from the day 1 to day 15 of the study. Average body weight at 15 th day was determined. The mean survival time and percentage increase of life span were calculated using the following formula [26].

Hematological profile evaluation
After the last dose and 18-hr fasting, blood was collected from six mice under mild anesthesia for hematological evaluation in each group which includes red blood cells (RBC), hemoglobin (Hb), total white blood cells (WBC), and differential count (neutrophils, lymphocytes, and monocytes) [27].

Tumor volume
After the treatment period, animals were dissected, and the ascitic fluid was collected from the peritoneal cavity and its volume, packed cell volume were determined [12], and viability was assessed using trypan blue exclusion assay.

Statistical analysis
The results were expressed as mean±SEM or mean±SD.

Acute toxicity
It was observed that treatment of animals with PEEPC, EAEPC, and EEPC did not show any changes in the autonomic or behavioral responses. Zero percent mortality was obtained for all the extracts of the WPC and hence the extracts were found to be non-toxic up to the dose of 2000 mg/kg.

Anticancer study
From Table 1, it was observed that the animals treated with EAC have increase in body weight, decrease in mean survival time, percentage increase in life span, and increase in tumor volume, packed cell volume, and viable cell count than the extract treated groups. On comparing within the extracts, group V and VI showed potential activity. Similar results were not found in other treatment groups. In Group II, animals were died after 18 days of inoculation whereas the life span of group V and VI was 6 weeks.

Hematological parameters
The haematological parameters namely RBC, Hb, Total WBC count and differential count were observed for various extracts of the WPC as furnished in Table 2. In group II, RBC, Hb, Lymphocytes and monocytes were found to be decremented whereas total WBC count and neutrophils were observed to be incremented. Group V and Group VI has restored RBC, Hb, total WBC count and differential count.
Estimation of lysosomal marker enzymes, liver markers, Na + /K + -ATPase and Mg + -ATPase, DNA, and RNA i. The Lysosomal Marker enzymes in liver of experimental groups were depicted in Table 3. In Group II, the activity of Cathepsin-D was elevated twice (41.19±1.75), than the Group I (21.73±0. 19). β-D glucuronidase activity was increased (38.78±1.25) by 56%in Group II when compared to Group I. The increase in the activity of acid phosphatase was 3-fold (9.14±0.24) when compared to Group I. In Groups III -VI, the lysosomal marker enzyme levels were decreased compared to Group II.
ii. Table 4 depicts the activity of liver marker enzymes 5'-nucleotidase and LDH in experimental groups. The level of 5'-nucleotidase in Group II was elevated thrice (6.46±0.25) when compared to Group I (2.58±0.10). Nucleotidase activity is increased when tumor occluding the bile ducts.

Abirami and Muthusamy
In Group III-VI, the marker enzyme levels were decreased compared to the Group II. LDH activity in the liver of Group II (0.41±0.12) was markedly declined in comparison to Group I (1.59±0.10). In Group III-VI, the increase in LDH level of liver was observed compared to the Group II.
iii. Table 5 illustrated that the effect of different extracts of WPC on the activities of ATPases, nucleic acids in liver of normal and experimental group of animals. When compared to Group I, Na + /K + -ATPase activity was decreased significantly by 2-fold; the decrement in the activity of Mg 2+ -ATPase was found to be 56% in Group II. The increment in enzymes was observed in other treatment groups. The level of DNA in Group II gets elevated by 4-fold; when compared to Group I. The increment in the levels of RNA in Group II was found to be 55% when compared to Group I. In Group III-VI, the marker enzyme levels were decreased compared to Group II.

DISCUSSION
The phytoconstituents were responsible for the therapeutic properties of the plants [24]. From the phytochemical screening, it was found that the numbers of phytoconstituents were more in ethanol extract followed by ethyl acetate extract and petroleum ether extract. All these extracts were found to be nontoxic up to the dose 2000 mg/kg in acute toxicity study. Based on the in vitro cytotoxic evaluation on various cell lines, the whole plant of P. corymbosa Lamk. exhibited potential activity against breast cancer cell line [22]. EAC is an experimental breast cancer derived from spontaneous mouse adenocarcinoma. EAC causes rapid buildup and accumulation of ascitic fluid [38] in the peritoneal cavity. In EAC bearing mice, the body weight, ascitic volume [39], and viable cell count was increased. Supplementation of WPC extract reverses the altered levels of body weight, MST, percentage of life span, tumor volume, PCV and incremented, the non-viable cell count indicating its cytotoxicity toward     Values are expressed as mean±SD (n=6). (1 unit = µ moles of π liberated/min) cancer cells. The order of efficacy of extracts wasEEPC > EAEPC > PEEPC. The EEPC has potential antitumor activity similar to that of reference drug, 5-Fluorouracil. On hematological investigation, the decrease in RBC and hemoglobin was found which may be due to the deficiency of iron or due to the hemolytic or myelopathic conditions in EAC mice [40]. The significant increase in WBC count and neutrophils in tumor bearing mice was due to its primary defense mechanism [41]. Decreased lifespan was due to the low Hb levels in cancerous condition [42]. The above conditions were reversed in drug treated groups which evidenced the protective action of WPC extracts on the hematopoietic system. Because of free radical generation in cancer cells, the leakage of lysosomal enzymes [43] occurs due to loss of membrane integrity. Lysosomal specific marker enzymes include Cathepsin-D [44][45][46], Acid phosphatase and β-D-glucuronidase [47][48][49][50]. Liver enzyme markers such as 5'nucleotidase [51], Lactate dehydrogenase [52][53][54], potential tumor markers used to assess the progression of the proliferating malignant cells. Among the various extracts of WPC, EEPC produces potential action which may be due to the presence of flavonoids which scavenges the free radicals and protects membrane integrity [36,38,55]. Treatment with EEPC elevates antioxidant enzymes such as serum total protein, superoxide dismutase, catalase, reduced glutathione, and glutathione peroxidase activity in CCl 4 induce hepatotoxicity [8] and alloxan-induced diabetic rats [7]. Oxidative stress accumulation causes significant decrease in the ion transport proteins [56] such as sodium potassium ATPase and magnesium ATPase in liver of cancer bearing mice [57,58]. Chemo resistance occurs by this mechanism due to efflux of drugs from transport proteins [59]. In the EEPC treated EAC-control, membrane bound ATPase levels were increased. Flavonoids influence the permeability of bio membranes by interacting with ATPase pumps in the animal cell thereby regaining their normal efficiency [59] and resumed the normal function [60]. Elevated levels of hepatic nucleic acids DNA and RNA were observed in disease control [61][62][63][64] but in treatment groups, reduction in their levels were observed. Attainment of nucleic acids levels to near normal was observed in EEPC treated groups. This proves that the drug has the potential to inhibit the proliferation of cancer cells, can stabilize, and restore the system to its normal physiological function.

CONCLUSION
From the above findings, it was concluded that P. corymbosa Lamk. possess antitumor activity. The mechanism by which it elucidates its anticancer potential has to be determined.