1. INTRODUCTION:

Natural products are important players in medical research and development and have been used for prevention or treatment of various human diseases from time immemorial^1.Phytochemicals are being explored for their potential chemopreventive action². Phytocompounds with antioxidant, anti-inflammatory, cell cycle modulatory, apoptotic and antiangiogenic activities have therapeutic potential in cancer chemoprevention^3-5.

Several phytocompounds with anti-inflammatory or antiangiogenic activities (e.g. curcumin and epigallocatechin gallate) are being studied for their chemopreventive action in various cancers^6-8.

An extremely promising strategy for cancer treatment today is chemoprevention, which is defined as the use of synthetic or natural agents (alone or in combination) to block the development of cancer in humans. Presently there is a growing interest in pharmacological evaluation of various plants used in Indian traditional system of medicine because of increased cure, low cost, least side effects and easy availability. One such plants is Cleome viscosa Linn. belonging to the family Capparidaceae. It is a weed distributed throughout the tropics of the world and the plains of India⁹. The plant is selected depending on the information gathered from traditional healers, reputed informants and various available ethnobotanical literature. It has been a part of traditional and folklore systems of medicine¹⁰. Sahu et al (2014) have reported the presence of phenolic and flavonoid compounds in the plant and detected the presence of myricetin, kaemferol-7-O-neohesperidoside, galacatechin, gallic acid, protocatechuic acid and beta-resorcylic acid¹¹. Ray et al (1980;1982) have shown that the seeds contain coumarou-lignins, cleomiscosin A and B^12,13. Naringenin glycoside¹⁴, kaempferide-3-glucuronide¹⁵, Glucosinolates¹⁶ and lupeol have also been isolated. It has been studied for various pharmacological activities like analgesic, anthelmintic, antidiarrheal, anti-inflammatory, antimalarial, anti-microbial, anti-ulcer, etc^17-24. However, this plant has not been explored for its anticancer effects. In the present study the anticancer property of Cleome viscosa Linn. on a panel of cancer cell lines in vitro has been evaluated. As we have found a potential cytotoxic activity in vitro, we have further studied the activity in liquid tumor model using Ehrlich ascites carcinoma cells in mice where it exhibited a significant anticancer activity. Our preliminary screening revealed that the extract has remarkable ability to inhibit cancer cell proliferation. Therefore, further bio-activity guided fractionation and the detailed mechanism of action for the activity need to be explored.

2. MATERIALS AND METHODS:

2.1 Chemicals and cell lines used:

Dulbecco’s modified eagle medium (DMEM), Hank’s balanced salt solution (HBSS), agarose, cytochalasin B, RNAse, proteinase K, nonidet P-40, ethidium bromide, Tris–HCl, acridine orange, hoechst 33342, methyl tetrazolium, sulforhodamine B, rhodamine 123, verapamil, cyclosporine A, mitoxantrone, reserpine and propidium iodide were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Absolute alcohol, dimethyl sulphoxide (DMSO), ethylene diamine tetra-acetic acid (EDTA), sodium bicarbonate, sodium chloride, potassium hydrogen phosphate, disodium hydrogen phosphate, potassium chloride and hydrochloric acid were purchased from Qualigens Fine Chemicals (Mumbai, India). Foetal bovine serum, antibiotic-antimycotic solution were purchased from Invitrogen, USA.

HeLa (Human cervical adenocarcinoma), U343 (Human neuronal glioblastoma), A549 (Human lung adenocarcinoma) and MCF-7 (Human breast carcinoma) supplemented with 10% foetal bovine serum, 100 U/mL penicillin and 100µg/mL streptomycin, in a humidified 5% CO₂ incubator at 37°C.

2.2 Preparation of plant extracts and fractionation

The whole plant of Cleome viscosa Linn. belonging to the family Capparidaceae was collected from Udupi district, Karnataka (India) in the month of October, and was authenticated by the head of the Botany department, Poorna Prajna College, Udupi, Karnataka. The authenticated specimen was submitted in Herbarium of Manipal College of pharmaceutical sciences, MAHE, Manipal. Shade dried and coarsely powdered plant (2 kg) was extracted in a soxhlet apparatus for 24h using methanol. The crude methanolic extract was concentrated in a rotary evaporator under reduced pressure for solvent recovery and collected. The concentrated extract was dried and stored in a desiccator for further use. The yield of crude methanolic extract was 4.2%. For fractionation, the dried crude extract (60 g) was resuspended in methanol-distilled water and extracted successively with solvents such as petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol in increasing polarity. The remaining fraction was considered as ‘Rest fraction’. Each fraction was concentrated in a rotary evaporator under reduced pressure, dried and stored in a vacuum desiccator. Crude alcoholic extract and fractions were named as CVA (alcoholic crude), CVP (petroleum ether), CVD (dichloromethane), CVE (ethyl acetate), CVB (n-butyl alcohol), and CVR (rest fraction). For in vitro studies, crude extract and all the fractions were dissolved in DMSO. For in vivo study suspension of each extract was prepared using 0.25% sodium CMC as suspending agent.

2.3 Methyl tetrazolium assay (MTT assay):

Percentage cell survival after treatment with CVA, CVP, CDV, CVE, CVB and CVR was measured using the MTT assay. Briefly, cells at the exponential growth phase were trypsinized and resuspended in the culture medium and seeded in a 96-well tissue culture plate at a density of 5000 cells per well. After 24h of incubation, defined concentrations of the drugs in culture media were prepared by serial dilution and treated in triplicates at concentrations of 25, 50, 100 and 200µg/mL. Methotrexate was used as the standard. After 48h treatment, 20µl of MTT reagent (from 5mg/ml stock) was added to each well and incubated for further 3h. The culture medium was removed and formazan crystals formed were dissolved in 200µl of DMSO. The plates were read in a 96 well microplate reader (Biotek-ELx-800) at a wavelength of 540nm. Percentage cell viability was calculated from the optical density values and plotted against concentration. IC₅₀ value for each extract or fraction.

2.4 Sulforhodamine B assay (SRB assay)

Percentage cell survival after treatment with CVA, CVP, CDV, CVE, CVB and CVR was measured using the SRB assay. Briefly, cells at the exponential growth phase were trypsinized and resuspended in the culture medium and seeded in a 96-well tissue culture plate at a concentration of 5000 cells/well. After 24h incubation, cells were treated with the drugs at concentrations of 25, 50, 100 and 200µg/ml with methotrexate as standard. Dilution was carried out in such a way that the concentration of DMSO in the wells did not exceed 0.5% (v/v).

The plates were then incubated at 37ºC for 48 h. At the end of treatment, cells in each well were fixed with 100µl of 10%w/v trichloroacetic acid (TCA) at 4ºC for one h. The plates were then washed thrice under running water and air-dried. The air-dried plates were stained with 100μl SRB (in 0.4% w/v in acetic acid) for 30 minutes at room temperature. The plate was then washed with 1% acetic acid thrice to remove the unbound dye. The plates were again air dried and 100µl of 10mM Tris base was added to each well for solubilisation of the dye. The plates were shaken for 10 minutes on rotary shaker to facilitate solubilisation. The plates were read in a 96 well microplate reader (Biotek ELx-800) at a wavelength of 540nm. Percentage cell viability was calculated and IC₅₀ value for each extract or fraction were calculated.

2.5 In vivo studies:

2.5.1 Animals:

8-week-old Swiss albino mice (20-25g) were used in the study. All the animals were acclimatized to the experimental room having temperature 25±2ºC, controlled humidity conditions, and 12:12 h light and dark cycle. The mice were fed with standard food pellets and water ad libitum. Study was conducted after obtaining ethical committee clearance from the Institutional Animal Ethics Committee of KMC, Manipal.

2.5.2 Acute Toxicity Studies:

Acute toxicity studies were conducted to determine the safe dose by up and down procedure according to OECD Test Guidelines 425.

2.5.3 Experimental design:

The EAC cells originally obtained from Dr. Ramdasan Kutan (Director, Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala, India), were maintained and propagated by serial i.p transplantation of EAC cells in an aseptic environment. The EAC cells propagated for 12-14 days were used in experiment²⁵. The ascitic fluid was drawn using an 18-gauge needle into sterile syringe. Tumour viability was determined by Trypan blue exclusion assay and cells were counted using haemocytometer. The Ascitic fluid was suitably diluted in normal saline to get a concentration of 1x10⁷ (ten million) cells/ml. From this stock suspension, 0.25ml (2.5 million cells/mice) was injected i.p to obtain ascitic tumour. Those fractions that showed significant activity in the in vitro cytotoxicity and antioxidant activities were chosen for this in vivo testing. On day 1, the animals were randomized and divided into eight groups (n = 10), and treated as shown in table 1.

Table 1: Grouping and treatment details.

S. No.	Group	Treatment	Dose
1	Sham control	Distilled water	-
2	EAC control	Sodium CMC	-
3	Standard	Cisplatin	3.5mg/kg
4	Test 3	CVP	200 mg/kg
5	Test 4	CVP	100mg/kg
6	Test 5	CVD	200mg/kg
7	Test 6	CVD	100mg/kg
8	Test 7	CVE	200mg/kg
9	Test 8	CVE	100mg/kg

Fractions were suspended in 0.25% sodium CMC and treatment was given on 3^rd, 5^th, 7^th, 9^th, 11^th, and 13^th day of tumour inoculation p.o.²⁶. Positive control group was treated with Cisplatin on 1^st day. The animals were sacrificed on day 14 by cervical dislocation and tumor volume, tumor cell count, spleen weight, tumour angiogenesis, hematological parameters and antioxidant status were assessed. The second set of animals was continued with the similar design so as to observe their life span.

2.5.4 Tumor Growth Response:

The antitumor effect of CV was assessed by change in body weight, ascites tumor volume, viable tumor cell count, mean survival time (MST) and percentage increased life span (%ILS)²⁷. The effect of the fractions on tumor growth was monitored by recording the mortality for a period of 40 days and percentage increase in life span was calculated as following:

First death + Last death in the group MST =--------------------------------------------------

MST of treated group – MST of control group

% ILS = ----------------------------------------------------------

MST of control group

Body weight of the animals was recorded both in control and treated groups at the beginning of the experiment and subsequently on every alternate day and percentage increase in body weight was calculated.

Animal weight on respective day

% Increase in bidy weight------------------------------------------------1 × 100

Animal weight on day 0

2.5.5 Hematological investigation:

Blood was collected from retro orbital plexus of animals before sacrifice and used for the estimation of hemoglobin (Hb) content, red blood cell count (RBC), and white blood cell count (WBC) using automated veterinary blood cell counter (Model: PC210; ERMA, TOKYO).

2.5.6 Biochemical Investigations:

The liver of animals was excised after perfusion with ice-cold saline transcardially, blotted dry, and weighed. A 10% (w/v) homogenates were prepared in ice cold potassium chloride solution (150mM) using tissue homogenizer (Yamato L.S. G L.H-21, Japan). The homogenate was used for biochemical investigations²⁸. Estimation of lipid peroxidation as described in Kumar and Muller (1999)²⁹, glutathione content (GSH) and Glutathione-s-transferase (GST)^30,31. Rest of the homogenate was centrifuged at 2500 × g for 15 minutes at 4^oC and superoxide dismutase (SOD) as described by Kuninaka et al. (2000)³² and catalase (CAT) according to Kaynar et al. (2005)³³. Total protein was estimated by BCA protein assay kit (Pierce®) according to the manufacturer’s method³⁴.

2.6 Statistical analysis:

The data was analysed using GraphPad Prism5 Version (GraphPad Software Inc., La Jolla, CA, USA) by one-way analysis of variance (ANOVA) and data that was found statistically significant were further analyzed by Tukey’s post hoc test. p<0.05 was considered significant. Data of cell cycle phase distribution and DNA content was analysed using Summit v4.3 software.

3. RESULTS AND DISCUSSION:

3.1 In vitro cytotoxicity screening:

Cytotoxicity was evaluated on a panel of cell lines, namely MCF7, U343, HeLa and A459 by both MTT and SRB assays and IC₅₀ was calculated for the fractions. The fractions were found to be potent in HCT116 cell line. Table 2 shows IC₅₀ values of fractions on various cell lines in both the assays. This result formed the basis for studying the in vivo anticancer for further testing of the fractions. Although slight differences have been found in the IC₅₀ values in MTT and SRB assays, the fractions, CVP, CVD and CVE have found to possess a significant activity in both the methods. Therefore, we have tested those fractions in the animal studies after performing the toxicity studies.

3.2 Acute toxicity study:

All the three fractions, CVP, CVD and CVE were found to be safe in the toxicity testing at a dose of 2000mg/kg (p.o.) and no signs of toxicity were observed. Therefore, two dose levels, 200mg/kg and 100mg/kg have been selected for the study.

3.3 Tumor growth response:

The EAC inoculated mice were found to gain body weight progressively till day 13. The maximum gain in tumour weight (37.13%) was observed on day 13 of tumour inoculation. Cisplatin has significantly reduced the elevated body weight as compared to EAC control. Since nutritional requirement is provided by the ascitic fluid, an increased ascitic fluid volume and a subsequent increase in the body weight was observed in the EAC control group. All fractions inhibited rise in body weight in pattern similar to the Cisplatin treatment. The change in body weight is shown in Figure 1.

Mean survival time in EAC inoculated mice was found to decrease significantly when compared to normal as well as with extracts treated mice. The MST of EAC control mice (12.2 days) was significantly (p<0.001) improved by the Cisplatin treatment (22.2 days). MST was improved in all fraction treated cells in dose dependant manner. CVP and CVD were significantly increased life span by 53% and 40% respectively.

Ascitic cell count and Ascitic fluid volume were found to be 15.42 million cells/mL and 5.32 mL respectively in the EAC control group. Treatment with Cisplatin significantly (p<0.001) reduced EAC count to 0.6 million cells/mL and EAC volume to 1.65mL. All the selected fractions at both the doses significantly (p<0.001) reduced EAC count and EAC volume compared to EAC control. These results indicate the cytotoxic effect of the fractions on tumor cells either by direct or indirect local effect involving macrophage activation and vascular permeability inhibition^35,36. Table 3 shows ascitic cell count and fluid volume in different groups and table 4 shows MST and %ILS.

Table 2: IC₅₀ values of the fractions on Hela, U343, MCF7 and A549 cell lines by MTT (left) and SRB (right) assays.

MTT	Fraction	Hela	U343	MCF7	A549	SRB	Fraction	Hela	U343	MCF7	A549
	CVA	218.8	199	192.6	146.8		CVA	182.79	155.6	148.3	122.7
	CVP	46.8	55.5	79.93	93.8		CVP	56.29	55.62	75.34	103.2
	CVD	49.7	34.71	68.29	164.8		CVD	39.74	35.55	63.20	138.20
	CVE	36.9	61.10	58.08	308		CVE	54.5	83.91	52.38	259.70
	CVB	429.6	149.5	151.6	428.2		CVB	443.2	161.10	183.8	432.2
	CVR	231.5	146.2	182.5	331.4		CVR	105.8	133.29	221.7	289.3

Fig 1: Percentage change in the body weight of the animals compared between groups every alternate day. ^ap<0.05, ^bp<0.01 & ^cp<0.001 compared to EAC control and, compared to sham control.

Table 3: Ascitic cell count and fluid volume in different groups.

Treatment	Dose (mg/kg, *p.o.*)	Ascitic fluid (1X106 cells/ml)	Ascitic fluid volume (ml)
Sham Control	Distilled water	-	-
EAC control	0.25% Na. CMC	15.42±0.93	5.32±0.31
Cisplatin	3.5	0.65±0.13^c	1.65±0.37^c
CVA	100	5.26±1.05^c	2.95±0.76
CVA	200	5.24±0.99^c	3.01±0.27
CVP	100	4.38±0.93^c	3.51±0.67
CVP	200	3.17±0.48^c	2.84±0.12^a
CVD	100	6.03±1.18^c	2.51±0.59^b
CVD	200	5.49±0.69^c	2.09±0.28
CVE	100	2023±0.27^c	3.73±0.77
CVE	200	2.38±0.53^c	3.44±0.45^b

All values are expressed as Mean±SEM. ^ap<0.05, ^bp<0.01 & ^cp<0.001 compared to EAC control.

3.4 Haematological parameters:

WBC count was found to increase in the EAC inoculated mice. RBC count and Haemoglobin content were reduced in EAC inoculated mice as compared to sham control. The WBC count has been restored in the standard and test groups. Treatment with Cisplatin and fractions were found to significantly reverse in RBC count and Hb content.

Table 4: Mean survival time and percentage increase in life span in different groups

Treatment	Dose	MST	%ILS
Sham control	Distilled water	25±6.23	-
EAC control	0.25% Na. CMC	12.2±6.26	-
Cisplatin	3.5	22.2 11.18	82.79±22.35^a
CVA	100	17.0±9.83	40.16±20.89^a
CVA	200	17.4±9.39	43.44±19.84^a
CVP	100	16.4±7.60	35.25±10.23^a
CVP	200	18.6±9.39	53.28±20.17^a
CVD	100	15.6±7.60	28.69±7.14^a
CVD	200	17±8.05	40.16±10.03^a
CVE	100	16.6±8.49	36.89±9.90^a
CVE	200	16.4±8.49	35.25±10.56^a

All values are represented as mean±SEM. ^ap< 0.05 compared to EAC control

3.5 Spleen weight and Angiogenesis:

Spleen weight increases in case of increased degradation of RBC. Spleen weight was found to be increased significantly in EAC control group as compared to sham control. Cisplatin treatment caused significant (p<0.001) reduction in spleen weight compared to EAC control. CVD at both the doses and CVE (200mg/kg) reduced spleen weight significantly compared to EAC control.

The total number of blood vessels present on the ventral peritoneal skin layer which is in direct contact with the liquid tumour were counted. The number was found to be high in case of EAC control animals. Cisplatin as well as the fractions were found to significantly lower the blood vessel formation (p<0.001) compared to EAC control. CVD fraction showed dose-dependent anti-angiogenic activity. This reduction in the angiogenesis formation is an essential trait of chemotherapeutic agents to prevent the supply of oxygen and nutrients to the tumor tissue. All the fractions have shown a reduction in this angiogenesis prompting their use in cancers.

Table 6: Effect of different fractions of CV on spleen weight and angiogenesis

Treatment	Dose *(mg/kg, p.o.)*	Spleen weight	Angiogenesis
Sham Control	Distilled water	0.12±0.01	8.33±0.33
EAC control	0.25% Na. CMC	0.24±0.02	20.20±0.86
Cisplatin	3.5	0.10±0.03^a	10.00±0.71^b
CVA	100	0.15±0.02	14.50±1.19^b
CVA	200	0.16±0.03	12.57±1.08^b
CVP	100	0.17±0.02	15.75±0.85^a
CVP	200	0.16±0.04	16.33±0.88^a
CVD	100	0.12±0.02^a	13.33±0.33^b
CVD	200	0.13±0.01^a	12.67±0.65^b
CVE	100	0.14±0.01	15.67±0.88^a
CVE	200	0.13±0.01^a	14.5±1.32^b

All values are expressed as mean±SEM. ^ap<0.05 & ^bp<0.01 compared to EAC control.

3.6 Biochemical investigation:

There was significant reduction in GSH, GST, SOD, Catalase and total thiol level in EAC control animals as compared to sham control. Cisplatin and fractions treatment significantly (p<0.05) improved reduced level compared to EAC control. The level of lipid peroxidation was significantly increased in the EAC control (p<0.001) compared to sham control. Cisplatin and found to significantly lower lipid peroxidation (p<0.001). The fractions also significantly reduced the lipid peroxidation.

A decrease in the antioxidant enzyme levels and an increase in the MDA levels in cancer is well documented. This was also reflected in the EAC inoculated mice. The antioxidant enzyme systems are essential in protecting the tissues against insults by free radicals and other compounds like hydrogen peroxide. These free radical scavenger systems are affected in cancer and it is necessary to restore their levels in the treatment of cancer. Chemotherapeutic agents help restore these enzyme levels thus providing defence against various insults. In the present study, cisplatin and the fractions, have shown to decrease the levels of MDA and increase the levels of SOD.

4. CONCLUSION:

Cancer is one of the fatal diseases and there has been an increased incidence of cancer in recent times. Chemotherapy remains as one of the major modalities for the treatment of cancer. However, an effective therapy depends upon its early diagnosis and stages of progression; a convincing cure is elusive especially at the later stage of disease. Traditionally, some plants are used for the treatment of cancers in India. However, no scientific data backing the evidence exists for the same. Among such plants is Cleome viscosa Linn., which is used in the Indian system of medicine for cancer treatment. Crude methanolic extract of the whole plant and its subsequent fraction by bioactivity guided fractionation, shows a significant cytotoxic activity in a panel of human cancer cell lines like glioblastoma, cervical cancer, breast cancer, and lung cancer. With this encouraging data, in vivo anticancer activity was taken up using the EAC model. In the present study, the fractions have shown a significant anticancer activity. However, further mechanistic studies need to be conducted to explore the exact mechanism of action and the chemical entities responsible for the activity.

ACKNOWLEDGEMENTS:

The authors are thankful to Dr. Gopalakrishna Bhat, Poorna Prajna College, Udupi, Karnataka for authenticating the plant. The authors also thank Manipal College of Pharmaceutical Sciences, MAHE for providing facility to carry out the research work.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 24.06.2020 Modified on 18.09.2020

Research J. Pharm. and Tech 2021; 14(10):5397-5404.

DOI: 10.52711/0974-360X.2021.00941

Treatment	*Dose (mg/kg, p.o.)*	WBC (1X103 cells/mm³)	RBC (1X106 cells/mm³)	Haemoglobin (gm%)
Sham Control	Distilled water	11.60±0.44	9.75±0.40	12.20±0.40
EAC control	0.25% Na. CMC	25.48±2.75^*	4.72±0.66	7.20±1.15^*
Cisplatin	3.5	14.60±0.84^a	9.03±0.59^b	11.47±0.63^a
CVA	100	12.90±3.35^a	7.23±0.12	7.96±0.50
CVA	200	19.34±2.92	7.03±0.12	8.57±0.38^a
CVP	100	18.32±3.32	7.31±0.54	6.64±0.42
CVP	200	21.66±2.56	7.40±0.43^a	9.76±0.26^b
CVD	100	15.40±2.69^a	8.60±0.48^b	8.60±0.36^a
CVD	200	14.36±1.39^a	9.00±0.23^b	10.46±0.64^b
CVE	100	21.86±3.10	8.01±0.14^a	8.03±0.33
CVE	200	17.52±3.19	7.13±0.86	8.05±1.25