INTRODUCTION:

In countries with a westernized lifestyle, about quarter of the deaths is caused by cancer. Colorectal cancer is the third leading cause of death out of more than two hundred types of cancer¹. Colon cancer is linked to several genetic and epigenetic factors². Although there are various approved drugs for cancer, they have a huge profile of adverse effects. Resistance in chemotherapy is emerging to peaks and treatment options for cancer are becoming narrow. Besides, patient compliance for chemotherapy is very low due to many adverse effects.

Hence, there is an urgent need to screen for newer anticancer drugs³. Developing countries are largely dependent on traditional medicines for various ailments since time immemorial. India, being home for a wide variety of plant species, traditional medicine is an integral part of the country and is being practiced for centuries. Hence, there is a need for screening newer compounds for anticancer activity in order to come out with cost-effective, lower side effect profile yet potent molecules. Natural products play a pivotal role in cancer therapy with substantial number of anticancer agents used (vincristine, irinotecan, etoposide, paclitaxel, etc.) being either natural or nature derived^4,5. India is a home for numerous medicinal plants out of which Clerodendrum is a genus of flowering plants belonging to the family Lamiaceae⁶. The important groups of chemical constituents present in the species are phenolics, flavonoids, terpenoids, oils and steroids⁷. Other plants in this genus are also being explored for anticancer activity⁸. The plant Clerodendrum indicum, commonly known as Bharangi has been reported to possess various medicinal properties like anti-nociceptive, anti-diarrhoeal, antimicrobial⁹, anti-rheumatic, antioxidant and anticancer^10,11activities. Positive result for cytotoxic property has been explored by brine shrimp lethality but no other significant work has been reported for its anticancer activity. The present study was aimed to investigate the anicancer properties of Clerodendrum indicum root by both in vitro and in vivo methods.

MATERIALS AND METHODS:

Plant material:

The plant Clerodendrum indicum (L.) Kuntze was collected from Pernankila, Udupi, Karnataka in the month of August and was authenticated by Dr. Gopalakrishna Bhatt, Professor of Botany, Poorna Prajna College, Udupi, Karnataka. Roots were separated from the plant, shade-dried at room temperature and powdered coarsely. The powder was packed in a soxhlet apparatus, macerated overnight with ethanol and extraction was performed till the solvent became colourless. After extraction, the solvent was collected and concentrated using Buchi rotavapor under reduced pressure. The yield obtained was found to be 32%. Dry extract was added to distilled water (15g to 150ml) and extracted with petroleum ether (PEF) followed by ethyl acetate (EAF). Each fraction was concentrated using rotavapor, freeze dried and stored till further use in a vacuum desiccator.

Estimation of total flavonoid content:

Aluminium chloride method was used for the determination of total flavonoid content of the fractions¹². In brief, 0.5ml of methanolic solution of the fractions was taken. To it, 0.1ml AlCl₃ (10%), 0.1ml of 1mol/L sodium potassium tartrate and 2.8ml distilled water were added and its absorbance was recorded at 415nm after 30min of incubation. A calibration curve was generated using quercetin as standard at concentrations 31.25, 62.5, 125, 250, 500 and 1000µg/ml. Total flavonoid content in the fractions was calculated and was expressed as mg quercetin equivalent per gram of the sample.

HPLC characterization of selected fractions:

HPLC analysis was carried out in an HPLC system (Scimadzu, Tokyo, Japan) that was equipped with dual pump LC-20AD binary system. The detector used is photo diode array SPD-M20A. Separation was achieved using reversed-phase column (I.D. 4.6mm x 250mm, 5µm). The samples were dissolved in HPLC grade methanol at a concentration of 2mg/ml. The volume of sample injected was 20µl by the autosampler. A two pump linear gradient program was set with pump A (Acetonitrile) and pump B (water + 0.1% formic acid). The program was set for 35 minutes with elution gradient of 90% B changing to 30% then washing for 25 min. The flow rate was 1ml/min. quercetin was used as the standard.

In vitro screening:

Cell lines:

In vitro cytotoxicity screening was performed on human colorectal carcinoma (HCT116), human breast adenocarcinoma (MCF7) and human lung carcinoma (A549) cell lines obtained from National Centre for Cell Sciences (NCCS), Pune, India.

Cytotoxicity assay:

MTT assay was carried out to evaluate the cytotoxicity of the extracts¹³. 5000 cells/well were seeded in a 96 well plate and incubated for 24h. After 24h incubation, the cells were treated with 100µl of test compounds (3-1000µg/ml) in triplicates and incubated for 48h. 20µl of MTT reagent was added to each well and incubated for 3h¹⁴. Media was aspirated and 200µl of DMSO was added to each well to stabilize the formazan crystals. Absorbance was measured at 540nm from which the percentage cell death was calculated and IC₅₀ was derived for each compound.

% Cell death = (Absorbance of control – absorbance of test/ Absorbance of control) *100

Apoptosis assay:

Apoptosis is an important property that an ideal anticancer compound should possess¹⁵. To assess the apoptosis causing potential of the fractions, 0.75X10⁶cells (HCT116) were seeded in a 6-well plate and incubated for 24h. Cells were treated with the fractions (EAF and PEF) and incubated for 48h. Medium was aspirated, wells were washed with (phosphate buffered saline) PBS and fixed with ice cold ethanol for 20min¹⁶. Ethanol was aspirated and the cells were washed with PBS. Acridine orange (10mg/ml) and ethidium bromide (10mg/ml) stains were prepared in PBS and mixed in 2:3 ratio. 200µl of dual stain was added to each well and the plate was kept for 20min at room temperature. Unbound stain was washed with PBS and the wells were observed under fluorescent microscope (Nikon TS 100F) at an excitation wavelength of 450-490nm. Apoptotic index was calculated as the number of apoptotic cells in the total number of cells observed.

Cell cycle analysis:

It is important to analyze which phase of the cell cycle is inhibited by an anticancer agent, for which flow cytometry is carried out¹⁷. Cells were seeded in 60mm dishes and incubated for 24h. The cells were then treated with the fractions and incubated again for 48h. Media was aspirated and the cells were washed thrice with PBS. Cells were trypsinized, media was added and centrifuged¹⁸. The pellet was resuspended in warm PBS 70% ethanol, incubated for 1 h at 4˚C and ethanol was aspirated. The cells were analysed for phosphotidylserine using annexin V and propidium iodide (PI) dual staining assay. Annexin V-FITC conjugate and PI solution was added along with RNAse and incubated for 20min in dark on ice. Cells were analysed in flow cytometer.

Anti-metastatis assay:

Cells were cultured in 6 well plate and checked for confluency as a monolayer. After the cells reached confluency, media was aspirated and a scratch (width 2mm) was created with a 200µl sterile microtip¹⁹. Cells were treated with the fractions and standard (Doxorubicin). Photographs were taken at 0, 6, 12, 24 and 48h using an inverted microscope under 40X magnification. The mean wound area was calculated and the result was expressed as percentage of recovery (%R).

In vivo screening:

Acute toxicity testing:

In order to assess the safety, acute toxicity study was performed as per OECD TG 425 guidelines.

Animals:

Male Wistar rats inbred at Central Animal Research Facility at Manipal Academy of Higher Education were used for the study. The animal care and handling were according to the CPCSEA guidelines. Animals were acclimatized to the experimental room bearing the temperature 23±3˚C, controlled humidity conditions, and 14:10 hour light and dark cycle. The animals were housed in sterile polypropylene cages containing sterile paddy husk bedding. The animal care and handling were carried out in accordance to guidelines put forth by the Institutional Animal Ethics Committee (IAEC), Manipal. The committee has cleared the study conducted and has issued the clearance certificate bearing the number, IAEC/KMC/81/2014.

DMH induced colon cancer model:

1, 2-dimethylhydrazine dihydrochloride (DMH) was used for colorectal carcinogenesis^20,21. DMH was dissolved in 1mM/L EDTA-normal saline and the pH was adjusted to 6.5 with 1M NaOH. DMH solution was freshly prepared before each dosing. The carcinogen was injected intra peritoneally (i.p.) at a dose of 20mg/kg once every week for the first eight weeks and 30mg/kg for the next eight weeks. After 16 weeks of carcinogen administration, the fractions were administered for 21 days. Suspension of fractions was prepared in 0.25% CMC.

Table 1: Treatment schedule for DMH induced colon cancer model for NC, DMH-C, 5-FU, EAF and PEF groups.

S. No.	Group	Number of animals	Dose	Dosing schedule
1	NC	9	0.25% CMC p.o.	Weekly for 16 weeks
2	DMH-C	9	30mg/kg, 20mg/kg i.p	Weekly for 16 weeks
3	5-FU	9	50mg/kg i.p.	Weekly for 4 weeks
4	EAF	9	100mg/kg p.o.	Daily for 21 days
5	PEF	9	100mg/kg p.o.	Daily for 21 days

NC: Normal control; DMH-C: Dimethyl hydrazine control; 5-FU: 5-Fluoro uracil; EAF: Ethyl acetate fraction; PEF: Petroleum ether fraction.

Major organ index:

Immediately after sacrifice, the heart was perfused with normal saline and major organs like spleen, liver, kidneys and heart were harvested and the weights were noted. Organ index was calculated per body weight of the animal.

Organ Index = (Weight of organ/body weight of the animal)

Hematological parameters:

Just before the sacrifice, blood was withdrawn by retro-orbital method using heparin coated rat capillary tubes and taken in micro centrifuge tubes with 10% EDTA. RBC count, WBC count, platelet count and haemoglobin level were estimated using automated veterinary blood cell counter (Model: PC210; ERMA, TOKYO).

Biochemical investigations:

Colons were dissected out after perfusion with an ice-cold saline transcardially. Colon was removed, dried, weighed and 10% homogenate was prepared with ice-cold potassium chloride (150mM) solution using tissue homogenizer (Yamato L.S. G L.H-21, Japan). Homogenate was used for biochemical estimations²². Total protein content was estimated by BCA protein assay kit (Pierce®) according to the manufacturer described method, reduced glutathione (GSH) by method described inGiustarini, Dalle-Donne et al. 2013²³, catalase by Kaynar H, Meral et al.²⁴,superoxide dismutase by Kuninaka, Ichinose et al.²⁵, lipid peroxidation by KC and Mulle²⁶and nitrite assay by Nathan S. Bryan, Matthew²⁷.

STATISTICAL ANALYSIS:

Statistical comparisons were performed using GraphPad Prism 5.03 Demo Version (GraphPad Software Inc., La Jolla, CA, USA) by one-way analysis of variance (ANOVA) and statistically significant data were further analyzed by Tukey’s post hoc test. Results were expressed as Mean ±SEM and p<0.05 was considered significant.

RESULTS:

Flavonoid content:

Total flavonoid content in PEF was found to be highest among the fractions which was 300.37 µg quercetin equivalent per mg fraction when estimated with quercetin as standard. The flavonoid content of EAF was found to be 235.14 µg quercetin equivalent per mg fraction

HPLC characterization:

The chromatogram of HPLC analysis performed with quercetin as reference showed a peak at a retention time of 27.526 at 254nm. PEF fraction had shown a peak at 27.540 at 254nm. Figure 1 shows the chromatograms for quercetin and PEF.

Figure 1: A) HPLC chromatogram for Quercetin. B) HPLC chromatogram of quercetin in petroleum ether fraction of the root.

In vitro Screening:

Cytotoxicity MTT assay:

IC₅₀was calculated for the fractions on HCT116, MCF7 and A549 cell lines. The fractions were found to be potent in HCT116 cell line as the IC₅₀values on the cell line were 72.83µg/ml for EAF and 31.33µg/ml for PEF. In other cell lines, the IC₅₀was found higher. Table 3 shows IC₅₀values of PEF and EAF on various cell lines. This result formed the basis for selecting the in vivo model for further testing of the fractions.

Table 2: IC₅₀ values of the fractions on different cell lines

Fraction	IC₅₀ value (µg/ml)
Fraction	HCT116	MCF7	A549
EAF	72.83	266.9	606.4
PEF	31.33	189.2	467.1

The results were expressed as µg/ml of each fraction in three different cell lines.

Apoptosis assay:

Number of apoptotic cells in each sample was observed and the apoptotic index was found to be more in the standard group. PEF was found to possess an apoptotic index of 30.36±1.26 which was significant compared to the control group (10.51±1.68). Table 3 shows the apoptotic indices on HCT116 cell line when treated with different fractions.

Table 3: Percentage apoptosis in HCT 116 cell line with different treatments

Group	Apoptotic index (Mean±SEM)
NC	10.51±1.68
5-FU	37.58±2.35^b
EAF	20.25±4.15
PEF	30.36±1.26^a

Results were expressed as Mean±SEM, ^bp<0.001 vs. Normal control group and ^ap<0.05 vs Normal control group.

Cell cycle analysis:

An increase in the percentage of cells in G₂/Mphase was observed both in EAF and PEF. The transition was found to be halted, which was evident from the cell cycle analysis. There was an increase in the percentage of cells in G₂/M phase from 18.2% (untreated) to 23.6% and 22.3% in EAF and PEF treated groups respectively. Figure 2 shows the graphs of flow cytometry.

5-FU

EAF

PEF

Figure 2: Graphs show percentage of HCT116 cells labelled with propidium iodide stain in different phases of cell cycle after treatments.

Anti-metastasis assay:

Table 4 shows the effect of treatments on the width of the scratch after 0 h, 24 h and 48 h. From the present study it is seen that the fractions did not possess any significant antimigratory activity at any point of time.

Table 4: Effect of various treatments on wound closure after 0h, 24h and 48h.

Group	Width (µm) (Mean±SEM)
Group	0h	24h	48h
NC	1236.44±17.44	1091.24±33.02	947.31±5.2
5-FU	1127.72±3.165	880.47±5.55	540.46±5.4
EAF	1136.5±7.33	741.66±13.04	328.03±5.22
PEF	1130.57±5.3	619.07±11.08	444.01±8.4

The results show the width of the wound in micrometer at different timepoints. Results were expressed as Mean±SEM.

In vivo Screening:

Acute toxicity test:

Both EAF and PEF fractions were found to be safe at a dose of 2000mg/kg (i.p.). No signs of toxicity were observed at specified dose and all the vitals were found to be normal.

Haematological parameters:

There was no significant change in the WBC count between the groups. There was also no significant change in the RBC count except in the standard group where it had decreased compared to the normal control. The haemoglobin content had decreased in the standard and PEF group compared to the disease control. The DMH (disease control) had significantly increased the platelet count compared to the normal control. Table 5 shows the values of various blood parameters in different groups.

Table 5: Effect of different treatments on hematological parameters

Treatment

WBC

(x10³cells/

mm³)

RBC

(x10⁶ cells/ mm³)

%Hb

(g/dl)

Platelets

(x10³ /µl)

10.45±

0.79

11.5±

0.79

14.6±

0.88

36166±

61.9

DMH-C

10.83±

0.48

10.18±

0.31

13.7±

0.94

636.23±

32.4^c

5-FU

10.62±

0.60

6.8±

0.89^a

10.65±

0.59^a

570.3±

15.0^a

EAF

10.54±

0.89

9.51±

0.31

12.7±

0.36

521.4±

57.29

PEF

10.93±

0.59

11.53±

0.64

10.9±

0.58^b

437.8±

50.52

All values are expressed as Mean±SEM, ^ap<0.0.01 vs. NC, ^bp<0.05 vs DMH-C, ^cp<0.01 vs NC

Major organ index:

There was no significant change in the spleen and heart index between the groups but there was a significant increase in the liver and kidney index in the disease control group compared to the normal control (NC) group. The kidney index was found to be reduced in the EAF group compared to the disease control group. Effect of fractions on major organ and liver indices are shown in Figure 3a and 3b respectively.

Figure 3: (A) Effect of fractions on liver index. (B) Effect of fractions on other major organ indices.

Values are mean ± SEM. *p<0.05 with respect to normal control. ^ap<0.05 with respect to DMHC. **p<0.01with respect to normal control. **p<0.01 Vs NC and *p<0.05 Vs DMH control.

Biochemical parameters:

Various biochemical parameters were measured in the colon tissue homogenate. There was a significant increase in the levels of MDA and nitrite levels in the disease control group. The levels have been reduced by the standard and the test groups compared to the DMH control group. The GSH and catalase levels were significantly decreased in the DMH group but there was no significant change in the test groups compared to disease control. No alteration in the SOD levels was observed between the groups. Figures 4 (A and B) and Figure 5 (A, B and C) show the lipid peroxidation, nitrite, GSH, catalase and SOD levels in different groups respectively.

DISCUSSION:

Despite having various drugs for the treatment of cancer, there is still a need to screen for newer compounds that are effective and possess lower side effect profile as the existing chemotherapy has many adverse effects and cause mental trauma in patients. Therefore, it is the hour to discover alternative ways to chemotherapy. India is known for its folklore medicines. There are many plants that have been investigated and are being investigated for their medicinal properties^28,29. One such plants is Clerodendrum indicum belonging to the family Lamiaceae (The Plant List, 2010)³⁰. Roots of the plant were extracted with ethanol and fractions were obtained by partition with petroleum ether (PEF) and ethyl acetate (EAF). Total flavonoid content was estimated using quercetin as the standard spectrophotometrically and the flavonoid content was found to be higher in ethyl acetate fraction. The presence of good quantity of flavonoids in both the fractions could be one of the reasons for the anticancer property. Further, characterization was done using HPLC and a peak nearer to the R_tof quercetin was detected in PEF. This could indicate the presence of quercetin in the fraction and may be responsible for the antioxidant property of the fraction that were reflected in the in vivo experiments. This is in support of a previous study that has also shown the presence of quercetin in the fraction³¹. Though the flavonoid content³² was found to be higher spectrophotometrically in EAF, we could not trace the peak for quercetin in the EAF fraction and this could probably be due to presence of a number of other interfering compounds in the fraction which might have caused quenching.Upon screening the fractions on different cancer cell lines namely HCT116, MCF7 and A549 by MTT method, both PEF and EAF were found to be more active against HCT116 cell line than in the other cell lines with an IC₅₀ value of 72.83 and 31.33 µg/ml respectively. This formed the basis for selecting DMH induced colorectal cancer as an in vivo model.

To examine if the mode of cell death was due to necrosis or apoptosis, AO/EB fluorescent dual staining assay was performed where the cells were stained with acridine orange and ethidium bromide. Acridine orange stains all the cells green but ethidium bromide displaces acridine orange in necrotic cells and appear red. This fluorescent staining assay revealed that the cell death was due to apoptosis. Since for an anticancer agent the ideal mode of causing tumor cell death is apoptosis, the fractions have shown the same mode of cell death indicating a good anticancer property. Cell cycle analysis was performed by flow cytometry inwhich propidium iodide dye was used which stains the DNA and the percentage of cells in different phases is measured. The results suggest that both the fractions have arrested the cell cycle at G₂/M phase owing to an increase in the percentage of cells in the phase. This is because the compounds may downregulate cell cycle promoters or upregulate the suppressor proteins³³. An ideal anticancer agent inhibits the metastasis of the tumors. Hence, anti-metastatic property of the fractions by in vitro antimetastatic assay was performed for measuring the 2D cell migration in the presence of fractions on HCT 116 cell line. The assay has shown that there was no significant antimigratory activity as the fractions did not inhibit the migration of cells in the scratch area.

DMH is a chemical that is most widely used for the colorectal cancer induction in rodents. As it is metabolically activated in the liver to its intermediates azoxymethane and methylazoxymethanol which are toxic in nature, an increase in the liver was observed³⁴. As major organ index can be a marker of toxicity, the organ indices of liver, spleen, kidneys and heart were measured and it was found that the liver index in DMH control group has significantly increased compared to the normal group indicating the obvious toxicity of DMH. The fractions did not show any signs of toxicity in any of the major organs. Hematological parameters did not show a significant change in the fractions treated groups indicating that there is no predictable toxicity on the hematological system. In biochemical estimations, the fractions have significantly decreased the levels of nitrite and lipid peroxidation indicating anti-inflammatory properties. As the antioxidant system of the body protects the cells from oxidative stress, we have studied the effect of the fractions on the antioxidant enzyme levels. GSH and catalase levels have decreased significantly in the DMH treated group. The levels were restored in the treatment groups but they were not found to be significant. There was no major change in the levels of SOD between the groups. Based on these findings, further exploration into the compounds present in the fractions will be useful.

CONCLUSION:

The present study was performed to evaluate the anticancer potential of the roots of Clerodendrum indicum (L.) Kuntze. The fractions have induced apoptosis in the tumor cells affecting mainly G2/M phase transition. But the fractions did not show good antimigratory activity in vitro. The fractions also did not have a significant effect on the in vivo antioxidant parameters. Further, characterization and isolation of active compounds in the fractions have to be performed to identify the exact molecules that were responsible for the activity.

ACKNOWLEDGEMENT:

We acknowledge Manipal College of Pharmaceutical Sciences for providing the infrastructure, chemicals and equipment required for carrying out the study.

We also acknowledge DST-FIST for providing auto analyzer for serum estimations.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

REFERENCES:

1. Young A, Hobbs R, Kerr D. ABC of colorectal cancer. BMJ books, US. 2011.Abei H. Catalase in vitro. Methods enzymol. 1984; 105: 121-126.

2. Rajalekshmi M, Shreedhara, CS, Lobo, R and Rao, 'The review on genetics, epigenetics, risk factors and diagnosis of colon cancer', Research Journal of Pharmacy and Technology, 2018; 11(11): 5147-5151.

3. Chandrasekar, Raju and Sivagami, B. and Babu, M. A Pharmacoeconomic Focus on Medicinal Plants with Anticancer Activity. Research Journal of Pharmacognosy and Phytochemistry. 2018; 10: 91.

4. Nobili S, Lippi D, Witort E, Donnini M, Bausi L, Mini E, Capaccioli S. Natural compounds for cancer treatment and prevention. Pharmacol Res. 2009; 59(6): 365-378.

5. Krishnasamy L, Selvam, Masilamani, Ravikrishnan, Bharathi. Anticancer property of Colchicine isolated from Indigofera aspalathoids. Research Journal of Pharmacy and Technology. 2016; 9: 386.

6. Sarmistha Rej, Madhurima Dutta, Shahid Jamal, Sumanta Das, Sabyasachi Chatterjee. Study of Phytochemical Constituents and Antibacterial Activity of Clerodendrum infortunatum. Asian J. Res. Pharm. Sci. 2014; 4(4): 187-195.

7. Shrivastava N, Patel T. Clerodendrum and healthcare: An overview. Medicinal and Aromatic Plant Science and Biotechnology. 2007; 1:142-150.

8. Habeela Jainab N., MK. Mohan Maruga Raja. In Silico Molecular Docking Studies on the Chemical Constituents of Clerodendrum phlomidis for its Cytotoxic Potential against Breast Cancer Markers. Research J. Pharm. and Tech. 2018; 11(4): 1612-1618.

9. Abouzid SF, Wahba HM, Elshamy A, Cos P, Maes L, Apers S, Pieters L, Shahat AA. Antimicrobial activity of some Clerodendrum species from Egypt. Nat prod Res. 2012; 27(11): 1032-1036.

10. Barua CC, Das AS, Sen S, Talukdar A, Barua AG, Baishya G, Nath SC. Clerodendron indicum: A repertoire of phytochemicals and its antioxidant activity. Int J Phytopharmacol. 2014; 5(4): 252-260.

11. Dash RR, Pattanaik S., Prusty SK, Bhatnagar S. Anti-oxidant and cytotoxic activities of leaf and bark extracts of Clerodendrum indicum (L.) Kuntze. Am. J. Pharmtech res. 2014; 4(3): 229-236.

12. Deleu QC, Gressier B, Vasseur J, Dine T, Brunet C, Luyckx M. Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour. J Ethnopharmacol. 2000; 72(1-2): 35–42.

13. Sadika Banu, Ramakrishnaiah T. N. Screening of Garcinia cambogia for in-Vitro Anti-Cancerous Activity against Colon Adenocarcinoma (Caco-2) Cell Line. Res. J. Pharmacognosy and Phytochem. 2018; 10(4): 272-276.

14. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(2): 55-63.

15. Hung RW, Chow AW. Apoptosis: Molecular mechanisms, regulation and role in pathogenesis. Can J Infect Dis. 1997; 8(2): 103-9.

16. Kasibhatla, S. Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis. Cold Spring Harbor Protocols, 2006(21).

17. Namrata Dwivedi, Bhavna Dwivedi, Skand Mishra, Yogeshwer Shukla. Lupeol Induced Apoptosis in Human Lung Cancer Cell Line: A Flow Cytometry Study. Research Journal of Pharmacology and Pharmacodynamics. 2014; 6(4): 197-203.

18. Sasaki K, Kurose A, Ishida Y. Flow cytometric analysis of the expression of PCNA during the cell cycle in HeLa cells and effects of the inhibition of DNA synthesis on it. Cytometry. 1993; 14(8): 876-882.

19. Sheryl P. Denker, Diane L, Barber. Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1. J Cell Biol. 2002; 159(6): 1087-96

20. Kangralkar, VA. and Kulkarni, AR Evaluation of effect of Piper betel, Centella asiatica and Aristolochia indica extracts on bacterial enzymes in 1, 2-dimethyl hydrazine induced colon cancer in Wistar rats. Research Journal of Pharmacy and Technology. 2014; 7: 151-154.

21. Nandagaon, VS. and Kulkarni, AR. Effect of Annona squamosa, Bacopa monneri and Baliospermum montanum alcoholic extracts on bacterial enzymes in 1, 2-dimethyl hydrazine induced colon cancer in rats. Research Journal of Pharmacy and Technology. 2013; 6: 379-383.

22. Sinha AK. Colorimetric assay of catalase. Anal biochem. 1972; 47(2): 389-94.

23. Giustarini D, Dalle-Donne I, Milzani A, Fanti P, Rossi R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat Protoc. 2013; 8(9): 1660-9.

24. Kaynar H, Meral M, Turhan H, Keles M, Celik G, Akcay F. Glutathione peroxidase, glutathione-S-transferase, catalase, xanthine oxidase, Cu-Zn superoxide dismutase activities, total glutathione, nitric oxide, and malondialdehyde levels in erythrocytes of patients with small cell and non-small cell lung cancer. Cancer Let. 2005; 227(2): 133-9.

25. Kuninaka S, Ichinose Y, Koja K, Toh Y. Suppression of manganese superoxide dismutase augments sensitivity to radiation, hyperthermia and doxorubicin in colon cancer cell lines by inducing apoptosis. Br J Cancer. 2000; 83(7): 928–934.

26. Kumar KC S, Muller K. Medicinal plants from Nepal; II Evaluation as inhibitors of lipid peroxidation in biological membranes. J Ethnopharmacol. 1999; 64(2): 135-139.

27. Nathan S, Bryan, Matthew B, Grisham. Methods to detect nitric oxide and its metabolites in biological samples. Free Radic Biol Med. 2007; 43(5): 645–657.

28. Pal A, Mahmud ZA, Akter N, Islam SM, Sitesh BC. Evaluation of antinociceptive, antidiarrheal and antimicrobial activities of leaf extracts of Clerodendrum indicum. Pharmacognosy Journal. 2012; 4(10): 41-46

29. Mia M M, Kadir MF, Hossan SM, Rahmatullah M. Medicinal plants of the garo tribe inhabiting the madhupur forest region of Bangladesh. Am.-Eurasian J. Sustain. Agric. 2009; 3(2): 165-171.

30. The Plant List (2013). Version 1.1. Published on the Internet; http://www.theplantlist.org.

31. Ho JCM, Zheng S, Comhair SA, Farver C, Erzurum SC. Differential expression of manganese superoxide dismutase and catalase in lung cancer. Cancer Res. 2014; 61(23): 8578-8585.

32. Khatiwora E, Adsul VB, Kulkarni MM, Deshpande N, Kashalkar R. Spectroscopic determination of total phenol and flavonoid contents of Ipomoea carnea. Int.J. ChemTech Res. 2010; 2(3): 1698-1701.

33. Zhang L, Zheng Y, Deng H, Liang L, Peng J. Aloperine induces G2/M phase cell cycle arrest and apoptosis in HCT116 human colon cancer cells. Int J Mol Med. 2014; 33(6): 1613–1620.

34. Perse M, Cerar A. Morphological and molecular alterations in 1,2 dimethylhydrazine and azoxymethane induced colon carcinogenesis in rats. J biotech biotechnol. 2011; 1-14.

Received on 03.09.2019 Modified on 07.10.2019

Research J. Pharm. and Tech 2020; 13(5): 2321-2328.

DOI: 10.5958/0974-360X.2020.00418.7