Int J Pharm Pharm Sci, Vol 8, Issue 1, 439-444Original Article

IN VITRO CYTOTOXIC ACTIVITY OF CLERODENDRUM INFORTUNATUM L. AGAINST T47D, PC-3, A549 AND HCT-116 HUMAN CANCER CELL LINES AND ITS PHYTOCHEMICAL SCREENING

MIR HARIS, RIAZ MAHMOOD^*, HASEEBUR RAHMAN, NAZNEEN RAHMAN

Department of Biotechnology and Bioinformatics, Kuvempu University, Shimoga 577451, Karnataka, India
Email: riaz_sultan@yahoo.com

 Received: 26 Oct 2015 Revised and Accepted: 08 Dec 2015

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ABSTRACT

Objective:Present investigation accounts for scientific evaluation of the plant Clerodendrum infortunatum L. for its medicinal efficacy which includes phytochemical screening and anticancer activities.

Methods:Phytochemical screening of Clerodendrum infortunatum extracts was performed for the qualitative detection of reducing sugars, terpenoids, flavonoids, saponins, tannins, alkaloids, phlobatannins, steroids, amino acids and glycosides using standard procedures. The sulforhodamine B (SRB), in vitro cytotoxic assay, was used to investigate the anticancer activity of hexane, chloroform, ethyl acetate and ethanol extracts of leaves and roots of Clerodendrum infortunatum against T47D (Breast), PC-3 (prostate), A549 (lung) and HCT-116 (colon) cancer cell lines.

Results:Secondary metabolites including alkaloids, flavonoids, terpenoids, steroids, tannins, and saponins are present in many extracts. Alkaloids and flavonoids found to be present in almost all the extracts. The best cytotoxic activity has been exerted by hexane of root exhibiting growth inhibition of 72.83 ± 0.44, 85.50 ± 0.29 and 68.17 ± 1.36 % against PC-3, A549 and HCT-116 at a concentration of 100 µg/ml. Further, hexane of root showed a moderate cytotoxic effect of 42.17 ± 0.17 % against T47D at 100 µg/ml. At similar concentration, the chloroform extract is also effective against these three cell lines showing 61.50 ± 0.76, 67.00±0.58, and 68.53 ± 0.80% growth inhibition against PC-3, A549 and HCT-116 cell lines respectively, whereas, T47D cancer cell line showed 46.43 ± 0.30 % growth inhibition. The results have indicated that all the leaf extracts, as well as ethyl acetate and ethanol extracts of root, have exhibited a poor response (≤ 40 %).

Conclusion:The present findings suggest that the Clerodendrum infortunatum extracts are rich in alkaloid, flavonoids and terpenoids. The hexane and chloroform root extracts of Clerodendrum infortunatum possess significant anticancer activities which may be due to the presence of these phytochemical groups.

Keywords:Alkaloids, Cancer cell lines, Clerodendrum infortunatum, Flavonoids, Growth inhibition.

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© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Cancer is one of the most malignant diseases in which deregulating propagation of abnormal cells plague and disrupt contiguous tissues [1]. Both developed and developing countries express serious public health problems in terms of cancer. It is a mortal disease and presently, about one in five of all deaths is because of cancer [2]. Normal human cells proliferate for a fixed number of generations and then enter a condition of replicative senescence, but cancer cells can multiply endlessly [3]. There are 12.7 million cases of cancer and 7.6 million cancer deaths recorded in 2008; of these 64% of deaths exist in the economically developing countries [4]. The detectable tumors can be evaluated by surgical means, but it may put down nest of cancer cells in the patient, which continue to flourish while radiation therapy provides an inaccurate healing as it can exterminate both cancer and normal cells. The limited success of these therapies in the treatment of cancer indicates that there is an imperative need of alternative strategies in cancer management [5]. The universal fatality rate may increase by nearly 80 % by 2030, with enormous probability in low-and-middle-income countries devoid of instant action cited by the World Health Organization (WHO). Novel therapeutic alternatives to analyze for cancer treatment are of elevated priority for most of the pharmaceutical industries and independent research organizations worldwide. Significant research activity is committed to the innovation of more potent treatments while reducing their toxic side effects. So, there is a precondition of cancer chemotherapy for the development of selective drugs that can kill malignant tumor cells or render them benign without affecting normal cells which may eventually direct to attain the goal [6].

The practical access to this difficulty is the use of terrestrial plants as an application for drug development. Plants have played a leading role in the development of sophisticated traditional medicine systems. About 80% of the populations in some Asian and African countries rely on traditional medicine for main health care as estimated by the WHO. The 70–80% of the populations of developed countries also decides on plant products as a complementary medicine.

Different species of Clerodendrum genus have been traditionally used over the centuries, and their antioxidant, and hepatoprotective potential have already been proved [7-9]. C. infortunatum is very common throughout the plains of India. The leaves are slightly bitter, cure inflammation, skin diseases and good in smallpox [10]. The plant found to contain triterpenes, steroids, and flavonoids [11, 12]. The antioxidant [13], antimicrobial [14], anti-malarial [15], anthelmintic [16] and analgesic [17] activities of the plant have further created an upsurge in investigations on the plant. Therefore, the objective of the present study was designed to evaluate the anticancer potential of different extracts of C. infortunatum against different cancer cell lines.

MATERIALS AND METHODS

Plant source and extract preparation

The plant C. infortunatum (Verbenaceae) was collected from the roadside of Bhadra River Channels, Shimoga, Karnataka, India. The plant was authenticated by Dr. V. Krishna, Professor, Department of Biotechnology, Kuvempu University, Karnataka, who is one of the known taxonomists. The leaves and roots (after cutting into small pieces) were shade dried for several days. The plant material was then oven dried for 24 h at a considerably lower temperature (40 °C) for moisture free and better grinding. The air-dried and finely ground leaf (1000g) and root (500g) material of the plant was extracted in a Soxhlet apparatus successively with hexane, chloroform, ethyl acetate and ethanol from low polarity to high polarity. A suitable solvent was added to the flask, and the setup was heated under reflux. The steam of the solvent which, when contacts with the material will dissolve metabolites and brings back metabolites to the flask. The extracts were filtered, pooled and concentrated to dryness under reduced pressure in a rotary evaporator (Buchi, Flawil, Switzerland) to yield dried hexane, chloroform, ethyl acetate and ethanol extracts. The extracts so obtained from each of solvents were labeled and yield was calculated in terms of grams/weight of the powdered material.

Solubility tests of plant extracts

Solubility tests were carried out for the analysis of solubility of crude extracts in different solvents like hexane, chloroform, ethyl acetate, acetone, DMSO, ethanol, methanol, water, 1N NaOH, and 1N HCl.

Phytochemical analysis of extracts

Phytochemical screening of C. infortunatum extracts was performed for the qualitative detection of reducing sugars, terpenoids, flavonoids, saponins, tannins, alkaloids, phlorotannins, steroids, amino acids and glycosides using standard procedures [18, 19].

Chemicals

The chemicals utilized in this analysis comprises RPMI-1640 (Roswell Memorial Park Institute), Minimum Essential Medium, fetal calf serum, trypsin, trypan blue, ethanol, penicillin, streptomycin, gentamycin, dimethyl sulfoxide (DMSO), sulforhodamine B (SIGMA), adriamycin, mitomycin-C, paclitaxel, 5-fluorouracil (5-FU; Sigma Chemical Co., St Louis, MO, USA), phosphate buffer saline (Merck, Darmstadt, Germany); trichloroacetic acid (TCA), distilled water, sodium hydroxide, Tris-EDTA buffer, Tris buffer (Hi-Media, Mumbai, India), acetic acid, sodium bicarbonate, hydrochloric acid (Rankem, New Delhi, India), isopropanol (Sisco, Mumbai, India), and Tris-acetate-EDTA buffer. All other chemicals used in this study were purchased locally and were of analytical grade.

Cell lines and cell culture

The human cancer cell lines used in this study, including T47D (Breast), PC3 (prostate), A549 (lung) and HCT-116 (colon) were obtained from the National Center for Cell Science, Pune, India. The properties of the cell lines under investigation as well as their culture conditions are summarized in table 1.

The cells were sustained in a CO₂ incubator with 5% CO₂ and 95% humidity and supplemented with desired medium and 10% fetal bovine serum (FBS). Glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 mg/ml) were also added to the medium to 1×final concentration from a 100 × stock. After attaining the confluent growth, the cells were trypsinized using Trypsin-EDTA (0.25%) and the number of cells required to perform the assay were seeded into sterile 96-well plates. Then the plates were incubated in a CO₂ incubator with 5% CO₂ and 95% humidity [20, 21].

Table 1: Review of cell lines under investigation, their origin, characteristics and culture conditions

Cell Line	Description	Seeding density (Per cm2)	Growth media	ER status	P53Status	Doubling time (h)
T47D	Breast	30,000	DMEM	+	Mu	32
PC-3	Prostate	50,000	DMEM	+	Null	40
A-549	Lung	40,000	RPMI 1640	+	Wt	22
HCT-116	Colon	30,000	RPMI 1640	+	Wt	21

Cytotoxic assay preparation

Trypan blue exclusion method has been employed to determine the viability of the cells. Concisely, 100 µl of cell suspension (10,000 cells) of each cell line was plated in each well of 96-well plates, and incubated for 24 h at 37 °C in a humidified CO₂ (5%) incubator. The growth inhibitory activity of the hexane, chloroform, ethyl acetate and ethanol extracts of both leaf and root were evaluated against four cell line panels consisting of T47D (Breast), PC3 (prostate), A549 (lung) and HCT-116 (colon) using the SRB assay[22]. The lyophilized extracts were dissolved in DMSO and filtrated by 0.2 µm cellulose acetate sterile filter. After 24 h of incubation time of cell lines, an amount of 1, 10 and 100 µg/ml of each extract was added. Several standard anticancer drugs were used as positive control for comparison. The drugs include Adriamycin (10µM) against T47D, mitomycin (1µM) against PC-3, paclitaxel (1 µM) against A549 and 5-Fluorouracil (20µM) against HCT-116 were added as a positive control against desired cancer cell lines per well and incubated for 48 h.

Sulpho rhodamine B assay

After 48 h of incubation with test samples, each well was fixed with 50 µl of 50% trichloroacetic acid solution to stop the reaction and incubated at 4^oC for 1 h followed by washing with distilled water. Excess water was drained off and the plates were air-dried for 24 h. Then cells were stained with 50 µl of 0.4% SRB solution in 1% acetic acid for 30 m at room temperature (27±2 ^oC). After incubation, the unbound SRB solution was discarded, and the plates were washed with 1% acetic acid. The plates were air-dried, and 100 µl of 10 mM Tris-base solution (pH 10.5) was added to each well to solubilize the dye and shaken for 30 min on a mechanical shaker. The optical density (OD) at 540 nm was determined by the micro plate reader (Bio-Rad, Model 680, USA). All the investigation was performed in triplicates. The percentages of the cell growth inhibition were determined using the following equation:

Statistical analysis

The data from biological assays were subjected to the One-way analysis of variance (ANOVA) procedures which were presented as mean±SEM. P ≤ 0.01 was considered as significant.

RESULTS

Physical characteristics and percentage yield of extracts

Extraction (as the term is pharmaceutically used) is the separation of medicinally active portions of plants using selective solvents through standard procedures. The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or (after removing the solvent) in dry powder form, and are intended for oral or external use. These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts or powdered extracts. The percent yield of both leaf and root extracts obtained from C. infortunatum and their description is outlined in table 2 and 3.

Table 2: Percent yield (w/w) and physical characteristics of C. infortunatum Leaf extracts

Samples	Quantity used for extraction		Nature of the extract	% Yield
	Powder (g)	Solvent (ml)
Hexane	1000	250	Black Sticky mass	3.0
Chloroform	1000	250	Dark Solid	2.0
Ethyl acetate	1000	250	Slightly light green solid mass	3.5
Ethanol	1000	250	Dark sticky brown paste	20.0

Table 3: Percent yield (w/w) and physical characteristics of C. infortunatum root extracts

Samples	Quantity used for extraction		Nature of the extract	% Yield
	Powder (g)	Solvent (ml)
Hexane	500	250	Light yellow mass	1.0
Chloroform	500	250	Brown sticky paste	1.5
Ethyl acetate	500	250	Orange red paste with acidic smell	1.0
Ethanol	500	250	Red paste with acidic smell	4.0

Table 4: Phytoconstituents present in the leaf extracts of C. infortunatum L.

Phytoconstituents	Hexane extract	Chloroform extract	Ethyl acetate extract	Ethanol extract
Alkaloids	+ve	+ve	+ve	-ve
Flavonoids	+ve	+ve	+ve	+ve
Terpenoids	+ve	-ve	+ve	-ve
Steroids	-ve	-ve	-ve	+ve
Tannins	-ve	-ve	-ve	+ve
Phlobtannins	-ve	-ve	-ve	-ve
Saponins	-ve	-ve	-ve	+ve
Carbohydrates	+ve	+ve	+ve	+ve
Glycosides	+ve	+ve	+ve	-ve
Amino acids	-ve	-ve	-ve	+ve

(+ve): Present; (-ve): Absent.

Table 5: Phytoconstituents present in the root extracts of C. infortunatum L.

Phytoconstituents	Hexane extract	Chloroform extract	Ethyl acetate extract	Ethanol extract
Alkaloids	+ve	+ve	+ve	-ve
Flavonoids	-ve	+ve	+ve	+ve
Terpenoids	+ve	+ve	-ve	+ve
Steroids	-ve	-ve	+ve	+ve
Tannins	-ve	-ve	-ve	+ve
Phlobtannins	-ve	-ve	-ve	-ve
Saponins	-ve	-ve	-ve	+ve
Carbohydrates	+ve	+ve	+ve	+ve
Glycosides	-ve	-ve	-ve	-ve
Amino acids	-ve	-ve	-ve	-ve

(+ve): Present; (-ve): Absent.

Table 6: Cytotoxic effect of leaf extracts of plant on different cancer cell lines

Tissue			Breast	Prostate	lung	Colon
Cell line type			T47D	PC-3	A549	HCT-116
S. No.	Sample	Conc. (µg/ml)	% Growth inhibition
1.	Hexane	1	15.73±0.64**	NT	NT	1.93±0.52**
		10	20.87±0.35**	1.73±0.43**	1.37±0.20**	3.03±0.15**
		100	28.43±0.49**	13.93±0.61**	43.83±0.55**	22.10±1.15**
2.	Chloroform	1	NT	NT	NT	NT
		10	NT	NT	NT	NT
		100	NT	14.20±0.44**	35.70±0.65**	1.67±0.24**
3.	Ethyl acetate	1	21.10±0.49**	NT	NT	5.83±0.90**
		10	20.87±0.58**	NT	2.37±0.33**	12.83±0.27**
		100	24.43±0.32**	11.33±0.33**	21.23±0.67**	12.27±0.68**
4.	Ethanol	1	NT	NT	NT	NT
		10	NT	NT	NT	NT
		100	18.07±0.58**	11.87±0.24**	10.13±0.09**	11.27±0.37**
5.	Adriamycin	10µM	63.27±0.37	-	-	-
6.	Mitomycin	1µM	-	67.60±0.56	-	-
7.	Paclitaxel	1µM	-	-	76.73±0.82	-
8.	5-Fluorouracil	20µM	-	-	-	50.60±0.31

NT: Non-toxic, Values are expressed as mean±SE of three values. **P<0.01; **p values were calculated comparing the experimental results to standard results.

From the above tables, it is estimated that the yield of hexane, chloroform, ethyl acetate and ethanol extracts of the leaf was found to be 3.0, 2.0, 3.5 and 20.0% respectively. Likewise, the yield of hexane, chloroform, ethyl acetate and ethanol extracts of root was found to be 1.0, 1.5, 1.0 and 4.0 % respectively. It is evident from the above tables that extraction with ethanol in both leaf and root yields the highest amount of extracts.

Qualitative detection of phytochemical constituents

All the solvent extracts from leaf and root were subjected to preliminary qualitative phytochemical analysis. The various groups of phytochemical constituents found to be present in these extracts are shown below (table 3 and 4). The presence of groups of all these different extracts includes alkaloids, flavonoids, terpenoids, steroids, tannins and carbohydrates. This analysis revealed that the plant possesses alkaloids, flavonoids, carbohydrates in high concentration.

In vitro cytotoxic activity of plant extracts

The in vitro cytotoxic effect of eight extracts of leaf and root (four from each part) derived from C. infortunatum were evaluated on four human cancer cell lines from different tissues of origin, namely; T47D (Breast), PC-3 (prostate), A549 (lung) and HCT-116 (colon) cancer cell lines. The cytotoxic activity of the extracts was compared with the activity of standard anticancer drugs. The results suggest that among the four different root extracts, hexane and chloroform showed a significant cytotoxic activity against PC-3, A549 and HCT-116 cell lines used and had shown moderate activity against the T47D cell line, whereas, the ethanol and ethyl acetate extracts did not show promising activity when compared to the respective standards used. The results of cytotoxic effects of both leaf and root extracts are summarized in tables 5 and 6. The best antiproliferative activity has been exerted by hexane of root exhibiting growth inhibition of 72.83±0.44, 85.50±0.29 and 68.17±1.36 % against PC-3, A549 and HCT-116 at a concentration of 100µg/ml. Further, hexane showed a moderate cytotoxic effect of 42.17±0.17 % against T47D at 100µg/ml. At a similar concentration the chloroform extract is also effective against these three cell lines showing 61.50±0.76, 67.00±0.58, and 68.53±0.80 % growth inhibition against PC-3, A549 and HCT-116 respectively, whereas, T47D cancer cell line showed 46.43±0.30 % growth inhibition. The results have indicated that all the leaf extracts, as well as ethyl acetate and ethanol root, have exhibited a poor response (≤40%).

Percent growth inhibition resulting from the standard drugs on different cell lines used in this study is found between 50 and 77%. The cytotoxic effect of some extracts is comparatively more than that of standard drugs.

Table 7: Cytotoxic effect of root extracts of plant on different cancer cell lines

Tissue			Breast	Prostate	Lung	Colon
Cell line type			T47D	PC-3	A549	HCT-116
Sl. No.	Sample	Conc. (µg/ml)	% Growth inhibition
1.	Hexane	1	NT	NT	NT	NT
		10	NT	25.33±0.34**	2.00±0.58**	14.60±0.31**
		100	42.17±0.17**	72.83±0.44**	85.50±0.29**	68.17±1.36**
2.	Chloroform	1	NT	1.33±0.33	NT	NT
		10	NT	23.00±0.58	NT	6.83±0.22
		100	46.43±0.30**	61.50±0.76**	67.00±0.58**	68.53±0.80**
3.	Ethyl acetate	1	12.27±0.82**	NT	NT	NT
		10	14.33±0.88**	NT	12.00±0.583	NT
		100	26.43±0.260**	29.80±0.42**	14.00±0.58**	15.07±0.41**
4.	Ethanol	1	26.43±0.47**	NT	NT	NT
		10	31.23±0.67**	NT	NT	NT
		100	39.83±0.44**	11.33±0.88**	NT	5.00±0.58**
5.	Adriamycin	10µM	63.27±0.37	-	-	-
6.	Mitomycin	1µM	-	67.60±0.56	-	-
7.	Paclitaxel	1µM	-	-	76.73±0.82	-
8.	5-Fluorouracil	20µM	-	-	-	50.60±0.31

NT: Non-toxic, Values are expressed as mean±SE of three values. **P<0.01; **p values were calculated comparing the experimental results to standard results.

DISCUSSION

Cancer is the prominent origin of death in efficiently developed countries and second largest death in developing countries [23]. It is estimated that new cases of cancer will rise approximately 25% in each decade, aiming 24 million new cases per year in the year 2050 [24]. The current threat linked with presently existing drugs comprises selectivity, toxicity, resistance, and development of a secondary malignancy. The downsides of these anticancer agents have encouraged the examination of the novel, competent and well resistant drugs against cancer, as natural products, mainly from plants. Keeping this in mind, the present investigations have been carried out to screen the cytotoxic potential of extracts of C. infortunatum against different human cancer cell lines.

The plant material was subjected for Soxhlet extraction using solvents of appropriate polarity following the principles of ‘like dissolves like’ thus non-polar solvents were used to solubilize most lipophilic compounds (e. g., alkanes, fatty acids, pigments, waxes, sterols, few terpenoids, alkaloids). Medium polarity solvents were used to extract compounds of intermediate polarity (e. g., some alkaloids, flavonoids) while more polar solvents were used for more polar compounds (e. g., flavonoid, glucosides, quaternary alkaloids, tannins, etc.). Based on this principle, four different solvents have been used with increasing polarity, i.e., hexane, chloroform, ethyl acetate and ethanol for successive extraction.

Phytochemical screening of C. infortunatum extracts was performed to determine the presence of phytoconstituents such as: alkaloids, flavonoids, terpenoids, steroids, carbohydrates, tannins, saponins, glycosides, amino acids. Among these groups alkaloids and flavonoids are found to be very rich in these extracts followed by terpenoids, while as, saponins, tannins, and steroids are present only in ethanol extract. The presence of these groups may direct to comprehend the potent cytotoxic activity of the plant. Earlier reports on medicinal plants suggest that alkaloids [25]and flavonoids [26] extensively play a vital role in the toxicity of cancer cells.

In this study hexane, C. infortunatum root is found to be significantly toxic against PC-3 (prostate), A549 (lung) and HCT-116 (colon) cancer cell lines exhibiting growth inhibition of 72.83±0.44, 85.50±0.29 and 68.17±1.36% respectively at a concentration of 100µg/ml in the SRB assay compared to the standard drug. Likewise, hexane, chloroform root extract at the same concentration of 100µg/ml also exhibited considerable growth inhibition of 61.50±0.76, 67.00±0.58 and 68.53±0.80% against same cancer cell lines. It is pertinent to note that, the percentage inhibition is greater than the standard drug used. Both of these extracts exerted moderate cytotoxic activity against the T47D cell line. The growth inhibition may be due to the presence of non-polar and medium polar type of compounds. Hexane and chloroform revealed higher cytotoxic activity than other extracts used which could be due to the high content of alkaloids and flavonoids or may be of terpenoids. Interestingly, both extracts which showed highest anticancer activity are from the root parts of C. infortunatum and unveil the presence of alkaloids, flavonoids as well as terpenoids.

There are many earlier reports suggesting the biological activities of several alkaloids viz., the anticancer effects of vinblastine [27], camptothecin (CPT), a famous topoisomerase-I (TopI) inhibitor [28]. Similarly, berberine inhibits the proliferation of multiple cancer cell lines by inducing cell cycle arrest at the G1 or G₂/M phases and by apoptosis [29, 30, 31]evodiamine exhibits anticancer activities both in vitro and in vivo by inducing the cell cycle arrest or apoptosis, inhibiting the angiogenesis, invasion, and metastasis in a variety of cancer cell lines [32, 33]. Piperine and matrine are also considered to be potent anticancer alkaloids [34, 35].

The role of flavonoids in cancer prevention indicates that flavonoids have important effects on cancer chemoprevention and chemotherapy [36]. The various flavonoids which showed anticancer activity on different human cancers include flavanones, daidzein, genistein, quercetin, luteolin are effective against breast cancer[37, 38]. Flavone, quercetin have also been proved to be potent against human lung cancer [39, 40]. Catechin, epicatechin, quercetin, kaempferol, luteolin, genistein, apigenin, myricetin, silymarin possess significant effect against human prostate cancer [41-43]. The flavonoids which are considered to be very effective against human colon cancer exclude flavone, quercetin, genistein, anthocyanin [44, 45].

In addition, of alkaloids and flavonoids, the diverse array of terpenoid structures and functions has provoked increased interest in their commercial use. Terpenoids have been found to be useful in the prevention and therapy of several diseases, including cancer [46].

Some Species of Clerodendrum genus that have been investigated in the anticancer study and have resulted in the innovation of promising drugs include C. serratumSpreng. [47, 48], C. bungei Study [49, 50], C. calamitosumL. [51]. Other species of Clerodendrum including C. phlomidis, C. inerme, C. colebrookianum and C. trichotomum also possess significant cytotoxic activity [52, 53]. Some investigations also identified the important cytotoxic activity of leaf extracts of C. viscosum [54].

Based on all this information the alkaloids and flavonoids or terpenoids present in C. infortunatum may be the actual source of cytotoxic effect against the human cancers investigated in this study.

CONCLUSION

Thus, the findings of this investigation suggest that the C. infortunatum extracts are rich in alkaloids, flavonoids, and terpenoids. The hexane and chloroform root extracts of the plant possess significant anticancer activities in vitro against PC-3 (prostate), A549 (lung) and HCT-116 (colon) cell lines which may be due to the presence of these alkaloids, flavonoids, and terpenoids. Hence, C. infortunatum could be considered as promising plant possessing anticancer properties. Further, extensive studies are required to identify, isolate and characterize specific phyto molecules responsible for bioactivities observed in this study and their inherent mechanism(s) of action.

ACKNOWLEDGMENT

The authors acknowledge the University Grants Commission for their financial support. The authors are grateful to. Dr. Ram A. Vishwakarma, Director IIIM (Indian Institute of Integrative Medicine, Jammu, India), to host the permission to carry out the part of this work. The immense support and guidance of Dr. Shashank Kumar Singh, Senior Scientist, and Iram Qayoom, Research Scholar, Cancer Pharmacology Division, IIIM, is also appreciated. We are also thankful to Mr. Bilal Ahmad Rah, Research Scholar, IIM, Jammu for his technical support.

CONFLICT OF INTERESTS

We declare that there are no conflicts of interest, financial or otherwise, pertaining to this study.

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