The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367

Studies on Phytoconstituents, In vitro Antioxidant and Cytotoxity Activity of Bark Extracts on Human Epidermoid Larynx Carcinoma Cell Line (HEp-2) R. Rajeswari1, S. Murugesh1*, D. Jegadeeshkumar2, B. Prakash3, V. Vinoth kumar4 1Department of Botany, School of Life Sciences, Periyar University, Periyar Palkalai Nagar, Salem-636 011, Tamil Nadu, . 2Chromopark Research Centre, Namakkal - 637001 Tamil Nadu, India. 3Department of Biotechnology, School of Life Sciences, Vels Institute of Science Technology & Advanced Studies, Chennai, Tamil Nadu, India. 4Department of Microbiology, Sree Narayana Guru College, Coimbatore-641105, India.

*Corresponding Author Email: [email protected] Phone: +91 9943364913 ORCID: 0000-0002-8382-8553 Abstract Cancer is major public issues and one of the leading causes of death in prosperous countries. Traditional are a valued source of novel cytotoxic agents and are still in performance better role in health concern. In the present study, the bark of methanol and chloroform extracts were subjected to preliminary phytochemicals screening was done using different biochemical tests. Among the 2 solvent extracts, methanol showed the highest number of phytochemicals, which extract was subjected to in vitro study of antioxidant activity by DPPH method and cytotoxicity activity against HEp-2 cell lines by MTT assay. Furthermore, DNA fragmentation and GCMS analysis were subjected. By the analsyis, following phyto-constituents like tannins, saponins, Steroids, Carbohydrate and Phenols, Flavonoids, Terpenoids, and quinine were observed. The methanol extract was found to be selectively cytotoxic in vitro to HEp-2 cell lines with IC50values was15.6 µg/ml. The GC-MS chromatogram of methanolic bark extract of Harpullia arborea confirmed the presence of various compounds. The present findings strengthen the potential of the selected as a resource for the discovery of novel antioxidant and anticancer agents. Keywords: Anticancer, Harpullia arborea, HEp-2, Catechol, DNA fragmentation. Introduction beneficial properties in the management of various Cancer is a severe metabolic syndrome, and cancers (3). one of the leading causes of death irrespective of Harpullia arborea (Blanco) Radlk, a native developments in disease diagnosis, prevention, and to Indo-Malayan, Western, and Eastern Ghats, treatment tools Cancer of the mouth and pharynx belongs to the family . Various parts of ranks the sixth most common cancer in the world this plant are used traditionally by Kerala, population (1). Most of the larynx carcinoma cancers Tamil Nadu and Karnataka peoples for hair wash, are develops from the squamous cells, which are the rheumatism and wound healing. The fruit majority of the laryngeal epithelium. Cancer can of Harpullia arborea was used as an appetizer and to develop in any part of the larynx, but the location of cure digestive problems (4). The plant is reported to the tumor affects the cure rate (2). The chemical drugs exhibit bioactivities such as larvicidal, antifungal, are the toxicity that is caused to the normal cells due antioxidant, antibacterial, and anticancer activity (5). to the inability of the chemical drugs to differentiate Although earlier studies reported between normal and cancerous cells. Traditional curative properties of active phytoconstituents from medicines have been tested and researched to obtain Harpullia arborea, According to our literature an effective drug against cancer. knowledge no one likes that anticancer activity of Numerous cancer studies for the bark extract of Harpullia arborea against human chemotherapeutic potential of medicinal plants were laryngeal epithelial cell line HEp-2. Thus, the present performed to discover new therapeutic agents that work intended to screen the methanol extract lack the poisonous consequences associated with of Harpullia arborea for testing their potential to modern therapeutic agents. Traditional medicine is inhibit the viability of cells in HEp-2 and determinate commonly used as an alternative remedy for most the antioxidant activity. cancers. About 60% of anticancer agents are Material and Methods originated from herbal plants and other natural Plant material and extraction sources, however, still a few numbers of plants that The bark of Harpullia arborea was used in have anticancer activity, they do this work, which was collected in the period of April 2017 in Gedamalai, Namakkal District, Tamilnadu, not have yet been completely analysis. In recent India. The plant was identified at the Botanical survey years, herbal plants and plant acquired products have of India, Coimbatore. The voucher number is also attracted researchers due to their varied range of BSIS/RC/5/23/2017/Tech/350. The bark sample was

Volume XII, Issue I, January/2020 Page No:1270 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367

cleaned and air-dried, and the powder (100gm) was examined in an inverted phase contrast microscope left in chloroform and methanol for 48 h during (6). which the container was stirred occasionally. The DCFH-DA staining test for detection content was filtered through 4-fold muslin cloth of intracellular reactive oxygen species (ROS) followed by Whatman filter paper (No. 1). The filtrate level: was evaporated to dryness. The crude leaf extract Microscopic fluorescence imaging was used obtained was stored in a refrigerator. to study ROS generation in HEp-2 cells after Antioxidant study exposure to different concentrations of extracts (8). DPPH radical scavenging assay After exposure, cells were incubated with DCFH- DPPH radical scavenging assay was carried DA (10 mM) as the fluorescence agent for 30 minutes out as per the method reported earlier, with slight at 37ºC. The reaction mixture was aspirated and modifications. Briefly, one ml of the test solution replaced by 200µl of PBS in each well. The plates (individual plant extracts) was added to an equal were kept on a shaker for 10 minutes at room quantity of 0.1 mM solution of DPPH in methanol. temperature in the dark. An inverted fluorescence After 20 minutes of incubation at room temperature, microscope with a CCD cool camera was used to the DPPH reduction was measured by reading the analyze intracellular fluorescence of cells. Increased absorbance at 517 nm. Ascorbic acid (1 mM) was intensity of intracellular fluorescence was indicative used as the reference compound (6). of increased level of generated ROS. Estimation of total phenolic content AO/EtBr staining The total phenolic content of Harpullia The AO (0.1 mg/mL) and EtBr (0.1 mg/mL) arborea was determined using the Folin-Ciocalteu were used to label nuclear DNA in HEp-2cells. Both reagent method of Wolfe et al., (7) with slight solutions were prepared in PBS buffer (pH 7.4). PBS modification. The 200 µl of extract was mixed with buffer was used to preserve normal physiological an equal volume of Folin-Ciocalteu reagent and activity for unicellular cells. For cell staining, HEp- incubated for10 minutes at 37 ºC. Then, 1.25 mL of 2cell samples (100 mL) were stained with aqueous sodium carbonate was added, and the AO/EtBr (5 mL) and observed under a fluorescence reaction mixture and incubated for 90 minutes at 37 microscope (9). ºC after addition of 1mL distilled water. The DNA fragmentation assay absorbance of the blue color was read at The DNA fragmentation assay was carried 760 nm spectrophotometrically using distilled water out based on the method of Sonia and Venkadasan (10) as a blank. Gallic acid is used as standard, and the with slight modification. After plant treatment, cells total phenolic content was expressed as were collected and washed with PBS at 4ºC. Then, mg/gm gallic acid equivalent (GAE). cells were centrifuged at 4500rpm for 3min. The obtained pellet was suspended with DNA lysis buffer Evaluation of cytotoxicity properties of methanolic and incubated on ice for One hr. After incubation extract of Harpullia arborea 20 µl of RNAse (20 mg ml-1) was added and Human laryngeal epithelial cancer cell line incubated for One h at 37ºC. Then, 20 µl of (HEp-2) was obtained from National Centre for Cell Proteinase K (20 mg /ml-1) was added. Follows, the Science (NCCS), Pune, India. Cells were placed into cell suspension were centrifuged at 12,000 rpm for 75 cm 2 tissue culture flasks and grown at 37ºC under 15min at 4ºC and the supernatant was discarded and a humidified, 5% CO2 atmosphere in Minimum transferred to sterile micro centrifuge tube. After, the essential medium (Eagle) supplemented with 10% DNA was precipitated with ice-cold absolute ethanol. Fetal bovine serum (FBS), 1% glutamine and Further, the precipitated DNA was washed with 70% 100µg/ml penicillin-streptomycin at 37 ºC in 5% CO2 ethanol and air dried for 20 min and eventually atmosphere. The HEp-2 oral cancer cells were seeded dissolved with sterile molecular grade water. Same in 96 well micro titer plates (5X103cells/well) and procedure followed for the control cells. The DNA of 0.1 mL of the test solution (7.8-1000µg) were added the sample (15.6µg/ml), control, and DNA ladder to the wells, with the plates kept in an incubator (5% were electrophoresed on an ethidium bromide CO2) at 37 ºC for 72 h. After 72 h, 20µl of MTT was containing agarose gel (1.5%). The gel was visualized added, and the plates were kept in the CO2 incubator and photographed in a gel documentation system. for two hours, followed by the addition Statistical analysis of propanol (100µl). The plates were covered with All experiments were performed in triplicate aluminum foil to protect them from light, and and all data was expressed at least three independent subsequently agitated in a rotary shaker for 10-20 evaluations, and the standard deviations (SD) were min, after wards the 27-well plates were processed on also calculated using Microsoft Excel 2008 software. an ELISA reader to obtain absorption data at 562 nm. Identification of bioactive constituents by GC-MS The percentage of cytotoxicity compared to the Samples of methanol extract were further untreated cells. All experiments were performed in analyzed the by Gas Chromatography — Mass triplicates. The morphology of the cells was then Spectrometry (GC-MS) to determine the species contained in the samples. The chemical components

Volume XII, Issue I, January/2020 Page No:1271 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367

were identified by matching their mass spectra with 85.14±6.52% at 1, 2, 3, 4 and 5mg/ml respectively. In those recorded in the mass spectral library. GC- the DPPH assay, the IC50 of ascorbic acid was 2.8 MS analysis was performed by using THERMO GC - mg/ml while that of the bark extract was 3.5 mg/ml TRACE ULTRA VER: 5.0 GC system, MS capillary (Fig 1). The scavenging ability of this extract was less standard non-polar column, flow rate of 1mL/min, than those of ascorbic acid. This present study carrier gas was helium, constant flow model, injector showed that the extract has proton donating ability temperature was 260ºC, injection volume was 1µL, and could serve as free radical inhibitors, possibly split injection technique, oven temperature was acting as primary antioxidants. In earlier report (4) programmed from 70ºC for two min with a found the antioxidant activity of leaf extract temperature increment rate of 6°C/min, and final of Harpullia arborea. Moustafa et al. (13) observed temperature of 280ºC for 2 minutes. Total running the DPPH radical scavenging potential from time was 30 minutes. two Harpullia species viz. H. cupanioides and H. Results and discussion pendula. Due to their diverse phytometabolic content The analysis of the cytotoxicity activity of with various biological activities, the use of herbal plant extract is important for safe treatment. The medicines in cancer treatment has gained growing antioxidant assay results further, directed the study attention in recent years. In the present study, plant towards cytotoxic assay. The results of was collected and identified according to their the cytotoxicity of a selected plant (methanol extract) taxonomical character as Harpullia arborea, which against the HEp-2cancer cells are summarized in Fig was analyzed for the determination of phytochemicals 2. Results were reported as the percent growth of the with two solvent extracts. treated cells when compared to the untreated control The Phytochemicals analysis of the methanol cells. It could also specify an indication of extract of Harpullia arborea revealed the presence possible cytotoxic properties of the tested plant of tannins, saponins, Steroids, Carbohydrate and extract of Harpullia arborea. MTT assay is based on Phenols. Whereas Flavonoids, Terpenoids, and the reduction of MTT by mitochondrial quinine were present in chloroform extract. The dehydrogenase and the entire extracts exhibit a presence of Tannin and Terpenoids were observed in gradual increase in antiproliferative activity with both extracts. In 2010, previous study also observed respect to dose dependent manner. the saponins and steroids from the seed of methanol The highest cytotoxicity of this extract extracts of Harpullia arborea(11). The obtained result against HEp-2cell was found in 200 and 100 µg/ml in this study is the first report on the bark extract concentrations with 97.58 and 88.92 percent of cell of Harpullia arborea. growth inhibition. It was found that the percentage of Among the different phytochemicals, growth inhibition to be reducing with increasing of phenolic compounds have been brought to the test compounds, and IC50 value of this assay was attention of various applications such as 15.6 µg/ml. According to a review of literature, no pharmaceutical, health, food, and cosmetic industries. one report about the anticancer activity of bark extract As part of our daily diet, these compounds are of Harpullia arborea against HEp-2 cells. Seham et abundant in the plant kingdom and attractive as al (14) reported that same genus of (Harpullia natural antioxidants(3), anticancer agents(12). In this cupanioides Roxb and Planch. Ex present study, the highest phyto constituents were F.Muell) plants were capable to active against various observed from methanol extract, therefore which was cancer cells. As far as we are aware, this is the first subjected for further study. Earlier reported that seed time to refer to the in vitro cytotoxic activity extract had saponins, resins, glycosides, and steroids of Harpullia arborea bark extract against the HEp- as the chemical class present in the extracts (11). 2cancer cell lines under investigation (Fig.3). Antioxidant activity plays a key role in The Fig. 4 was illustrated that plant treated reducing chronic diseases such as cancer and cancer cells stained with DCFH-DA become more cardiovascular disease (CAD). DPPH assay is based fluorescent with increased dosage indicative of on the ability of the stable free radical 2,2-diphenyl-1- significant intracellular ROS accumulation inducing picrylhydrazyl to react with hydrogen donors apoptosis. Cells treated with extract emitted bright including phenolics. The removal of DPPH solution fluorescence with cripple morphology because of increases linearly with increasing amount of extract in disturbance in the integrity of plasma membrane due a given volume. The DPPH method revealed that the to ROS generation. scavenging of the free radicals was found to be 10.2 Furthermore, Plant extract treated HEp-2 ±0.78%, 21.21±1.62%, 41.01±3.12%, 59.13±4.53% cells were subjected to AO/EB staining. AO stain are and 75.14±5.75% at 1, 2, 3, 4 and 5 mg/ml entered the nucleus and marks the nuclei green, respectively. and EB will penetrate the nucleus of dead cells, is The antioxidant capacity of the plant extract mainly taken up by the cells when membrane was correlated with ascorbic acid. The scavenging of integrity is lost and stains the nuclei red. Since the free radicals was found to be 15±1.14%, AO intercalates in the DNA but only interacts with 30.42±2.33%, 55.11±4.2%, 76.34±5.84% and the RNA, viable cells do not uptake EtBr and these

Volume XII, Issue I, January/2020 Page No:1272 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367

cells exhibit green nuclei. In the present study, early the data library. From the chromatogram peaks, found apoptotic cells were appeared as a granular yellow- different bioactive leads which include phenolic green with AO nuclear staining and necrotic cells compounds and fatty acid and the detailed of each revealed uneven, orange-red fluorescence at their compound was listed in Table 1. Among them, periphery without chromatin fragmentation (Fig. 5). following cancer inhibitory substance were observed In this study, DNA fragmentation assay was including catechol, 1, 2, benzene dicarboxylic acid, carryout with the plant extract for elucidate the and Phthalic acid. According to a review of literature mechanism of cells death. The DNA fragmentation no one was found the anticancer activity of catechol clearly demonstrates the role of apoptosis in plant from Harpullia arborea. treated cells. The DNA migrated as separate bands The catechol, also known as pyrocatechol, is which were compared to DNA markers and untreated a naturally-occurring compound found in fruits and cell. From this it is revealed that this DNA fragment vegetables such as onions, apples and olive oil (16 % shows that the methanolic extract of Harpullia 17). This was showed anti-cancer activity by directly arborea has anticancer activity in the HEp-2cell lines. binding with signaling molecules important According to previous studies, no one likes was in carcinogenesis. The previous report (18) of were reported that DNA fragmentation of HEp-2cell line inhibited the lung cancer cells and latterly (19) were with Harpullia arborea (Fig.6). reported the anticancer against Human HepG-2 From this study showed antioxidant cancer. However, no report has yet explained the anti- and cytotoxicity of tested plant extract against HEp- carcinogenic effects of catechol against HEp-2cancer 2cells, which indicate there could be cells. In view of this study, catechol may be inhibited some phyto compounds in this extract. Several the HEp-2 cells and exhibiting anti-cancer properties. authors reported that phenolic, acids, flavonoids, In conclusion, it was observed that the steroids, terpenoids are known to be bioactive plant of Harpullia arborea consists of a broad range principles. Baba and Malik (15) was reported that of secondary metabolites that hold robust antioxidant close relationship between total phenolic contents and and anticancer capability based on the experiments antioxidative activity of plants. In this present study, performed which add scientific evidence to conduct phenolic content was studied in the bark extract of in addition research, investigate the phyto compounds Harpullia arborea, where in the methanolic extract current in the plant, consider its anticancer achievable exhibited 35.20±0.14mg/gm GAE. on in vivo animal model and put forward a try to In the present study, GC-MS chromatogram carry out trails on human beings. of methanolic bark extract of Harpullia arborea Conflicts of interest showed number of peaks, which indicates the The authors declare that they have no competing presence of various compounds. The mass spectral interests. fingerprint of each compound can be identified from

Table 1 GC-MS analysis of Methanol bark extract of Harpullia arborea

Volume XII, Issue I, January/2020 Page No:1273 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367

100

80 60 Methanol 40 extract 20

% % ofactivity A.acid 0 1 2 3 4 5 Concentration of extracts (µg/ml)

Fig. 1 DPPH radical scavenging activity

110 100

90 80 70 60 50 40

% % of viability 30 20 10 0 3.12 6.25 12.5 25 50 100 200 Concentration of extract (µg/ml) Fig.2 In vitro Cytotoxicity of Methanolic bark extract of Harpullia arborea against HEp-2 Cell line

Fig.3 Morphological changes of HEp-2cell lines after MTT assay treatment. A- 200µg of extract, B - 50 µg of extract, C - 12.5 µg of extract, D - Control (Without plant treatment).

Volume XII, Issue I, January/2020 Page No:1274 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367

Fig. 4 DCFH-DA staining test for detection of MECD induced intracellular ROS level: Control - Healthy HEp2 cancerous cells without fluorescence (no ROS), Low- Extract treated cells with less fluorescence, IC50- Extract treated cells with more fluorescence.

Fig. 5 Hep2 cells showing red fluorescence indicated that they are undergoing apoptosis with Acridine Orange/Ethidium Bromide stain and control cells showed green fluorescence

Fig.6 DNA fragmentation of plant extracts, M- 1KB DNA marker, 1-Control cells, 2-Low concentration of extract, 3- IC50 Concentration References 3. C. Shridhar, Ghagane, I. Sridevi, Puranik, M. 1. Krishnaveni, K. Suresh, Induction of apoptosis Vijay, Kumbar, B. Rajendra, Nerli, S. Sunil, by quinine in human laryngeal carcinoma cell Jalalpure, B. Murigendra, Hiremath, line. Int.J.curr.Res. Acc.Rev 3(3):169-178 Shivayogeeswar Neelagund, Ravindranath (2015). Aladakatti. In vitro antioxidant and anticancer 2. R. Sanjay, Patel, P. Ashok, Suthar, M. Rajesh, activity of Leea indica leaf extracts on human Patel, In Vitro Cytotoxicity Activity of prostate cancer cell lines. Integr Med Res 6(1): Semecarpus anacardium Extract against HEp- 79–87(2017). 2Cell Line and Vero Cell Line. International 4. H. Raghavendra, T.R. Prashith Kekuda, Karthik, Journal of PharmTech Research 1(4):1429-1433 G.N. Ankith, Antiradical, antibacterial and (2009). antifungal activity of Harpullia arborea

Volume XII, Issue I, January/2020 Page No:1275 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367

(BLANCO) RADLK. (SAPINDACEAE). Int J absinthium L. extract. Cell Mol Biol 64(3):25- Curr Pharm Res 9(5): 32-36 (2017). 34 (2018). 5. T.R. Prashith Kekuda, K.S. Vinayaka, 13. S.M.A. Moustafa, B.M. Menshawi, G.M. Inhibitory activity of Harpullia arborea Wassel, K. Mahmoud, M.M. Mounier, (Blanco)Radlk. and Hydnocarpus pentandra Screening of some wild and cultivated Egyptian (Buch.-Ham.) Oken against seed-borne fungi. plants for their free radical scavenging activity. Journal of Medicinal Plants Studies 5(4): 114- Int J PharmTech Res 6:1271-8 (2014). 117 (2017). 14. M.A. Seham, Moustafa, M. Bassem, Menshawi, 6. Rafik Shaikh, Mahesh Pund, Ashwini Dawane, M. Gamila, Wassel, Khaled Mahmoud, Marwa, Sayyed Iliyas. Evaluation of Anticancer, M. Mounier, Screening of some Plants in Egypt Antioxidant, and Possible Anti-inflammatory for their Cytotoxicity against four Human Properties of Selected Medicinal Plants Used in Cancer cell lines. Int.J. PharmTech Res Indian Traditional Medication. Journal of 6(3):1074-1084 (2014). Traditional and Complementary Medicine 15. S.A. Baba, A.S. Malik, Determination of total 4:253-257(2014). phenolic and flavonoid content, anti-microbial 7. K. Wolfe., X. Wu, R.H. Liu. Antioxidant and antioxidant activity of a root extract of activity of apple peels. Journal of Agricultural Arisaema jacquemonti Blume. J. Taibah Univ. and Food Chemistry 51:609-614(2003). Sci. 9, 449–454 (2015). 8. M. Ahamed, D. Ali, H.A. Alhadlaq, M.J. 16. N. Schweigert, A.J. Zehnder, R.I. Eggen, Akhtar, Nickel oxide nanoparticles exert Chemical properties of catechols and their cytotoxicity via oxidative stress and induce molecular modes of toxic action in cells, from apoptotic response in human liver cells microorganisms to mammals. Environ (HepG2) Chemosphere. ; 93:2514–2522 (2013). Microbiol 3:81–91 (2001). 9. C. Ciniglia, G. Pinto, C. Sansone, A. Pollio. 17. M. Brenes, C. Romero, A. Garcia, F.J. Hidalgo, Acridine orange/ Ethidium bromide double M.V. Ruiz-Mendez, Phenolic compounds in staining test: A simple In-vitro assay to detect olive oils intended for refining: formation of 4- apoptosis induced by phenolic compounds in ethylphenol during olive paste storage. J Agric plant cells. Allelopathy Journal ;26 (2):301-8 Food Chem 52:8177–8181(2004). (2010). 18. Do Young Lim, Seung Ho Shin, Mee-Hyun 10. Sonia Sarkar and Venkatesan Kotteeswaran. Lee, Margarita Malakhova, Igor Kurinov, Qiong Green synthesis of silver nanoparticles from Wu, Jinglong Xu, Yanan Jiang, Ziming Dong, aqueous leaf extract of Pomegranate (Punica Kangdong Liu, Kun Yeong Lee,Ki Beom Bae, granatum) and their anticancer activity on Bu Young Choi, Yibin Deng,Ann Bode, and human cervical cancer cells. Adv. Nat. Sci.: Zigang Dong, A natural small molecule, Nanosci. Nanotechnol. 9:1-10 (2014). catechol, induces c-Myc degradation by directly 11. Shyamala and Vasantha. Solvent Based targeting ERK2 in lung cancer. Oncotarget Effectiveness of Antibacterial and 7(23): 35001–35014 (2016). Phytochemical Derivatized from the Seeds of 19. H.T. Abdel-Mohsen, J. Conrad, K. Harms, D. Harpullia arborea (Blanco) Radlk. Nohr, U. Beifuss, Laccase-catalyzed green (Sapindaceae. J Appl Sci Environ synthesis and cytotoxic activity of novel Management13:99-101 (2010). pyrimidobenzothiazoles and catechol thioethers. 12. Koyuncu, Evaluation of anticancer, antioxidant RSC Advances 7:17427-17441 (2017). activity and phenolic compounds of Artemisia

Volume XII, Issue I, January/2020 Page No:1276