Identification and Quantification of Quercetin, a Major ...with cancer and 6.7 million of them are dying of cancer every year. Parkin DM [2] reported that a gradually increasing ratio

Research ArticleVolume 6 Issue 3 - September 2017DOI: 10.19080/AIBM.2017.06.555690

Adv Biotech & MicroCopyright © All rights are reserved by Jalal TK

Identification and Quantification of Quercetin,a Major Constituent of Artocarpus altilis byTargeting Related Genes of Apoptosis and Cell Cycle: in vitro Cytotoxic Activity against Human Lung Carcinoma Cell Lines (A549)

Jalal TK*Department of Biomedical Science, International Islamic University Malaysia (IIUM), Malaysia

Submission: June 12, 2017; Published: September 27, 2017

*Corresponding author: Jalal TK, Department of Biomedical science, International Islamic University Malaysia (IIUM), Kulliyyah of Allied Health Sciences, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia, Email:

IntroductionCancer is an irregular growth of cells caused by genetic

alterations that lead to the deregulation of cell proliferation and cell death. Sridharan V [1] reported that cancer is a genetic disorder of the cancerous cells. Cancer is initiated by external causes (tobacco, infectious organisms, chemicals, and radiation) and internal causes (inherited mutations, hormones, immune conditions, and mutations originating from metabolism). In 2000 approximately 10 million people were diagnosed with cancer when the global population was approximately 6 billion which 5.3 million patients were men and 4.7 million were women. In 2014 approximately 176,000 of the estimated 585,720 cancer deaths were caused by tobacco consumption. According to the World Cancer Research Today 24.6 million people are living with cancer and 6.7 million of them are dying of cancer every year. Parkin DM [2] reported that a gradually increasing ratio

of older people in the world will result in approximately 50% increase in new cancer cases over the next 20 years. More men than women suffer from the cancer of the lung, stomach, throat and bladder. In wealthier countries, prostate, breast and colon cancers are more common than in poor countries [3]. External factors such as tobacco consumption, exposure to chemicals and radiation, infectious organisms and internal factors such as inherited mutations, hormones, and immune status can cause cancer. These dangerous causes may act together or in sequence to initiate or promote carcinogenesis. The American Cancer Society reported that more than 175, 000 cancer deaths were caused by tobacco the consumption in year 2005 [4]. A study in 2015 reported that approximately 14.1 million incidences and 8.2 million mortality related to cancer occurred in 2012. Globally, cancer (lung and breast) has been in top frequently diagnosed

Adv Biotech & Micro 6(3): AIBM.MS.ID.555690 (2017) 0065

Abstract

Nine phenolic compounds were identified and quantified in Altilia A fruit. One of the main compounds was quercetin, which is the major class of flavonoids which has been identified and quantified in pulp part of A. altilis fruit of methanol extracts. The aim of this study was to evaluate the in vitro cytotoxic assay study of the flavones: quercetin, on A549 (human lung carcinoma cells). Furthermore, the pulp of the fruit was down regulated the expression of anti-apoptosis gene BCL-2 and up-regulated the expression of pro-apoptosis gene BAX. CASPASE-3 was also activated by the fruit which started a CASPASE-3-depended mitochondrial pathway to induce apoptosis. It was also found that the pulp of methanol extracts was arrested the cells in G0/G1 and G2/M phases. As the results, the pulp was the most active in terms of all tests due to high amount of quercetin in pulp part 78% of total flavonoids. Taken together, these findings suggested that A. altilis induces apoptosis in a mitochondrial-dependent pathway by releasing and up-regulating CYTOCHROME C expression and regulates the expression of downstream apoptotic components, including BCL-2 and BAX. Pulp part of the methanol extracts can be a potent and promising medicine as anticancer agent.

Keywords: Quercetin; Apoptosis; Cell cycle; CASPASE- 3

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How to cite this article: Jalal TK. Identification and Quantification of Quercetin, a Major Constituent of Artocarpus altilis by Targeting Related Genes of Apoptosis and Cell Cycle: in vitro Cytotoxic Activity against Human Lung Carcinoma Cell Lines (A549). Adv Biotech & Micro. 2017; 6(3): 555690. DOI: 10.19080/AIBM.2017.06.555690

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Advances in Biotechnology & Microbiology

cancer for both man and woman respectively [5]. Thus noticing the global pattern, this study choose lung and breast cancer to understand the effects of porcupine bezoar in using both cancer cells as a model. In the lung cancer study in 2015 incidence reported as the second leading in male (14%) and female (13%). In 2016, though the percentage rate of incidence retains but the cases had increased by 2310 for man and 880 cases for woman [6]. It seems the lung cancer mortality rate was highest for male and female for both years. In 2015 the mortality rate of lung was 28% (total case of 312 150) for male and 26% (total case of 277 280) for female. The rate was decreased in 2016 for male by 1% (total case of 314 290) while for female remains 26% (total case of 281400) [6]. It has been discovered that flavonoids have many beneficial actions on body cells by enhancing the activity of many enzyme systems, effective in inflammation, arteriosclerosis, bleeding, allergy and swellings. It is also known to be associated with reduced risk of certain types of cancers. However the major problem associated with the use of quercetin are the very low bioavailability biological activities of flavonoids such as anti-allergic, anti-toxic, anti-microbial, anti-cataract and anti-cancer.

Materials and MethodsMaterials

The plant sample was collected from Taman Pertain Sultan Haji Ahmad Shah, Kuantan and Pahang. The parts of A. altilis fruits were prepared for extraction by washing off all dirt and soil residues then, peel off all the skin. Cut the pulp to even parts. Then the dried pulp of A. altilis were ground to a fine powder and then stored in a cold room at 4 °C until further analyses. The ground plant materials were extracted using a Soxhlet apparatus according to Akbar E et al. [7]. A sample mass of 250g pulp of Artocarpus altilis were extracted by filling the absorbent cellulose thimble and placing it in the thimble chamber of the Soxhlet apparatus. Three solvent systems (hexane, dichloromethane and methanol) were used for 12-18h in sequence in order of increasing polarity to obtain three different types of extracts i.e. hexane extract of pulp; DCM extract of pulp and methanol extract of pulp. In the current study only the methanol extract of the pulp going to be used. The solvent was removed by rotary evaporation at 60 °C in vacuo.

Cell line and cell cultureHuman lung carcinoma (A549) was obtained from American

Type Culture Collection (ATCC). The preparation of complete growth media (CGM) used were Dulbecco’s modified Eagle medium (DMEM), L-glutamine supplemented with 10% fatel bovine serum (FBS) and 100U/ml penicillin, 100mg/ml streptomycin. Media were liquated into 50ml centrifuge tube kept in 4 °C and pre-warmed to 37 °C prior to use. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C and 95% air. All handling processes of the cells were performed under strictly aseptic techniques inside the class II bio safety cabinet and performed as rapidly as possible to minimize contamination.

Determination of inhibitory concentration 50% (IC50)A stock solution of test sample (100mg/ml) was prepared by

dissolving tested sample of pulp of methanol extracts in DMSO (0.1g/1000µl). To minimise the DMSO effect in the sample, 1mg from the stock solution (100mg/ml) was prepared by aspirating 10µl from the stock and dissolved into 990µl of complete culture medium. It was then gently shaken using vortex shaker. This 1mg sample is the working solution and is kept at 4 °C until use. It is preferable to prepare these fresh at the time of the experiment. Setting approximately 5 x 104 cells per well, in the 6-well plates of A549 cell lines. After 24h, the partial monolayer was formed; the supernatant was cleared off, washed 1-2 times with PBS (Gibco BRL, UK). Serial dilutions of 1mg solution were prepared in the culture medium in 6-well plates ranged between 12.5-200µg. Table 1 shows the working solution concentrations of methanol pulp extract solution which was freshly prepared prior to use for further assays. Test sample at the appropriate concentrations was added to individual wells as mentioned, while only adding complete DMEM growth medium for the control (untreated no extracts added). As for screening a series of concentration range from higher to lower concentration of the extract of pulp of A. altilis were carried out in triplicate. The plates were then incubated at 37°C in 5% CO2 incubator for 72h [8]. After 72h incubation the medium was aspirated, the cells harvested and the IC50 concentration was determined using TBEA method briefly using a haemocytometer which is a device used to count cells with a special type of microscope slide consisting of two chambers by uploading 10µl of diluted cell suspension (10µl) is a mix of try pan blue and cell suspension (1:1) [9]. The experiment was repeated in triplicate and analysed using GraphPad Prism 6.01 software.

Table 1: List of concentrations of pulp extracts used in this study.

Volume that Added to the Well from Stock Solution

(1mg/Ml)

Volume of CGM/Well/µl

Final Concentrations

(µg/Ml)

400 1600 200

200 1800 100

100 1900 50

50 1950 25

25 1975 12.5

Identification and quantification of phenolic compounds using UHPLC-MS/MS

Analyses of the photochemical components of A. altilis of pulp part was performed on an AB Sciex 3200QTrap liquid chromatography-tandem mass spectrometry (LCMS/MS) (AB Sciex, Toronto, Canada) coupled to Perkin Elmer Flexar FX15 ultra-high-performance liquid chromatography (UHPLC) system (Massachusetts, USA), operated by AB Sciex analyst software. Reference standards were also used for comparison. The retention times of the extract with those of the reference standards were compared for further confirmation and



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identification. The external standard method was used for the quantification of the individual phenolic compounds. Contents of 9 phenolic compounds such as quercetin, rutin, ascorbic acid, p-coumaric acid, ferulic acid, gallic acid, 4-hydroxybenzoic acid, protocatechuic acid and sinapic acid, were calculated with the regression equations from the standard curves. The standard calibration curve of each of the standard was used to quantify the amount of phenolic compounds present in the various crude extracts.

Flow cytometric analysis of apoptosis and cell cycleIn order to determine the level of apoptosis in cells, Guava

Nexin reagent staining, a pre-made cocktail containing Annexing V-PE and 7-AAD in buffer. All adhering and floated cells were harvested and pipette 100µl of each sample into appropriate well using 96-wells then 100µl Guava Nexin reagent was added to each well, incubated for 20 minutes at room temperature in the dark. Sample was acquired on Guava flow cytometry system. The results were analysed using guava Incited software, version 2.7 which 2000 cells were collected for the analysis. While for cell cycle after cells were harvested and transferred to sterile centrifuge tube, then discard the supernatant and washed with PBS centrifuge the supernatant was removed and the pellet resuspended with 70% cold ethanol (400µl) and kept at -20 °C for 1-2h. The cells were washed with PBS, centrifuge and discard the supernatant. Then 200µl of PI kit (containing 1mg/ml RNAse) was added to the cell pellets and incubated in the dark for 30minutes at room temperature. The cells were then analysed by Guava flow cytometry, 5000 cells were collected for analysis using guava Incited software, version 2.7.

Quantitative real-time PCR Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis RNA extraction and CDNA preparation

The process of exerting RNA and converting to CDNA were carried out according to manufacturer’s instructions. Total RNA was isolated from selected treated and untreated cell lines using innuPREP RNA Mini Kit (Konrad-Zuse-Strasse, Germany) after the appropriate drug incubations for 72h of both control and treated cells. Total RNA 100ng was used for CDNA synthesis in a final volume of 20μl by using reverse transcription system Sense FAST™ bioline kit, performing the CDNA step by setting in the thermal cycler. The thermal cycler was fixed up at the following program: 25 °C for 10min (primer annealing); for the reverse transcription, the program set up was at 42 °C for 15min; for inactivation it was set at 85 °C for 5min; and finally set at 4 °C to keep the product cold. Using the comparative CQ method, mRNA expression levels of target genes were normalized to the expression of glyceraldehydes phosphate dehydrogenase (GAPDH) and β-action (ACTB) as endow genes.

Statistical analysisData of replicates were analysed using a one way ANOVA

analysis and were expressed as means± standard error (SE) mean of the triplicates. Statistical differences between the

reference and the sample groups were evaluated by ANOVA (one way) using LSD (least significant differences) test p <0.05 using SPSS 20.0 software. And used for calculating IC50 Graph pad prism version 6.01.

ResultsDetermination of pulp of methanol extracts that effectively inhibits 50% of A549 cells

Figure 1 shows the graph obtained by the log value of the agonist concentration (in M) versus the percentage of cells viability as compared to the untreated cells to determine the IC50 value of the methanol extracts on A549 cells, following incubation for 72h. The IC50 of the pulp of the fruit extract was found to be 23.10±0.71µg/ml.

Figure 1: Concentration-dependent effect of methanol extracts of pulp part on the percentage of cells viability of A549 cells. Data presents the mean±standard error mean (S.E.M.) of mean of triplicate analyses of two independent experiments (n = 6).

Identification of various phenolic compounds in A. altilis of pulp extractusing uhplc-Ms/Ms

The identification of phenolic compounds was classified into flavonoids and phenolic acids. Two of the compounds were identified as flavonoids and seven as phenolic acids.

Flavonoids: Flavonoids have attracted widespread attention recently because of their broad range of beneficial effects on human health. The best-described characteristics of almost every group of flavonoids are their ability to remove free radicals and inhibit other oxidation reactions [10]. Compounds belonging to various flavonoid classes (quercetin and ruin) were detected in A. altilis samples of pulp analysed. Peak 1, (Figure 2) presented spectral characteristics of quercetin [11]. Quercetin’s spectral characteristics are: US-via spectrum at 360nm, M-H at m/z 301 and fragment ion at m/z 151 (Table 2). The standard chromatograph of quercetin is shown in Figure 2. Lepley DM [12] and Sergediene E et al. [13] reported that numerous phenolic compounds have been shown to exhibit ant proliferative and cytotoxic effects on several tumour cells, and presented toxic effects that specifically target cancer cells rather than normal cells. Quercetin is a powerful antioxidant, and the most abundant in dietary flavones. It is found in abundance in fruits, vegetables, beverages and berries [14]. This compound was said to have very powerful chemo preventive and chemotherapeutic (anticancer)



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potential Ko WG et al. [15] Kamaraj S et al. [16] reported quercetin’s anticancer properties against benzo (a) pyrene- induced lung carcinogenesis in mice which was attributed to its free radical scavenging activity. Peak 2 Figure 2 was identified as rutin based on its M-H at m/z 609 and fragment ion at m/z 301 which was also previously reported by [17]. They also provided

the reference chromatogram of rutin Figure 2. Rutin is found in citrus fruits [18] and has been associated with potential health benefits. Additionally, aside from their antioxidant properties, the flavonoids are powerful agents against chronic diseases such as cardiovascular diseases, atherosclerosis, and malignancies [19-21].

Figure 3: Apoptosis induced by A. altilis fruit extracts on A549 cells. Cells were treated with a concentration of IC50 for 72h. Data presents as the mean±standard error mean (S.E.M.) of mean of triplicate analyses. Different letters indicate significant differences between treated cells upon IC50 of methanol extracts and relative respective control of each group (untreated) at p < 0.05.



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Figure 2: The chromatograms of phenolic compounds presented in pulp part of A. altilis methanol fruit extracts.

Table 2: Identification of phenolic compounds in Altilis A RT: Retention Times; UV: Ultraviolet; M-H: parent ion; MS2: Fragment Ions.

Compound Identity Standard RT (Min) UV (Nm) M-H(M/Z) MS2 (M/Z)

Quercetin 4.2 360 301 151

Ferulic acid 3.55 232 193 178

4-Hydroxybenzoic acid 2.9 280 137 93

Sinapic acid 3.57 323 223 193

Protocatechuic acid 2.53 219 153 109

Rutin 3.38 255, 352 609 301

p-Coumaric acid 3.34 223, 309 163 119

Gallic acid 2.06 271 169 125

Ascorbic acid 1.31 265 175 87

Phenolic acids: Phenolic acids (p-coumaric acid, ferulic acid, gallic acid, 4-hydroxybenzoic acid, protocatechuic acid, sinapic acid and ascorbic acid) were the most identified in A. altilis extracts. Figure 2 showed peak 3 in with M-H at m/z 193 and fragment ion at m/z 178 (Table 2) were identified as ferulic acid according to the mass spectral library and reference standard. In addition the mass spectrum was in agreement with those reported by Gómez-Romero M et al. [22]. Paiva LBD et al. [23] reported that ferulic acid-rich dates has been shown to have antioxidant, anti-microbial, anti-inflammatory, hepatoprotective, neuroprotective, ant carcinogenic, anti-diabetic and anti-cholesterolemic properties. The MS Peak 4 values at m/z 137 (M-H) and UV-via spectrum 280nm unambiguously identified it as 4-hydroxybenzoic acid. This is based on a similar compound reported in Taraxacum Formosa num, a Chinese medicinal herb grown in Taiwan [24]. The retention time and fragment ion (m/z 93) were also identical to those of 4-hydroxybenzoic acid standard. In a study by Seidel C [25], 4-hydroxybenzoic acid did not only arrest cell cycle progression but also triggered apoptotic cell death in cancer development. Peak 5 is shown in Figure 2 was characterized as sinapic acid based on UV-via spectrum

at 323 nm and M-H at m/z 223 [26]. In addition the fragment ion at m/z 193 was in agreement with a reference standard. This compound is one of the biologically active components of many fruits, vegetables, cereal grains, medicinal plants, and spices. Sinapic acid had been reported for its various biological activities such as anti-inflammatory [27], antibacterial [28] and anti diabetic [29] activities. It has been shown to possess anticancer effects in different cancer cell lines such as anti proliferative, anti apoptotic properties and is also able to arrest the cell cycle [29]. Peak 6 was identified as a protocatechuic acid. This compound was previously detected in abundance in edible fruits and vegetables and is thus one of the anti oxidative components of normal human diet [30]. Protocatechuic acid isolated from the Chinese herb Salviamiltiorrhiza played a crucial role against inflammatory cytokines of atherosclerosis. Hu L [31] reported that protocatechuic acid has the ability of inhibiting both the invasion and metastatic potential of malignant carcinoma cells. Peak 7 is shown in Figure 2 showed a mass spectral characteristic of p-coumaric acid having M-H at m/z 163 and fragment ion at m/z 119. Some identified phenolic acids including p-coumaric, ferulic and sinapinic acids have been



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earlier shown to inhibit the growth of some cancer cell lines [32-34]. The peaks were identified as p-coumaric acid and was confirmed by comparing these values with that of the standard p-coumaric acid. Peak 8 show M-H at m/z 169 and fragment ion at m/z 125. These were identified as gallic acid [26]. Gentisic acid, p-coumaric acid, ferulic acid and gallic acid elicited a significant increase in the activities of several antioxidant enzymes such as glutathione peroxidise (Gap), superoxide dismutase (SOD) and catalyse (CAT) in rats [35]. By comparing the UV-via spectrum at 265nm, M-H at m/z 175 and fragment ion at m/z 87 with those reported in literature [36], ascorbic acid peak 9, was identified. It is a naturally occurring organic compound with a powerful antioxidant properties and immune response activation. It is involved in wound healing and ontogenesis. It can be found in fresh vegetables and citrus fruits [37].

Quantification of phenolic compounds In A. altilisCalibration curves were generated from the reference

standards after the identification of phenolic compounds in the extracts. Linear regression equation obtained from the calibration curve of each standard was used for the quantification of phenolic compounds of A. altilis extracts. Concentrations of individual and total phenolic compounds (expressed in mg

per kg dry weight), linear regression equations and regression coefficients R2 are presented in Table 3 Concentrations (mg/kg dry weight), regression equations and regression coefficients R2 of phenolic compounds in A. altilis extract. Flavonoids varied from 12.20 to 43.20mg/kg dry weight for pulp. The range for phenolic acids in the present study was from 0.17 to 11.40mg/kg dry weight for pulp (Table 3). Quercetin was the most concentrated and abundant flavonoid in pulp amounting to 78.00% followed by MW and ML at 40.96% and 32.47% of the total flavonoids concentration, respectively. Rutin was (20.02%) of the total flavonoids concentration. Protocatechuic acid was the most concentrated phenolic acid present in plup part which accounted for 43.36% of the total phenolic acids. Followed by p-coumaric acid in pulp was 31.70% of the total phenolic acids concentration. Ferulic acid was 4.09%. Both the sinapic acid and ascorbic acid were the least components found in pulp part (1.06% and 0.41%). Gallic acid was at 4.00%. While the percentage of 4-hydroxybenzoic was 5.93%. These results indicate that the composition of phenolic compounds can vary between different parts of the same fruit. To date, this is the first report that investigates crude extracts of A. altilis fruit showing important bioactive compounds and the analysis of pulp of methanol extract of A. altilis fruit using LC-MS UHPLC.

Table 3: Concentrations (mg/kg dry weight), regression equations and regression coefficients (R2) of phenolic compounds in A. altilis extract.

Concentrations

Compound pulp Regression equations R2

Flavonoids

Quercetin 43.2 Y = 4.04e+006 x +4.38e+005 0.9987

Rutin 12.2 Y = 8.64e+005 x + 4.73e+004 0.9997

Total 55.4

Phenolic acids

Ferulic acid 3.29 Y = 2.09e+006 x + 2.32e+005 0.999

4-Hydroxybenzoic acid 1.56 Y = 7.81e+006 x + 2.19e+006 0.9995

p-Coumaric acid 8.33 Y = 5.27e+006 x + 1.01e+006 0.9997

Protocatechuic acid 11.4 Y = 5.69e+006 x + 1.12e+006 0.9996

Sinapic acid 0.28 Y = 9.86e+005 x + 6.38e+004 0.9997

Gallic acid 1.24 Y = 3.53e+006 x + 4.86e+005 0.9987

Ascorbic acid 0.17 Y = 5.03e+005 x + 6.64e+004 0.9984

Total 26.27

Altilis induced apoptosis studied by nexin stainingTo determine whether cytotoxic effect of A. altilis was due to

apoptotic induction, methanol extracts using IC50 were induced early and late apoptosis at 72h Figure 3. The percentages of both early and late apoptosis were induced by pulp part of the fruit on A549cell line for early apoptosis 22.98±0.10 and 32.26±0.26% compared to untreated cells 3.22±0.11% at p <0.05.

Effects of A. altilis extracts on cell cycle activity arrest on A549 cells upon treatment with IC50 at 72h

Figure 4 show the cell cycle arrest of A549 cells at 7h. At 24h the treated cells showed some decrease in cells population at

G0/G1 and S phases 38.01±0.56 and 8.75±0.24% compared to untreated cells 48.21±0.13 and 16.50±0.52%, respectively, at p < 0.05. While at G2/M phase the cells showed increment mediated by pulp 49.43±0.72%, compared to untreated cells 33.72±0.26%.

Induction of apoptotic and cell cycle gene expressions in a549 cells by A. altilis methanol extract

Critical exposure of the methanol extract of AltaVista A A549 cells moulded different expression profiles for apoptotic and pro-apoptotic genes (CASPASE-3, 8, 9, BAX, BCL-2, FAS-L, CYTOCHROME C and cell cycle gene (p21, CYCLIN-A1, CYCLIN-B1 andCDK1) mRNA levels. As shown in Figure 5 the mRNA level of



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CASPASE-3 and 8were induced at highest level than other genes and also as compared to the control as consider is one < 0.05. Figure 5 shows the expression level of methanol extracts of pulp induced apoptotic signals via intrinsic pathway on A549 cell lines by up regulating FAS-L and CASPASES-8 with 20.44±1.17 and 36.23±3.41. As can be observed from the results a high fold ratio was obtained by pulp giving opportunity for mitochondrial pathway via up regulation and release of CYCS1 of 29.70±2.80. This in turn up regulated CASPASE-9 to 21.59±0.60. Whereas the relationship of the CASPASE-3 and P21 is very evident, as can be observed from the results that when the ratio expression level of CASPASE-3 is high the p21 also high 27.81±2.65 and 36.23±3.41 CASPASE-3 and p21, respectively. As observed from the results the fold changes of the genes’ expression up regulated due the exposure to the treatment of IC50 for 72h. The pulp arrested the A549 cells at G2/M by down regulating the CYCLIN B1 and its kinas CDK1. P21 is able to influence and block this binding process at G2/M checkpoint by interfering with the CDK1/CYCLIN B1, Figure 5. Figure 6 presents the proposed schematic representation of the death signals of A549 cells after the

treatment. It followed the extrinsic pathway via activation of the death receptor FAS-L over CASPASE-8 then cleaves BID into tBID to initialize the mitochondrial pathway activating the BAX then release the CYTOCHROME C activation of CASPASE-9 and finally CASPASE-3 [6].

Figure 3a: Effects on apoptosis induction of A549 upon treatment of IC50 of A. altilis at 72h. A (untreated) cells and B (treated) cells.

Figure 3: Apoptosis induced by A. altilis fruit extracts on A549 cells. Cells were treated with a concentration of IC50 for 72h. Data presents as the mean±standard error mean (S.E.M.) of mean of triplicate analyses. Different letters indicate significant differences between treated cells upon IC50 of methanol extracts and relative respective control of each group (untreated) at p < 0.05.

Figure 4a: Effects of cell cycle arrest of A549 cells upon treatment of IC50 of A. altilis at 72h. A (untreated) cells and B (treated) cells.



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Figure 4: Cell cycle arrested by A. altilis fruit extracts upon treatment of IC50 on A549 cells for 72h. Data presents as the mean±standard error mean (S.E.M.) of mean of triplicate analyses. Different letters indicate significant differences between treated cells upon IC50 of methanol extracts and relative respective control of each group (untreated) at p < 0.05.

Figure 5: Real-time PCR analyses of selected genes that were modulated in A549 cells by pulp methanol extract treatment according to cDNA. The effects of fruit part on the expression levels of apoptosis genes FAS-L, CASPASE-3, CASPASE-8, CASPASE-9, BAX, BID, BCL2, CYCS1, and cell cycle genes p21, CYCLIN A1, CYCLIN B1, CDK1 and CDK2 are shown. Total RNA samples were isolated from A549 cells treated with IC50 µg/mL of pulp methanol extract for the 72 h. The modulations of mRNA expression levels were expressed as fold change based on calculation using GAPDH and ACTB as HKGs, assigning the ratio in untreated cells as 1. Data are presented the mean±standard error mean (S.E.M.) of mean of triplicate analyses of NRQs. *Indicate significantly different from control at p < 0.05.



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Figure 6: Proposed schematic model for apoptosis induction and cell cycle regulation in A549 cells affected by pulp extract for 72h.

DiscussionThe impact of new and novel agents from potential bioactive

plants or their extracts for disease treatment and prevention is still massive, even though the research contribution by synthetic chemistry and chemists, as a method of drug discovery and drug manufacture is intense [38]. Many studies have focused on a new strategy in the fight against cancer due to the chemo protective possibilities of plants having ant carcinogenic properties. Photochemical, including specific phenolic compounds from plants, seems to play a significant role in suppressing all three stages of tumour formation, i.e. initiation, promotion, and progression [39]. Natural products show promise for cancer chemoprevention using medicinal plants. These medicinal plants provide non-cytotoxic nutrients or pharmacological agents to enhance the physiological mechanisms that protect the organism against mutant clones of malignant cells [40]. Earlier studies on Artocarpus plants have shown various responses to extracts. Wang Y [41] reported that five greenly dihydrochalcones from the ethyl acetate extract of A. altilis leaves had cytotoxic effects on some human cancer cell lines, such as human lung adenocarcinoma (SPC-A-1 cells), human colon carcinoma (SW-480 cells), and human hepatocellular carcinoma (SMMC-7721 cells). Plant extracts and the bioactive compounds present in them which are responsible for anticancer activity have to be screened for valuable information and classification into various classes of anticancer agent. Natural photochemical can be categorised into arytenoids, phenolics, alkaloids, nitrogen-containing compounds and organosulfur compounds. These photo chemicals are responsible for various pharmacological actions like antioxidant, and anticancer properties [42,43]

reported that quercetin have been known to induce strong anti proliferative properties of a wide range of cancers such as prostate, cervical, lung, skin, stomach and colon cancer cells in three leukemic cell lines (CEM, K562 and Nalm6) two breast cancer cell lines (T47D and EAC) compared to other tested polyphones [44-47]. As reported earlier (Table 2) (Figure 2) pulp contained high percentage of quercetin and is considered the major compound 78% of total flavonoids. Thus, pulp promotes more activity towards selected cell lines. Results show pulp contain rich flavonoids mainly quercetin. The presence of a hydroxyl group for the methoxy group at the meta position of the phenyl ring significantly enhanced the proliferative activity [48]. The second major compound that was identified and quantified in pulp was proto catechuic acid (phenolic acids) which had been previously shown to inhibit the growth of some cancer cell lines such as human breast cancer MCF-7 cells, lung cancer A549 cells, HepG2 cells, cervix HeLa cells, and prostate cancer LINPAC [49,50] reported that p-coumaric acid (a naturally occurring bioactive phenolic acids in fruits) the third major compound that was identified and quantified in pulp 31.70% of the total phenolic acids concentration was found to induce cytotoxicity and increased anti proliferative activity against human colon cancer cell HT-29, HCT116 and HCT-15, cervix HeLa cell, breast cancer cells MDA-MB 468 and MCF-7. From the results it can be concluded that the pulp part has potential to be used in the treatment of cancer. Apoptosis is a physiological procedure to maintain homeostasis in healthy tissue and suppression of damaged cells. There are many chemo preventive agents, which result in cancer cell death by induction of apoptosis [51]. In this section flavonoids induced cellular cytotoxicity through apoptotic signalling, cell cycle regulation mechanisms by one of



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the major classes of flavonoids, i.e. quercetin. FITC Annex in V staining follows the loss of membrane integrity which attends the latest stages of cell death resulting of the apoptotic processes. Thus, staining with FITC Annex in V is normally used in combination with a vital dye such as presidium iodide (PI) or 7-amino-actinomycin (7-AAD) to allow the investigator to detect early apoptotic cells. In the present study, the effect of A. altilis fruit extract on apoptosis was distinguished from living cells with early apoptotic cells by Annex in V-FITC+/7-AAD- and late apoptotic cells by Annex in V-FITC+/7-AAD+ on selected cancer cell line in a time dependent manner. Acquired data results were displayed on the computer screen in a dot-plot format with a user-controlled quadrant marker (Figure 3a). This was further confirmed by a flow cytometric analysis using annex in V and 7-amino-actin (7-AAD) double staining. Recent report by Sun NJ [52] mentioned that quercetin induced apoptosis in both the human breast cancer (SK-BR-3) and the human epidermis carcinoma (A-431) cell lines with concentrations starting with 50 and 75μg/ml. While in gastric cancer cell lines (BGC-832) the concentration was 90 or 120μg/ml. Quercetin led to increase in CASPASE-3 and BAX and reduction in BCL-2 expression [53]. Numerous mechanisms can explain the ability of quercetin to induce apoptosis. It has been reported that apoptotic effect of quercetin in colon and breast cancer cells was allied to the ability to generate reactive oxygen species which in turn could lead to DNA damage resulting in cell death [54]. As stated that quercetin induced G0/G1, G2/M and S phases arrest followed by apoptosis in three leukemic cell lines (CEM, K562 and Nalm6) two breast cancer cell lines (T47D and EAC) and colorectal carcinoma [55-57]. Previously reported in this study that contain of quercetin in pulp 78%. Hartwell LH [58] reported that the induction of apoptosis might be due by the inhibition of DNA replication caused by the inability of the cells to replicate damaged DNA caused by the plant extracts [59,60]. Reported that when cells arrested at G2/M checkpoint and prevent entry into mitosis due to incomplete replication of DNA; it has been shown that quercetin treatment caused cell cycle arrests such as G2/M arrest or G0/G1 arrest in different cell types [61]. Demonstrated that A549 cells were arrested at G2/M phases by pulp, which was associated with induction of p21 and controlled by decrease in the expression of downstream cell cycle regulatory proteins [62] mentioned that type of phenolic acids causes cells arrest at G2/M such as quercetin, gallic and ferulic acids. As chemo preventive agents, flavonoids are active at different stages of cancer development, interfering with the overall process through various mechanisms such as up/ down regulation of apoptotic and cell cycle genes. Flavonoids act to inhibit the activity of DNA topoisomerase I/II [63], release of CYTOCHROME C from mitochondria and following activation of CASPASES-3, 8 and 9 [64,65] down regulate BCL-2 expression and up regulate BAX expression leading to apoptosis [65]. Quercetin is known as one of the major classes of flavonoids and in this study it was found that pulp contained high quantity of quercetin which resulted in pulp mediating downstream intrinsic pathway but

with high fold of gene expression change [66]. Mentioned that once BAX was activated, it promotes CYTOCHROME C release and mitochondrial breakdown. ATP in turn releases cytokines which suppress the inflammatory response, which triggers the activation of APAF1 into an apoptosome activating the CASPASE-9 molecules (upon cleavage of the bound zymogene procaspases-9). This in turn activated CASPASE-3 and CASPASE-9 [67,68] demonstrated that tBID activates BAX molecules which leads to the release of CYTOCHROME C from the mitochondria. BAX has been shown to be unnecessary in tBID induced CYTOCHROME C release. A home-like structure containing protein CYTOCHROME C located on the outside of the inner mitochondrial membrane (proteins control mitochondrial membrane permeability) binds to Apaf-1 which is released into the cytosol. Together with ATP it binds to apoptotic protease activating factor (Apaf1) and forms the apoptosome complex [69]. Makiuchi T [70] Stated that metronome is a highly reduced form of mitochondria that do not contain any genome and have lost the capacity to generate energy. CASPASE-9 becomes activated in the apoptosome and then activates CASPASE-3 [71]. Reported that the intrinsic pathway which is also called the mitochondrial path way is regulated by Bcl-2 family of proteins (BH, BCL2-homology). Induction or post-translational activation resulted in the significant inhibition of the expression of BCL-2 in all treated cell lines used in this study. It also resulted in changes in cell cycle phase’s inactivation of some BCL-2 family members. Up and down regulation of cell cycle gene expressions will lead to clear figures of the downstream arrest of treated cancer cell lines induced by the fruit extracts stimuli. A number of studies reported that involvement of cyclins and CDKs are important for regulation of cell cycle checkpoints in eukaryotes Murray AW [72]; Marinelli M [73] mentioned that a large number of studies have shown that p21 plays a role in tumour suppression in cancer. P21 can promote apoptosis depending on particular cellular stresses when it results in up regulation and is a common CYCLIN-CDK complex inhibitor that is activated in response to different stress stimuli and could act as cell cycle suppressor [74]. This study demonstrated that the molecular mechanisms for atilis A fruit extract induced cell growth inhibition and the occurrence of apoptosis in selected human cancer cell lines. Furthermore, it shows that the mode of action of the cell death signalling can be separated into two pathways [75]. Reported that quercetin can induce apoptosis via the mitochondrial pathway as against the harmful effects of carcinogenic chemicals by offering a defence system, by damaging DNA and initiating pro-oxidant property in cells as in A549 and HT-29 cells [76,77] reported that CDK1/CYCLIN B1 complexes promote the transition in mitosis through the phosphorylation of some substrates as APC (Anaphase Promoting Complex), responsible for the transition from metaphase to anaphase. Finally, p21 can act on G2/M checkpoint through the down regulation of Emi1 (Early mitotic inhibitor). Emi1 regulates mitosis by inhibiting APC/C ubiquity ligase complex (Anaphase Promoting Complex/Cyclo some) during S and G2 phases allowing a correct mitotic



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entry and preventing re-replication. It has been shown that Emi1 repression results in APC/C-dependent degradation of CYCLIN A1 and B1 and G2 phase arrest activated for the mitosis transition. Charrier-Savournin FB [78] suggested that p21 can block this progression by binding the complex and interfering with CDK1 dephosphorylation on Thr14 and Tyr15 and with CYCLIN B1 phosphorylation on Thr161. Therefore, pulp arrested the treated cells at G2/M resulting in disruption of normal chromatic disjunction during mitosis in mammalian cells and disrupting the breaking of the chromosome by the mitotic spindle during mitosis. Tumour cell lines increase sensitivity to anticancer agents. For example, several flavonoids, such as quercetin which had been shown to inhibit tumour growth in animal models were able to interrupt cell cycle progression in either G1 or G2 phase [79,80]. The current studies indicate that polyphenols induction of cellular cytotoxicity may be occurring through a non-classical apoptotic mechanism. It also showed that flavonoids are cytotoxic to cells which lack CASPASE-3 and CASPASE-8. However more extensive research into the role of flavonoids in each signalling pathway is still required. These findings provide potential effectiveness and a theoretical basis for the therapeutic use of A. altilis fruit extracts in the treatment of malignancies. A. altilis fruit extracts induced cellular apoptosis, apparently by acting on the functions of many intracellular genes important for this apoptotic process. In addition, comparing the apoptotic mechanisms of the three parts of the fruit on four cancer cell lines was found to be a potent inhibitor of apoptosis in response to a variety of cytotoxic stimuli.

ConclusionOne of the remarkable results of the present work was to

identify and quantify some phenolic compounds present in the methanol extracts of A. altilis by using the UHPLC-MS/MS based approach. Nine phenolic compounds were present, broadly identified as flavonoids. Phenolic acids were detected and characterized on the basis of their chromatographic retention time, UV-via spectra and mass spectra in the negative-ion mode and data from the literature. Overall pulp presented the highest concentrations of total flavonoids and phenolic acids, with ruin and protocatechuic acid being the major compounds. Quercetin and protocatechuic acid were the most abundant flavonoid and phenolic acid quantified in the pulp part. Indeed, the results indicate qualitatively and quantitatively that A. altilis fruit is an abundant source of phenolic compounds for use in the food and pharmaceutical industries. The fruit extracts inhibited the growth of the cells through induced apoptosis and cell cycle arrest at G0/G1, S and G2/M. Quercetin significantly inhibited the expression of particular tumour growth and angiogenesis by inducing apoptosis and regulating at G(1), S, G(2) and M phases of the cell cycle. Additionally, it has been shown to up regulate the expression of several tumour suppressor genes, via initiation of tumour suppressor genes. Inhibition of expression of tumour cell activity inhibited the cell cycle and reducing the risk of cancer occurrence. The methanol extract of pulp of A. altilis fruit inhibited the proliferation of selected cancer cells in vitro by

causing cell cycle arrest at G2/M inducing apoptosis mediated, at least in part via CASPASE-3 and mitochondrial apoptosis pathways in both capsize-dependent and independent manner. The molecular mechanisms involved are the up-regulation of the BAX/BCL-2 ratio, the activation of CASPASE-8 and CASPASE-3. The apoptosis were mediated by pulp via mitochondria pathway on A549 cells. The variation in the mechanism of the three parts of the fruit-induced cell cycle arrest and apoptosis in selected cancer cell lines may be dependent on the genetic profile of the cells.

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Identification and Quantification of Quercetin, a Major ...with cancer and 6.7 million of them are dying of cancer every year. Parkin DM [2] reported that a gradually increasing ratio