{"id":20453,"date":"2018-06-25T11:52:09","date_gmt":"2018-06-25T11:52:09","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=20453"},"modified":"2020-04-23T06:48:27","modified_gmt":"2020-04-23T06:48:27","slug":"anti-tumour-effect-of-two-persicaria-species-seeds-on-colon-and-prostate-cancers","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol11no2\/anti-tumour-effect-of-two-persicaria-species-seeds-on-colon-and-prostate-cancers\/","title":{"rendered":"Anti-Tumour Effect of two Persicaria Species Seeds on Colon and Prostate Cancers"},"content":{"rendered":"<p><strong>Introduction<\/strong><\/p>\n<p>In Egypt, the <em>Persicaria<\/em> species (Polygonaceae) have been used traditionally since many years for the cure of various ailments. In Egypt, medicinal herbs <em>P. salicifolia<\/em> (Brouss ex Wild) Assenov and <em>P. senegalensis<\/em> (Meism) <em>Sojak<\/em> (Polygonaceae) are two of the seven species of the genus.<sup>1<\/sup> They are perennial herbs commonly present along borders of watercourses of Nile Delta, shores of lakes, drains, and canals.<sup>2<\/sup> The major groups of phytochemical compounds include; phenolics and flavonoids.<sup>1<\/sup> Phenolics and flavonoids demonstrate pharmacological features in the form of antifungal, antibacterial, anti-inflammatory, and anti-tumour properties.<\/p>\n<p><em>Persicaria<\/em> species play a significant role in alternative medicines as they are used for treating various skin conditions (abscesses, boils, and scabies), colic pain, and inflammatory conditions (rheumatic pain, gout, knee pain, amenorrhea, and menstrual pain), since long. These species comprise of active biochemical ingredients, which possess anti-tumour, antioxidant, analgesic, antileukemic, antimicrobial, and tyrosinase inhibiting properties.<sup>3<\/sup> Moreover, they are considered as traditional medicines to treat disorders; like dyspepsia, haemorrhoids, diarrhoea, and itchy skin.<sup>3<\/sup> These species contain phenolic acids and flavonoid compounds and have shown potential therapeutic effects.<sup>4,6<\/sup><\/p>\n<p><em>Persicaria <\/em>species are known to possess anti-tumour and antioxidant properties; however, the constituents and anti-tumour activity of seeds alone of two <em>Persicaria <\/em>species have not been studied before. Therefore, the phytochemical analysis and anti-tumour effects of both seeds have been investigated in the present study. The present study has mainly focused on the evaluation of the anti-tumour effects of active constituents, presented in <em>P. salicifolia<\/em> and <em>P. senegalensis <\/em>seeds against human cancer cell lines. The determination of anti-tumour effects of the two investigated <em>Persicaria<\/em> seeds has been performed for the first time in this study against two kinds of cancer cells.<\/p>\n<p><strong><em>Materials and Methods<\/em><\/strong><\/p>\n<p><strong><em>Plant Material<\/em><\/strong><\/p>\n<p>Seeds of two selected plant species were collected from September 2016 to October 2016 from different localities of banks in Alexandria and Al- Mansoura, Egypt. The plants were identified according to the description reported by Boulos.<sup>1<\/sup><\/p>\n<p><strong><em>Preparation of Seed Extract<\/em><\/strong><\/p>\n<p>The seeds were washed, dried, and powdered in shade. The dried powder of seeds of two investigated <em>Persicaria<\/em> were extracted as detailed by Moustafa et al.<sup>7<\/sup> However, there was a slight alteration that was made in the seed extraction conducted in this study. The seeds under investigation (75g) were percolated in 450 ml methanol (50%), and then fully extracted by percolation at ambient temperature. Following this, the extracts underwent a filtration process using Whatman No.1 paper. A temperature of 40 \u00b0C was set out for the concentration process of these extracts. These extracts were dried using high vacuum. The extracts were kept in a refrigerator at 4\u00b0C until used for the experiment.<\/p>\n<p><strong><em>Extraction and Identification of <\/em><\/strong><strong><em>Flavonoids<\/em><\/strong><\/p>\n<p>The total content of the flavonoids was ascertained via the utilization of the aluminium chloride colorimetric method.<sup>8<\/sup> The results were then denoted in the form of milligram of catechin equivalents (CE). A spectrophotometer (Hewlett \u2013 Packarol, model 8452A, Rockville, USA) and the Folin-Ciocalteu method was used to establish total content of the polyphenols at 750 nm. The gallic acid equivalents (GAE) per gram DW was extracted from these results.<sup>9<\/sup><\/p>\n<p><strong><em>Extraction and Identification of the phenolic acids<\/em><\/strong><\/p>\n<p>The phenolic acids were isolated from the seeds of two investigated <em>Persicaria <\/em>species<sup>10<\/sup> and separated by reverse-phase HPLC instrument (Knauer, Germany). A model 7125 injection valve was used to supply the instrument (Rheodine, Cotati, CA, USA). The procedure of a 50 \u00b5l sample loop was handled using a computer (Knauer, HPLC version 211 a). It was seen that the flow rate was 1.0 ml \/ min. The detection of the phenolic acids was carried out via UV at 280 nm.<\/p>\n<p><strong><em>Separating and Identifying Lipid and Phenolic acids<\/em><\/strong><\/p>\n<p>The methanol extracts of the two investigated seeds (4g) were dissolved in water and defatted with n-hexane. The hexane extract was concentrated and analysed by Gas Chromatography-Mass Spectrometry (GC\/MS) using Shimadzu GC\/MS- QP 5050A software class 5000. [Column: DBI, 30 m, 0.53 mm ID, 1.5 \u00b5m film. Ionization mode: EL (70 eV). Carrier gas: Helium (flow rate 1 ml\/min).]. The temperature program was set at 40 \u00b0C (static for 2 min), which was progressively increased (160\u00b0C at a rate of 2\u00b0C \/min) to a level of 250\u00b0C (static for 7.5 min). Both the temperature of the injector and the temperature of the detector was set at 250 \u00b0C. The qualitative identification of hexane extract was achieved through library searched data Wiley 229 LIB.<sup>11<\/sup><\/p>\n<p>The defatted 50% methanol extract of two studied seeds was concentrated under reduced pressure till it dried completely. It was introduced to Liquid chromatography-mass spectrometry (LC\/MS). An Agilent (Waldbronn, Germany) Model 1100 quaternary pump was used to conduct the LC analysis and to supply an autosampler and a diode-array detector (DAD). The analysis of the data obtained was carried out using a chemstation HP Rev.A.08.03. Furthermore, the analysis of the results obtained was carried out using a Luna C18 column (150 d 2.1 mm i.d., 5m) (Phenomenex, Torrance, CA, USA). The identification of the compounds\u2019 structure was carried out via spectroscopic means. The UV spectral data of the separated compounds were measured through the inclusion of visible and ultraviolet absorption spectrometer (UV-VIS, Labomed Inc.). The ultraviolet and visible absorption spectrometer was in range of 200-500 nm.<\/p>\n<p>MS analysis was performed by mass spectrometer, Water Corporation, Milford, and MA01757 U.S.A. Electrospray ionization (ESI-MS Positive) was utilized for the ionization of the analytes. MS source parameters were optimised to: ionization potential, 70 eV, ion source temperature 290\u00b0C, scan speed 200 amu\/s, solvent delay 4.0 min, EV voltage 300 volts and scan range 30-600 amu was used. The process of ion acquisition was then conducted via the use of a XEVO TQD triple quadruple instrument [Column: ACQUITY UPLC-BEH C18 1.7um \u2013 2.1x 50 mm. Flow rate: 0.2 ml\/min, solvent system: consisted of A- water containing 0.1% Trifluoroacetic acid (TFA) and B- acetonitrile containing 0.1 % TFA]. Mobile phase gradient methods were optimised to allow maximum separation of analyte (Table 1)<\/p>\n<p><strong>Table 1: <\/strong><strong>LC\/MS mobile phase gradient methods<\/strong><\/p>\n<table style=\"width: 95%;\" border=\"1\" cellspacing=\"0\" cellpadding=\"4\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\" width=\"213\"><strong>Time (min)<\/strong><\/td>\n<td style=\"text-align: center;\" width=\"213\"><strong>% Mobile phase A<\/strong><\/td>\n<td style=\"text-align: center;\" width=\"213\"><strong>% Mobile phase B<\/strong><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"213\">0<\/td>\n<td style=\"text-align: center;\" width=\"213\">70<\/td>\n<td style=\"text-align: center;\" width=\"213\">30<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"213\">18<\/td>\n<td style=\"text-align: center;\" width=\"213\">0<\/td>\n<td style=\"text-align: center;\" width=\"213\">100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"213\">23<\/td>\n<td style=\"text-align: center;\" width=\"213\">0<\/td>\n<td style=\"text-align: center;\" width=\"213\">100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"213\">25<\/td>\n<td style=\"text-align: center;\" width=\"213\">70<\/td>\n<td style=\"text-align: center;\" width=\"213\">30<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"213\">30<\/td>\n<td style=\"text-align: center;\" width=\"213\">70<\/td>\n<td style=\"text-align: center;\" width=\"213\">30<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>&nbsp;<\/p>\n<p><strong>Cell viability assay<\/strong><\/p>\n<p><strong><em>Cell lines<\/em><\/strong><\/p>\n<p>Human prostate carcinoma (PC3) in the epithelial cells was procured from the site of metastasis on the bone and the disease was a grade IV level adenocarcinoma. Additionally, the human colon carcinoma (CaCO-2) in the epithelial cells was procured from the colon and the disease was colorectal adenocarcinoma. The American type culture collection (ATCC, Rockville, MD) was approached to procure both PC3 and CaCO-2 cells.<\/p>\n<p><strong><em>Cell culture and MTT assay <\/em><\/strong><\/p>\n<p>The sterility of the procedure was maintained by conducting it in a sterile area via the use of a laminar air flow cabinet that was at a biosafety level of class II. The culture was maintained in Roswell Park Memorial Institute medium (RPMI 1640) that contained a 1% antibiotic-antimycotic mixture (10,000\u00b5g\/ml streptomycin sulphate, 25\u00b5g\/ml amphotericin B and 10,000U\/ml potassium penicillin), 1% L-glutamine, and was augmented with a fetal bovine serum which was 10% heat-inactivated.<sup>7<\/sup> Culturing and sub-culturing were performed according to Thabrew et al.<sup>12<\/sup><\/p>\n<p>To assess the cell viability, the mitochondrial dependent reduction of yellow MTT was used (3-(4,5- dimethylthiazol-2-yl\u2013 2,5- diphenyl tetrazolium bromide). This yellow MTT undergoes a mitochondrial reduction to form purple formazan.<sup>13<\/sup> The 96 well tissue culture plate were inoculated with 1X105 cells \/ml (100 \u00b5l \/ well) and incubated at 37\u00b0C for 24 hours to develop a complete monolayer sheet. Growth medium was decanted from 96 well micro titer plates after the formation of confluent sheet of cells. Later, the cell monolayer was washed twice with wash media. Serial dilutions of both extracts of seeds and aerial parts of <em>Persicaria salicifolia<\/em> and <em>P. senegalensis<\/em> were prepared by dissolving each extract in dimethyl sulfoxide (DMSO) followed by dilution with RPMI\u20131640 medium to give a final concentration, 78.12, 156.25, 312.5, 625, 1250, 2500, 5000 and 10.000 \u00b5gml<sup>-1<\/sup>.\u00a0 0.1 ml of each concentration was assayed in triplicate in different wells leaving 3 wells as control, receiving only maintenance medium (RPMI-1640) with 2% serum.The treated cells was incubated at 37\u00b0C for 24 hour and examined. Cells were checked for any\u00a0 physical signs of toxicity, e.g. partial or complete loss of the monolayer, rounding, shrinkage, or cell granulation.\u00a0 MTT solution at 5 mg \/ml\u00a0 was dissolved in PBS (Bio Basic Canada Inc.) and 20 \u00b5l of\u00a0 it was added to each of the 96 wells. The solutions were placed on a shaker at 150 rpm for 5 minutes to mix the MTT into the media thoroughly and incubated at 37 \u00b0C (5 % CO2) for 4 hours to allow the MTT to be metabolized. The media was dump off and dry plate on paper towels was used to remove residue if necessary. The resuspended formazan (MTT metabolic product) in 200 \u00b5l DMSO was place on a shaker at 150 rpm for 5 minutes to thoroughly mix the formazan\u00a0 into\u00a0 the solvent. The optical density was recorded using a micro plate reader\u00a0 at 560 nm.<sup>13<\/sup><\/p>\n<p><strong><em>Determination of IC50 values<\/em><\/strong><\/p>\n<p>GraphPad prism version 5 software, Inc., California, U.S.A was used to calculate IC50 values of extracts of seeds and aerial parts of two Persicaria species against PC3 and CaCO- 2 cell lines. The log concentrations of extracts were plotted along the horizontal axis and the cells viability percentages were plotted up the vertical axis. Therefore, the decrease of cells viability percentages over the increased of concentrations of extracts can be expressed by the general equation of a straight line (1)<\/p>\n<p>Y = m. X + C \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0&#8230;(1)<\/p>\n<p>Thus<\/p>\n<p>Y (the value plotted up the vertical axis) will be the cells viability percentages<\/p>\n<p>m the gradient of the line.<\/p>\n<p>X (the value plotted along the horizontal axis) will be the log concentrations of the extracts.<\/p>\n<p>C the intercept on the vertical axis<\/p>\n<p>So, IC50, the half maximal inhibitory concentration can be calculated from equation (2)<\/p>\n<p>X (IC50) = (C-50)\/m \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0&#8230;(2)<\/p>\n<p><strong>Results <\/strong><\/p>\n<p>Through the phytochemical examination, a variety of phenolic acids were found in the seeds of two <em>Persicaria<\/em> species. The seeds constituents were analysed by LC\/MS and revealed several phenolic acids and flavonoid compounds between the two studied <em>Persicaria<\/em> species (Table 2)<em>. <\/em>The total phenolics of<em> P. salicifolia <\/em>were 46.3 mg GAE\/g DW and flavonoids was 5.04 mg CE\/g DW; while, the total phenolics of <em>P. senegalensis<\/em> were 37.6 mg GAE\/g DW and flavonoids were 18.2 mg CE\/g DW. The results have shown that the seeds of both <em>Persicaria<\/em> species contain five free phenolic acids; ferulic, caffeic, fumaric, coumaric and cinnamic. While, Gallic acid, chlorogenic acid and 3,4,5 methoxy cinnamic were only detected in seeds of <em>P. senegalensis<\/em> (Table 2). The phenolic acid of 50 % methanolic extracts of P.<em> salicifolia <\/em>and <em>P. senegalensis <\/em>have revealed 13 and 12 different flavonoid compounds, respectively. The analysis showed the presence of five flavonoids in both the seeds including; Luteolin (Figure 1), apigenin, kaempferol, quercetin, and rutin. Moreover<em>, P. salicifolia<\/em> seeds were characterized by the presence of a high amount of 2\u2019-O-methylcajanone, which is a new isoflavonone separated for the first time from <em>P. salicifolia<\/em> seeds (Figure 2)<em>.<\/em><\/p>\n<p><strong>Table 2: <\/strong><strong>The phenolic acids; the chemical constituents that were found in <em>P. salicifolia and P. senegalensis <\/em>seeds<em>. <\/em>LC\/MS was used for detection and identification<\/strong><\/p>\n<table style=\"width: 95%;\" border=\"1\" cellspacing=\"0\" cellpadding=\"4\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\"><strong>Phenolic acids<\/strong><\/td>\n<td style=\"text-align: center;\" width=\"27%\"><strong><em>P. salicifolia<\/em><\/strong><\/td>\n<td style=\"text-align: center;\" width=\"27%\"><strong><em>P. senegalensis<\/em><\/strong><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Ferulic<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Caffeic<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Fumaric<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Coumaric<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Gallic<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Chlorogenic<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">3,4,5 methoxy cinnamic acid<\/td>\n<td style=\"text-align: center;\" width=\"27%\"><\/td>\n<td style=\"text-align: center;\" width=\"27%\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\"><strong>Flavonoids:<\/strong><\/td>\n<td style=\"text-align: center;\" width=\"27%\"><\/td>\n<td style=\"text-align: center;\" width=\"27%\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Luteolin (Figure 1)<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">\u00a0(-)- Epicatechin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Luteo 6-glucose 8-arabinose<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Apigenin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Apigenin 6-glucose 8-rhamnose<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">kaempferol<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Kaempferol 3,7 dirhamnoside<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Dehydrokaempferol<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Quercetin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Rutin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Pentamethoxy quercetin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">2`-o- methylcajanone (Figure 2)<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Coumarin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">2&#8220;,5&#8220;,4`,5,6&#8220;,7methoxy isovitrixin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Sciadpitysin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Diosmetin 7-o- glucoside<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Narigenin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Hespertin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"45%\">Hesperidin<\/td>\n<td style=\"text-align: center;\" width=\"27%\">&#8211;<\/td>\n<td style=\"text-align: center;\" width=\"27%\">+<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>&nbsp;<\/p>\n<p>(+): Present; (-): Absent.<\/p>\n<table style=\"width: 70%;\" border=\"1\" cellpadding=\"5\">\n<tbody>\n<tr>\n<td><img decoding=\"async\" class=\"alignnone size-thumbnail wp-image-20457\" src=\"https:\/\/biomedpharmajournal.org\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig1-150x150.jpg\" alt=\"Figure 1: LC\/MS detection of luteolin.\" width=\"150\" height=\"150\" srcset=\"https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig1-150x150.jpg 150w, https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig1-256x256.jpg 256w, https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig1.jpg 751w\" sizes=\"(max-width: 150px) 100vw, 150px\" \/><\/td>\n<td><strong>Figure 1:<\/strong><strong> LC\/MS detection of luteolin.<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p><a href=\"http:\/\/biomedpharmajournal.org\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig1.jpg\" target=\"_blank\">Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>&nbsp;<\/p>\n<p>Luteolin is an example of flavonoid which was found in <em>P. salicifolia <\/em>and <em>P. senegalensis<\/em> seeds: (a) HPLC trace demonstrating luteolin detection at 280 nm, retention time (Rt) 10.02 minutes. (b) Mass spectral characteristics supporting correct identification of luteolin as a single charged ion, <em>m\/z<\/em> 286.<\/p>\n<table style=\"width: 70%;\" border=\"1\" cellpadding=\"5\">\n<tbody>\n<tr>\n<td><img decoding=\"async\" class=\"alignnone size-thumbnail wp-image-20458\" src=\"https:\/\/biomedpharmajournal.org\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig2-150x150.jpg\" alt=\"Figure 2: LC\/MS detection of 2\u2019-O-Methylcajanone.\" width=\"150\" height=\"150\" srcset=\"https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig2-150x150.jpg 150w, https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig2-256x256.jpg 256w, https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig2.jpg 736w\" sizes=\"(max-width: 150px) 100vw, 150px\" \/><\/td>\n<td><strong>Figure 2: LC\/MS detection of 2\u2019-O-Methylcajanone.<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p><a href=\"http:\/\/biomedpharmajournal.org\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig2.jpg\" target=\"_blank\">Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>&nbsp;<\/p>\n<p>2\u2019-O-Methylcajanone is an example of flavonoid which was found in <em>P. salicifolia <\/em>Seeds: (a) HPLC trace demonstrating 2\u2019-O-Methylcajanone detection at 280 nm, retention time (Rt) 14.25 minutes. (b) Mass spectral characteristics supporting correct identification of 2\u2019-O-Methylcajanone as a single charged ion, <em>m\/z<\/em> 436.<\/p>\n<p>GC\/MS was utilized to analyse polylipids of n-hexane extract of seeds that were obtained from the <em>Persicaria <\/em>species. As a result, <em>P<\/em>. <em>salicifolia<\/em> demonstrated 52 compounds; whereas, 29 compounds were found in<em> P<\/em>.<em> senegalensis<\/em>. Firstly, the major components of<em> P<\/em>. <em>salicifolia<\/em> were gamma-sitosterol (14.7 %), bis (2-ethylhexyl) phthalate (12.9 %), 9, 12-octadecadionoic acid, ethyl ester (9.7 %), hexadecanoic acid, ethyl ester (5.8 %), and ethyl oleate (1.9 %). Secondly, <em>P<\/em>.<em> senegalensis<\/em> contained hexadecanoic acid (33.3 %) (Figure 3), oleic acid (16.3 %), 1-[(2-aminoethoxy) hyroxyphosphinyl [oxy] methyl]-1, 2-ethanediylester (16.5 %), octadecanoic acid, 2, 3-dihydroxypropyl ester (6.25 %), and lucenin (3.6 %). While, other compounds recorded the lowest values in the two seeds of <em>Persicaria <\/em>species.<\/p>\n<table style=\"width: 70%;\" border=\"1\" cellpadding=\"5\">\n<tbody>\n<tr>\n<td>\u00a0<img decoding=\"async\" class=\"alignnone size-thumbnail wp-image-20459\" src=\"https:\/\/biomedpharmajournal.org\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig3-150x150.jpg\" alt=\"Figure 3: GC\/MS detection of hexadecanoic acid.\" width=\"150\" height=\"150\" srcset=\"https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig3-150x150.jpg 150w, https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig3-256x256.jpg 256w, https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig3.jpg 704w\" sizes=\"(max-width: 150px) 100vw, 150px\" \/><\/td>\n<td><strong>Figure 3:<\/strong><strong> GC\/MS detection of hexadecanoic acid.<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p><a href=\"http:\/\/biomedpharmajournal.org\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig3.jpg\" target=\"_blank\">Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>&nbsp;<\/p>\n<p>Hexadecanoic acid is an example of polylipid which was found in <em>P. senegalensis<\/em> seeds: (a) HPLC trace demonstrating hexadecanoic acid detection at 280 nm, retention time (Rt) 21.52 minutes. (b) Mass spectral characteristics supporting correct identification of hexadecanoic acid as a single charged ion, <em>m\/z<\/em> 256.<\/p>\n<p>The use of the MTT assay analyse the anti-tumour activity of methanol extract of seeds and aerial parts of two <em>persicaria<\/em> species. Human colon carcinoma (Ca Co-2) and prostate carcinoma (PC 3) cell lines were used. IC50, the half maximal inhibitory concentration is a measure of a compounds effectivity used in hindering biochemical or biological activity and to estimate seeds extract cytotoxicity.<\/p>\n<p>The results revealed that <em>P. salicifolia <\/em>seeds possessed anti-tumour activity against prostate carcinoma. In contrast, <em>P. senegalensis <\/em>seeds showed higher anti-tumour activity as compared to its aerial parts against CaCo-2 (Table 3) (Figure 4).<\/p>\n<table style=\"width: 70%;\" border=\"1\" cellpadding=\"5\">\n<tbody>\n<tr>\n<td>\u00a0<img decoding=\"async\" class=\"alignnone size-thumbnail wp-image-20460\" src=\"https:\/\/biomedpharmajournal.org\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig4-150x150.jpg\" alt=\"Figure 4: Cytoxic effects of P. salicifolia and P. senegalensis seeds and aerial parts extracts against PC3 and CaCO-2 cell lines.\" width=\"150\" height=\"150\" srcset=\"https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig4-150x150.jpg 150w, https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig4-256x256.jpg 256w, https:\/\/biomedpharmajournal.org\/staging\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig4.jpg 703w\" sizes=\"(max-width: 150px) 100vw, 150px\" \/><\/td>\n<td><strong>Figure 4:<\/strong><strong> Cytoxic effects of <em>P. salicifolia<\/em> and <em>P. senegalensis<\/em> seeds and aerial parts extracts against PC3 and CaCO-2 cell lines.<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p><a href=\"http:\/\/biomedpharmajournal.org\/wp-content\/uploads\/2018\/05\/Vol11No2_Ant_Ahm_fig4.jpg\" target=\"_blank\">Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><strong><br \/>\nTable 3: <\/strong><strong>Cytotoxicities of seeds and aerial parts of two <em>persicaria<\/em> <em>species<\/em><\/strong><\/p>\n<table style=\"width: 95%;\" border=\"1\" cellspacing=\"0\" cellpadding=\"4\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\" width=\"148\"><strong>Species<\/strong><\/td>\n<td style=\"text-align: center;\" width=\"148\"><strong>Plant part used<\/strong><\/td>\n<td style=\"text-align: center;\" colspan=\"2\" width=\"295\"><strong>IC50 (\u00b5g\/ml)<\/strong><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"148\"><\/td>\n<td style=\"text-align: center;\" width=\"148\"><\/td>\n<td style=\"text-align: center;\" width=\"148\">PC3<\/td>\n<td style=\"text-align: center;\" width=\"148\">CaCO-2<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"148\"><em>P.<\/em> <em>salicifolia<\/em><\/td>\n<td style=\"text-align: center;\" width=\"148\">Aerial parts<\/td>\n<td style=\"text-align: center;\" width=\"148\">1.1 \u00b1 0.15<\/td>\n<td style=\"text-align: center;\" width=\"148\">0.5 \u00b1 0.011<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"148\"><em>P.<\/em> <em>salicifolia<\/em><\/td>\n<td style=\"text-align: center;\" width=\"148\">Seeds<\/td>\n<td style=\"text-align: center;\" width=\"148\">0.6 \u00b1 0.018<\/td>\n<td style=\"text-align: center;\" width=\"148\">1.0 \u00b1 0.009<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"148\"><em>P<\/em>.<em> senegalensis<\/em><\/td>\n<td style=\"text-align: center;\" width=\"148\">Aerial parts<\/td>\n<td style=\"text-align: center;\" width=\"148\">2.3 \u00b1 0.03<\/td>\n<td style=\"text-align: center;\" width=\"148\">2.0 \u00b1 0.03<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\" width=\"148\"><em>P<\/em>.<em> senegalensis<\/em><\/td>\n<td style=\"text-align: center;\" width=\"148\">Seeds<\/td>\n<td style=\"text-align: center;\" width=\"148\">3.5 \u00b1 0.06<\/td>\n<td style=\"text-align: center;\" width=\"148\">1.5 \u00b1 0.03<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>&nbsp;<\/p>\n<p>IC50: The half maximal inhibitory concentration; PC3: human prostate carcinoma; CaCO-2, human colon carcinoma<\/p>\n<p><strong>Discussion<\/strong><\/p>\n<p>Phytochemical analysis of both seeds of <em>Persicaria<\/em> species revealed several phenolic, flavonoid, and polylipid compounds that were present in their aerial parts. For example, phenolic acids found in the present study for seeds of the two <em>Persicaria<\/em> species were correlated to the aerial parts of four species of Polygonaceae (2 <em>Periscaria<\/em> + 2 <em>Polygonum<\/em>). These species included; apigenin, quercetin, and kaempferol.<sup>5<\/sup> Luteolin, apigenin, kaempferol, quercetin, and rutin showed potential antioxidant, anti-tumour, and antihyperlipidemic effect.<sup>14,17<\/sup> These five compounds were found in both seeds of <em>Persicaria<\/em> species in the present study (Table 1). Luteolin compound was present in high values that is 25.2% in seeds of <em>p. senegalensis<\/em>; while, 2\u2019-O-methylcajanone (27.1 %) was present in <em>P. salicifolia<\/em> seeds. These two compounds showed anti-tumour activity<sup>18,19<\/sup> The presence of anti-oxidative compounds in the form of gallic and caffeic acid was noted in the two <em>Persicaria<\/em> seeds. These compounds are noted to have potential anti-tumour activity<sup>20<\/sup> Therefore, the identified compounds were confirmed by conducting a comparison of the data that was obtained and published in literature of aerial parts of <em>Persicaria<\/em> and <em>Polygonum<\/em> species.<sup>5,16,21,22<\/sup><\/p>\n<p>The cytotoxic activity against J82 (Bladder transitional carcinoma), HL60 (Human leukemia), P338 (Murine lymphocytic leukemia), LL2 (Lewis lung carcinoma) cancer cells, HepG2 (Hepatocellur carcinoma), and MCF7 (Human breast cancer) were analysed through the use of hexane fractions and chloroform with respect to <em>Polygonum bistora <\/em>sub-fractions. A moderate to very good activity against LL2 cancer cell lines, P338, and HL 60 were demonstrated through the hexane functions, sub-fractions and the chloroform.<sup>23<\/sup> Through the methanol extract of <em>Polygonum avicular<\/em>, the apoptotic and cytotoxic influence on Hela-s cervical cell line was demonstrated.<sup>24<\/sup> Reportedly, the n-butanol extract of <em>Polygonum bellardii<\/em> possesses the greatest cytotoxicity in HepG-2, Hela, and MCF-7 cells, with IC50 values of 30.09, 15.26, 50.66 \u00b5g\/ml, respectively.<sup>25<\/sup> In addition, myricetin \u20133-o\u2013(5 acetyl \u2013 a \u2013 arabinofreranoside) demonstrated a notable cytotoxicity in HepG\u20132 (IC50 41.03 \u00b5g\/ml) and Hela (IC50 75.04 \u00b5g\/ml) cells.<sup>25<\/sup> Another study conducted by Intisar et al. <sup>26<\/sup> demonstrated the anti-tumour activities of phenolic acids from <em>Polygonum bistora<\/em> L. that worked to resist the human hepatocellular carcinoma cell line (HCCLM3). They reported that eleven fractions demonstrated good to excellent levels of cytotoxicity, falling in the range of 200 \u00b5g\/ml\u2013800 \u00b5g\/ml. However, the lack of any activity even at levels of 800 \u00b5g\/ml were noted in two fractions and no anti-tumour component was detected in these. In the present study similar results were obtained for <em>Persicaria <\/em>species.<\/p>\n<p>The crude methanol extract of <em>P. glabrum<\/em> demonstrated significant cytotoxic activity with highest lethality IC50 value 0.74 \u00b1 0.045 \u00b5g\/ml.<sup>27<\/sup> The successive fraction of aqueous ethanol of <em>P. salicifolia<\/em> was conducted via the use of petroleum ether, methylene chloride, ethyl acetate, and n-butanol. The cell viability assays of these extractions were performed against MCF \u20137 and PC3 cell lines.<sup>21<\/sup> As per the results, ethyl acetate and methylene chloride fractions were demonstrative of the greatest activity against breast carcinoma (IC50 6.01 \u00b5g\/ml). Furthermore, the petroleum ether extract was demonstrative of the greatest activity against the prostate carcinoma (IC50 61.1 \u00b5g\/ml) cell line.<sup>21<\/sup><\/p>\n<p><strong>Conclusion<\/strong><\/p>\n<p>The results showed that there was a potential cytotoxicity of two <em>Persicaria <\/em>species seeds against two human cancer cell lines. Further investigations are recommended to identify the active principle responsible for anti-tumour activity of these plants. Therefore, seeds of <em>P. salicifolia<\/em> and <em>P. senegalensis<\/em> need to have a potential curative effect for prostate cancer and colon cancer, respectively.<\/p>\n<p><strong>Acknowledgement<\/strong><\/p>\n<p>Ahmed Mohamed Mohamed Youssef and Zeinab Ahmed Said El-Swaify designed the article, wrote this manuscript, participated in the literature search and conducted the data analysis.<\/p>\n<p><strong>Conflict of Interest<\/strong><\/p>\n<p>The research has no conflict of interest and is not funded through any source.<\/p>\n<p><strong>References<\/strong><\/p>\n<ol>\n<li>Boulos L. Flora of Egypt, vol. 1. Cairo: Al Hadara Publishing. 1999:417.<\/li>\n<li>Shaltout KH, Galal TM, El-Komi TM. 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Doi: 10.3329\/bpj.v17i2.22341.<br \/>\n<a href=\"https:\/\/doi.org\/10.3329\/bpj.v17i2.22341\" target=\"_blank\">CrossRef<\/a><\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"<p>Introduction In Egypt, the Persicaria species (Polygonaceae) have been used  [&#8230;]<\/p>\n","protected":false},"author":9,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[57],"tags":[],"class_list":["post-20453","post","type-post","status-publish","format-standard","hentry","category-vol11no2"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/20453","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/9"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=20453"}],"version-history":[{"count":6,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/20453\/revisions"}],"predecessor-version":[{"id":32210,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/20453\/revisions\/32210"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=20453"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=20453"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=20453"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}