In Vitro Anticancer Effects of a Traditional Sri Lankan Polyherbal Decoction and Its Individual Components Against Colorectal Cancer Cells

In Vitro Anticancer Effects of a Traditional Sri Lankan Polyherbal Decoction and Its Individual Components Against Colorectal Cancer Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In Vitro Anticancer Effects of a Traditional Sri Lankan Polyherbal Decoction and Its Individual Components Against Colorectal Cancer Cells Nipunika U. Rupasinghe, Nirwani N. Seneviratne, Harshini Kularathna, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7141234/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Colorectal cancer is globally the third highest prevalent and second highest mortality malignancy. Modern treatments were failed to reduce the cancer deaths to the expected level via producing exceptional side effects. On the contrary, traditional herbal medicine is gaining more attention in cancer remedies mainly due to its high efficacy and fewer side effects. The current study was aimed at investigating the anticancer and antioxidant properties of a poly-herbal decoction prescribed in Sri Lankan traditional cancer treatments. A polyherbal decoction (PHD) prepared using the Adenanthera pavonina , Thespesia populnea , Tinospora cordifolia , and Withania somnifera , and decoctions prepared from these four individual plants were used commonly in Sri Lankan traditional medicine for colorectal cancer treatments. Methods investigated for their total polyphenolic content, anti-oxidant activity, in vitro anti-colon cancer and apoptosis inducing activity (in HCT116 cell line) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, Ferric Reducing Antioxidant Power (FRAP) assay, hydroxyl radical scavenging assay, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, caspase activity assay and Real-time quantitative Polymerase Chain Reaction (RT-qPCR) respectively. Results Out of the five decoctions, W. somnifera decoction (WSD) exhibited strong anti-colon cancer activity (EC₅₀ = 83.1 ± 1.1 µg/mL) while PHD and other decoctions exhibited limited anti-cancer activity (EC₅₀ >100 µg/mL). WSD suppressed colony formation and induced caspase dependent programmed cell death, and lactate dehydrogenase (LDH) leakage. Up regulation of tumor suppressor p53 gene expression and down regulation of expression of key oncogenic and anti-apoptotic genes MMP-2, MMP-9, STAT3, JAK2, Cyclin D1, VEGF, and Bcl-XL were observed. Conclusions Overall findings validates the traditional use of Poly Herbal Decoction and W. somnifera decoction against colon cancer. Poly herbal decoction Withania somnifera Anti-cancer Anti-oxidant Phytochemicals Colorectal cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1.0 Introduction Colorectal cancer is a significant global health concern globally. In 2020, approximately 1.9 million new colorectal cancer cases were diagnosed, accounting for 9.6% of all cancer incidences, making it the third most commonly occurring cancer worldwide (WHO, 2025). In the same year, colorectal cancer led to about 900,000 deaths, representing the second leading cause of cancer mortality globally (WHO, 2025). Chemotherapy and radiotherapy remain the currently practiced treatments for colorectal cancer. However, these therapies often result in significant adverse effects. While short-term side effects such as nausea, fatigue, and stomatitis typically subside within a few months, long term complications including infertility, cardiac dysfunction, and secondary malignancies like leukemia can persist for years (Nurgali et al., 2018 ; Partridge et al., 2001 ; Yin et al., 2013 ). Given these limitations, there is an urgent need to explore alternative therapeutic strategies that are both effective and safe. Natural products, particularly plant-based compounds, have gained considerable attention in cancer research due to their diverse bioactive properties, including immune modulation and enhanced survival benefits (Kumar et al., 2016; Wang et al., 2017 ; Jafarian et al., 2014 ). Sri Lanka has a long-standing tradition of herbal medicine, with historical records indicating the use of medicinal plant extracts and decoctions for over 3,000 years (Weragoda, 1980 ). Many of these formulations are reputed to have anticancer properties (Jayasinghe et al., 2017 ; Luo et al., 2010 ). Notably, a decoction derived from the bark of Adenanthera pavonina and Thespesia populnea , the stem of Tinospora cordifolia , and the tuberous root of Withania somnifera have been incorporated into Sri Lankan traditional medicine and have claimed to be effective in cancer therapy with particular emphasis against colorectal cancer (Kuruppu et al., 2019 ). However clinical studies or other forms of in vivo or in vitro efficacy studies has not been conducted for poly herbal decoction (PHD). Present study aims to evaluate the anticancer, cytotoxic and apoptosis inducing activity of the PHD and its individual components in colorectal cancer cells. Additionally, the study seeks to determine the individual contributions of each plant component to the overall bioactivity of the decoction as well as total phenolic, flavonoid, and tannin content and the antioxidant properties. By providing scientific validation for the traditional use of this formulation, this research contributes to the growing body of evidence supporting herbal medicine as a complementary approach in cancer therapy. 2.0 Methodology Collection and identification of plant materials All plant species of Tinospora cordifolia (TC), Thespesia populnea (TP), Adenanthera pavonina (AP) were collected from Kotte, Western Province, Sri Lanka. Withania somnifera (WS) was purchased from a registered ayurvedic material vendor (Sridhara ayurvedic shop, Malabe, Western Province, Sri Lanka) in Sri Lanka. Each plant species was identified by a botanist at Bandaranaike Memorial Ayurvedic Research Institute (BMARI), Sri Lanka and voucher specimen were deposited at BMARI (voucher specimen numbers2093, 2094 and 2095 for AP, TP and TC respectively). Preparation of plant decoctions The aerial parts of TC, the bark of TP, the bark of AP and the rhizome of WS were freeze-dried and the poly-herbal decoction was prepared as of the methods given by the traditional medical practitioners. The individual decoctions were prepared by using 15 g of each freeze-dried aerial parts of TC, the bark of TP, the bark of AP and the rhizome of WS. In brief, the plant material was added with 1600 mL of deionized water and was heated till the volume reduced to 1/8th (200 mL) of the initial volume (Lindamulage and Soysa 2016 ). The decoction was centrifuged (5000 rpm, 15 min) and the supernatant was freeze-dried to obtain the dried powder dissolved in deionized water/phosphate-buffered saline (PBS) to obtain known concentrations of plant extracts. Hereafter, decoctions of TC, TP, AP and WS will be indicated as TCD, TPD, APD and WSD respectively. Poly-herbal decoction will be indicated as PHD. Estimation of Total phenolic, tannin and flavonoid contents Estimation of Total Phenol Content (TPC) TPC of the decoctions was determined by Falin-Ciocalteau method (Kaur and Kapoor 2002 ). Gallic acid was used as the standard and a standard curve was obtained at a concentration range of 5–65 µg/mL of gallic acid. The TPC of the decoctions were calculated from the standard curve (Fernando et al, 2015 ). All determinants were carried out in triplicates. Estimation of Total Flavonoids Content (TFC) The aluminium chloride colorimetric method was carried out to determine the total flavonoid contents of the decoctions (Fernando and Soysa 2015 ). Quercetin was used as the standard and the standard curve was obtained at a concentration range of 10-1000 µg/mL of quercetin. The TFC of the decoctions were calculated from the standard curve. All determinants were carried out in triplicates. Estimation of Tannin content (TAC) The tannin content (TAC) of the plant decoctions was determined according to the following method described (Pulipati et al. 2017 ). A volume of 500 µL of the decoction (20 mg/mL) was mixed with deionized water (500 µL) and 50 mg of polyvinyl polypyrolidone (PVPP) and it was vortexed and refrigerated (4 \(\:℃\) , for 15 min). The reaction mixture was subjected to vortex again and was centrifuged at 3000 g, for 10 min. A concentration series was prepared at 70-5000 µg/mL from the supernatant to estimate the tannin content. The rest of the reaction method was performed according to the phenolic content estimation. Tannic acid was used as the standard and a standard curve was obtained at a concentration range of 20–73 µg/mL of tannic acid. The tannin content of the decoctions was calculated from the standard curve and all determinants were carried out in triplicates. Determination of antioxidant properties Free radical scavenging properties DPPH radical scavenging assay (Soysa and Silva 2011 ), Ferric reducing antioxidant power assay (FRAP Assay) (Jemli et al. 2016 ) and Hydroxyl radical scavenging assay (Halliwell et al. 1987 , Poorna et al. 2013) were performed according to the standard methods given in the literature. All determinations were carried out in triplicate. Protein and lipid oxidation inhibition Inhibition of Protein oxidation was carried out using protein oxidation inhibition assay (Poorna et al. 2013; Martnez et al. 2001) and inhibition of lipid oxidation was performed using egg yolk lipids (Kizil et al. 2010 ; Perera et al. 2016 , Dhar et al. 2013 ) to the standard methods reported in previous study. Cell cultures HCT116 colorectal cancer cell line was used to determine the cytotoxicity of the targeted plant decoctions. HCT116 cell line was acquired from the Medical Research Institute, Colombo, Sri Lanka. It was cultured in Modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS), 3% L-glutamine, 7% Sodium bicarbonate, streptomycin (100 µg/mL), and penicillin (100 U/mL). Cells were incubated in humidified air at 37 \(\:℃\) . Exponentially growing (80% confluent) cells were passaged every 2–3 days using 0.25% trypsin–EDTA solution (Perera et al. 2008 ; Razak et al. 2019 ; Vichai and Kirtikara 2006 ; Fernando et al. 2015 ). Cytotoxicity assays Dimethylthiazol-diphenyltetrazolium bromide (MTT) assay Cytotoxicity was determined using MTT cell viability assay after being subjected to the 24 h treatment of the PHD and individual decoctions to HCT116 cells (Wageesha et al. 2017). Experiments were carried out in triplicates. WSD decoction was subjected to 24 h, 48 h, and 72 h after the initial screening. Preparation of cell lysate Cell plates were prepared and treated according to the previously described method (Fernando et al. 2015 ). Each well was washed with PBS (100 µL, pH 7.4) three times. Thereafter, Triton-X-100 (100 µL, 0.1 v/v) was added to each well and subjected to sonication for 5 seconds. The cell lysates were then collected and centrifuged at 3000 rpm for 5 min to remove cell debris. LDH leakage Cells were treated with WSD decoction, assay was performed according to the manufacture’s description (LDH SCE mod. liquiUV, Human Diagnostics, Germany) for both the treated medium and cell lysate. The percentage LDH leakage was determined by the equation given below; \(\:Percentage\:LDH\:leakage\:\:=\:\frac{LDH\:activity\:in\:the\:supernatant}{Total\:LDH\:activity}\) ×100 (Wageesha et al., 2017) Colony formation assay Trypsinized cells were seeded in a culture dish and after 24 h cells were treated with the WSD as previously described. The colonies were stained with 6% glutaraldehyde and 0.5% crystal violet. The colonies were counted using stereomicroscope (Franken, 2006). Caspase assay Cells were seeded and treated with WSD as previously described, and the assay was performed according to the manufacturer’s description (Caspase 3 Activity Assay Kit, China (E-CK-A311). \(\:Caspase\:activity\:\:=\:\frac{{Absorbance}_{sample}-{Absorbance}_{blank}}{{Absorbance}_{negative\:control}-{Absorbance}_{blank}}\) ×100 Ethidium bromide/acridine orange (EB/AO) assay Decoction-treated cells were stained with a 1:1 mixture of Ethidium bromide and acridine orange, 100 µg/mL, prepared in PBS and observed in UV under a fluorescent microscope (Wageesha et al., 2017). Morphological criteria were used to evaluate cell injury. Cells containing normal nuclear chromatin exhibit green nuclear staining. Cells containing fragmented nuclear chromatin due to apoptosis exhibit orange to red nuclear staining. DNA fragmentation Treated cells were trypsinized and were collected in microcentrifuge tubes (4×10 5 per microcentrifuge tube). Cells were pelleted by centrifuging (2000 rpm, 5 min, 4 \(\:℃)\) . TES lysis buffer (20 µL) was added, and the pellet was carefully resuspended. RNase was added (10 mg/mL, 10 µL) added and incubated for 2 hrs at 37 \(\:℃\) . Thereafter, Proteinase K (20 mg/mL, 10 µL) was then added and incubated at 50 \(\:℃\) overnight. The sample (10 µL) was mixed with 2 µL of DNA loading dye and was run in 1.5% agarose gel(Kasibhatla et al., 2006)(Wageesha et al., 2017). Gene expression analysis Based on the cytotoxicity, oxidative, and morphological changes results, a hypothetical gene expression pathway that might have expressed in driving cell death (apoptosis) was postulated. In that gene expression pathway, 9 genes were targeted (with the house keeping gene, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and primers were designed to analyze the relative gene expression during cell death. Some primers were manually designed following the standard guidelines (Bustin & Huggett, 2017 ; Dieffenbach et al., 1993 ). Either the RefSeq transcripts or all the transcript variants were considered in primer designing. A few primers were acquired from published literature. Primer sequence, alignment, position of annealing, nucleotide length, melting temperature and GC percentage of each primer are included in Table 1 . RT-qPCR and Gene expression analysis RT-qPCR analysis of the genes was performed in Rotor Gene Q (Qiagen, Germany), following the given amplification parameters (40 cycles for Initial denaturation at 95 ℃ for 3 minutes, Denaturation at 95 ℃ for 30 seconds, Annealing at 60 ℃ for 30 seconds, Extension at 72 ℃ for 1.0-minute, Final extension at 72 ℃ for 1.0 minute, 4 ℃, ∞). Reaction mixtures were prepared as follow GoTaq® qPCR Master Mix, 2X; 10.0 µL (1X), Forward Primer (20X); 1.0 µL (250 nM), Reverse Primer (20X); 1.0 µL (250 nM), Nuclease-Free Water; 5.0 µL, cDNA template; 3.0 µL. Melting curves were obtained and the relative gene expression was evaluated. A small aliquot (10 µL) of each RT-qPCR product was loaded along with the loading buffer. The gel was run in ethidium bromide stained, 0.8% w/v agarose gel at 90 V. It was observed under UV after running for 15 minutes and 30 minutes. Statistical data analysis The data were represented as mean ± standard deviation. For comparison, data were analyzed by regression and Pearson analysis. A value of p < 0.05 was considered statistically significant. Table 1 Primers used in gene expression analysis Target gene Primer name Primer sequence Primer length Annealing site Melting Temperature GC content (%) Product Length (cDNA) Reference Matrix metalloproteinase 2 MMP2_F 5' TACAGGATCATTGGCTACACACC 3' 23 661–683 63.9 48 90 (Dai et al., 2017) MMP2_R 5' GGTCACATCGCTCCAGACT 3' 19 732–750 63.3 58 (Dai et al., 2017) Matrix metalloproteinase 9 MMP9_F 5' TTGACAGCGACAAGAAGTGG 3' 20 1155–1174 62.5 50.0 179 (Uehara et al., 2017) MMP9_R 5' GCCATTCACGTCGTCCTTAT 3' 20 1314–1333 62.1 50.0 (Uehara et al., 2017) 18 S (house-keeping gene) 18S_F 5' CATTCGTATTGCGCCGCTAG 3' 20 937–956 63.3 55.0 135 Newly designed 18S_R 5' CTACGACGGTATCTGATCGTC 3' 21 1051–1071 61.1 52.4 Newly designed B-cell lymphoma-extra large Bclxl_F 5' GCGTAGACAAGGAGATGCAG 3' 20 595–614 62.2 55.0 136 Newly designed Bclxl_R 5' GCATTGTTCCCATAGAGTTCCAC 3' 23 708–730 62.3 47.8 (Taghavi et al., 2019) Signal transducer and activator of transcription 3 STAT3_F 5' AGCAGTTTCTTCAGAGCAGGT 3' 20 755–774 60.7 50.0 134 Newly designed STAT3_R 5' GCGTGATTCTTCCCACAGGC 3' 21 868–888 60.1 47.6 Newly designed Cyclin D1 Ccnd1_F 5' GATACCAGAAGGGAAAGCTTC 3' 21 1141–1161 60.2 47.6 189 Newly designed Ccnd1_R 5' ATCTGTAGCACAACCCTCCTC 3' 21 1309–1329 62.3 52.4 Newly designed Vascular endothelial growth factor VEGF_F 5' CTACCTCCACCATGCCAAGT 3' 20 1084–1103 63.7 55.0 109 (Du et al., 2015) VEGF_R 5' GCAGTAGCTGCGCTGATAGA 3' 20 1173–1192 63.8 55.0 (Du et al., 2015) p53 P53_F 5' TATGAGCCGCCTGAGGTTGG 3' 20 1653–1672 60.7 50.0 104 (Hübner et al., 2018) P53_R 5' CGCACCTCAAAGCTGTTCCG 3' 20 1738–1757 60.5 50.0 (X. M. Li et al., 2020) Glyceraldehyde 3-phosphate dehydrogenase (house-keeping gene) GAPDH_F 5' GGAGCGAGATCCCTCCAAAAT 3' 21 554–574 63.9 52.4 197 (Fu et al., 2019) GAPDH_R 5' GGCTGTTGTCATACTTCTCATGG 3' 23 728–750 63.0 47.8 (Fu et al., 2019) Janus Kinase 2 JAK2_F 5' TTGGAGCTTTGGAGTGGTTC 3' 20 3784–3803 62.4 50.0 178 Newly designed JAK2_R 5' ATCTGGGCATCCATCTGGTC 3' 20 3942–3961 63.6 55.0 Newly designed 3.0 Results and Discussion Estimation of total phenolic, flavonoid and tannin contents Estimated Total phenolic (TPC), total flavonoid (TFC) and total tannin contents (TAC) of the poly herbal decoction and individual plant decoctions are summarized in Table 2 . According to the results, APD and TPD have shown higher TFC and TAC contents. However, in TPC estimation, the highest value (49.01 ± 4.9 mg GAE/g) was observed in PHD while TPD and WSD resulted in considerably lower values (1.9 ± 0.1 mg GAE/g, 0.4 ± 0.0 mg GAE/g respectively). The estimation of TPC considers flavonoids and tannins, which encompasses a broad range of compounds including other phenol-type derivatives, this may be the reason for the deviations in TPC estimation (Muflihah, 2021). Table 2 Total Phenolic Content, Total Flavonoid Content and Tannin Content of the PHD and individual plant decoctions; APD, TPD, TCD and WSD. The values are mean of three independent experiments ± standard deviation (SD) Decoctions TPC (mg GAE/g) TFC (mg QCE/g) TAC (mg TAE/g) PHD 49.1 ± 4.9 1.0 ± 0.1 24.7 ± 2.2 APD 44.6 ± 2.6 1.8 ± 0.4 64.9 ± 5.8 TPD 1.9 ± 0.1 7.8 ± 0.8 74.2 ± 7.6 TCD 5.1 ± 0.2 0.1 ± 0.0 1.1 ± 0.3 WSD 0.4 ± 0.0 0.1 ± 0.0 1.1 ± 0.3 Antioxidant assays In the present study, three different antioxidant assays are used to accurately confirm the antioxidant ability of each decoction. DPPH assay The present findings indicate that all decoctions induced a colorometric transition of DPPH confirming the antioxidant capacity. PHD exhibited antioxidant activity with an EC 50 value of 44.2 ± 0.7 µg/mL, while TCD and WSD resulted with EC 50 value of 1025.5 ± 11.6 µg/mL 1011.9 ± 15.9 µg/mL respectively. APD displayed significantly stronger antioxidant activity (EC 50 value of 15.0 ± 0.5 µg/mL) as given in Table 3 . Hydroxyl radical scavenging assay All the decoctions produced a characteristics pink chromogen upon thermal treatment, indicating the presences of phenolic compounds. PHD exhibited antioxidant activity with an EC 50 of 36.5 ± 0.2 µg/mL and TPD had the most potent antioxidant activity (EC 50 value of 13.2 ± 0.2 µg/mL), while WSD resulted in weakest anti-oxidant activity with EC 50 858.1 ± 14.3 µg/mL (Table 3 ). Table 3 Comparative Antioxidant activities of PHD and individual plant decoctions. The values are mean of three independent experiments ± standard deviation (SD) Decoction/ Standard EC 50 values (µg/mL) DPPH Hydroxyl radical scavenging assay PHD 44.2 ± 0.7 36.5 ± 0.2 APD 15.0 ± 0.5 13.2 ± 0.2 TPD 15.5 ± 0.2 12.4 ± 0.1 TCD 1025.5 ± 11.6 816.3 ± 16.6 WSD 1011.9 ± 15.9 858.1 ± 14.3 Ascorbic Acid 5.42 ± 0.10 Ferric ion reducing power assay The dose-response curves of ferric ion reducing power assay are presented in Fig. 1 . TCD (200–600 µg/mL of concentration range in between 0.300-1.000 of absorbance) and WSD (with 175–600 µg/mL of concentration range in between 0.300–0.600 of absorbance) were excluded in the figure. Results from all three antioxidant assays, confirmed significant anti-oxidant activities in all tested decoctions with varying potency. APD and TPD demonstrated strongest antioxidant properties whereas TCD and WSD with the weakest antioxidant properties PHD exhibited intermediate anti-oxidant properties due to its composition containing individual components, PHD was prepared as a mixture including all the individual components together where it can be expected to have the highest antioxidant properties as a collective of all the herbal decoctions. However, this effect may be affected by the interactions and reactions of different components which may not undergo when they are in separate individual extracts and also no correlation was observed between antioxidant (DPPH) and TPC, TFC and TAC. Oxidation assays Inhibition of Protein oxidation The maximum inhibitory activity (lowest EC 50 ) was demonstrated in PHD at 223.7 ± 1.8 µg/mL, while TCD exhibited a marginal inhibitory effect at EC 50 value of 511.8 ± 4.5 µg/mL (Table 4 ). Table 4 Inhibition of protein oxidation and lipid peroxidation shown by PHD and other individual plant decoctions; APD, TPD, TCD and WSD. The values are mean of three independent experiments (n = 3) ± standard deviation (SD) Decoction EC 50 values (µg/mL) Protein oxidation Lipid peroxidation PHD 223.7 ± 1.8 383.2 ± 8.7 APD 343.8 ± 0.9 206.4 ± 2.7 TPD 260.5 ± 2.2 207.6 ± 3.5 TCD 511.8 ± 4.5 523.7 ± 6.2 WSD 295.0 ± 10.1 156.9 ± 12.4 Inhibition of Lipid Peroxidation Lipid peroxidation was quantified spectrophotometrically by measuring the chromogenic transition in the reaction medium. WSD demonstrated the maximal inhibitory activity(EC 50 156.9 ± 12.4 µg/mL), while APD (EC 50 206.4 ± 2.7 µg/mL) and TPD (EC 50 207.6 ± 3.5 µg/mL ) exhibited the minimal marginal inhibitory activity as given in Table 4 . All decoctions exhibited ac considerable anti-oxidant activity against both protein oxidation a lipid oxidation. However, no significant correlation was observed between these protective effects, suggesting distinct mechanisms of action for each oxidative pathway. Cytotoxicity assays MTT assay was employed in determining the cytotoxicity of the decoctions against the colorectal carcinoma. Level of cytotoxicity of the PHD and its individual decoctions against HCT 116 and HEK 293 cells were determined by MTT assay after 24 h of treatments (Table 5 ). As shown in Table 5 , the highest cytotoxic effect was observed with WSD (EC 50 value; 83.1 µg/mL), However, no significant e cytotoxic effect was detected in TCD decoction, while PHD (EC 50 383.1 ± 2.7 µg/mL) resulted in a comparatively higher EC 50 value than TPD (EC 50 268.3 ± 1.3 µg/mL ) the least active individual component (Fig. 2 ). The EC 50 values of the individual components resulted in a wide range from 83.1 µg/mL to 268.3 µg/mL. A positive correlation was observed in PHD, APD, TPD and TCD decoctions, however a significant correlation was not observed in WSD, which demonstrated a strong cytotoxicity comparatively to other decoctions. While WSD exhibited potent anti-cancer activity, due to its low total phenolic, flavonoid and tannin content a significant correlation was not observed. Comparatively WSD exhibited highest anti-cancer activity against HCT 116 cells than individual components decoction and PHD. Based on this activity, WSD was selected for further studies. Table 5 Level of cytotoxicity of the PHD and its individual decoctions against HCT 116 and HEK 293 cells by MTT assay after 24 h of treatments, expressed as mean ± SD of three individual experiments (n = 3). *n = 2 Decoctions Cell line EC 50 values (µg/mL) PHD HCT 116 383.1 ± 2.7 APD HCT 116 191.2 ± 2.7 TPD HCT 116 268.3 ± 1.3 TCD HCT 116 Not detected WSD HCT 116 83.1 ± 1.1 WSD HEK 293 534.5 ± 16.0* * The value is a mean of two independent experiments (n = 2) ± standard deviation (SD) In consistent to our study, A prior research reported the dose dependent and time dependent anti-cancer activity in Withania somnifera crude extract against human malignant melanoma cells (Halder, 2015). Evidently a study performed with Withania somnifera fruit extract resulted in cytotoxicity against HCT 116 cells with a LC 50 value of 410.2 µg/mL, and HepG2 cells with a LC 50 value of 164.7 µg/mL as the highest cytotoxicity demonstrated (Abutaha, 2015 ). Table 6 Cytotoxicity effects of the WSD after 24, 48 and 72 h of treatments. Time EC 50 values (µg/mL) 24 h 81.2 ± 0.7 48 h 17.4 ± 0.1 72 h 3.1 ± 0.3 Figure SEQ Figure \* ARABIC 3. At 24 hours, EC 50 of WSD was 81.2 ± 0.7 µg/mL. When the incubation period was extended for 24 and 72 hours, the EC 50 values decreased to 17.4 ± 0.1 µg/mL and 3.1 ± 0.3 µg/mL against HCT 116 cells respectively. LDH leakage Quantification of membrane integrity damage was demonstrated upon WSD treatment within 24 h, a significant LDH leakage (59.5 ± 2.1%) was exhibited resulting in dose dependent cytotoxicity as demonstrated in Fig. 4. LDH leakage and increased level of LDH release were previously reported in Withania somnifera extract-treated on androgen-independent prostate cancer 3 cells (PC3) exhibiting the anti-proliferative effect. (Balakrishnan et al., 2017 ). Similarly, in another study Withania somnifera root powder was reported to have a significant suppression in lysosomal and cytoplasmic enzyme release in polymorphonuclear leucocytes cells (Rasool, 2006 ). Colony forming assay As of the obtained data, the WSD decoction treatments have significantly reduced cell proliferation and differentiation into colonies (262 ± 20). The negative control resulted in higher number of colonies after 7 days of the treatments (595 ± 22), while the positive control resulted in 2 ± 2 colonies, as shown in Fig. 5 . Sumantran et al 2007 reported that aqueous extract of Withania somnifera roots had a dose dependent inhibition in colony formation with Chinese Hamster ovarian cell line. It also reported that Withania somnifera root extract induce long term growth inhibition of CHO cells which was cell density dependent upon the drug treatment (Sumantran et al 2007 ). Ethidium bromide/acridine orange (EB/AO) assay Acridine orange (AO) is a chromatin staining dye that emits green fluorescence upon intercalation and enters live cells with normal membrane potentials. Hence, it is important in live, healthy and proliferating cells. Ethidium bromide (EB) penetrates only the cells that have lost their membrane integrity and emits a fluorescence of red upon DNA intercalation. Further, it dominates over AO. This makes the live cells in green colour and apoptotic and dead cells in orange-red colour (Ribble et al., 2005). Nuclei stained with green colour indicate live cells, while greenish yellow shows early apoptotic cells. Condensed orange red nuclei demonstrate late apoptotic cells, whereas red colour indicates dead cells. As shown in Fig. 6 , the EB/AO assay resulted in prominent apoptotic morphology in the HCT 116 cells upon treating with WSD for 24 hours. As shown in Fig. 6 A, orange colour and red colour cells were observed, indicating the late apoptotic cells and dead cells respectively. When the cells were observed in high power, fragmented nuclei were observed as in 6B. The images 6C and 6D indicate the negative and positive control respectively. The morphological changes of the images indicate the ongoing apoptosis in the cells upon treatment. DNA fragmentation As shown in Fig. 7 , HCT 116 cells resulted in DNA fragments upon treatment after 24 hours. According to the gel image visualization, faint bands of DNA fragments provide evidence of active apoptosis. As the negative control is free of such DNA fragments, it can be concluded that the WSD treatment has initiated apoptosis and DFF function, resulting DNA fragments in the HCT 116 cells. DNA fragmentation is a main feature of apoptosis and thus is considered as a marker of apoptosis. During apoptosis, double-stranded DNA is cleaved at A and T-rich regions by the DNA fragmentation factor (DFF). The 40 kDa catalytic subunit (DFF40) provides the endonuclease activity of the DFF for DNA cleavage, while 45 kDa subunit (DFF45) provides the regulatory functions (Majtnerová & Roušar, 2018). At normal stages, DFF40 remains inactive, which is regulated by DFF45. Upon activation of Casapse3, the DFF40-DFF45 complex is cleaved. Active DFF40 is released, and hence, DNA fragmentation is started. It cleaves DNA in about 180 bp and its multiples (360 bp, 540 bp and 720 bp), resulting a ladder pattern upon agarose gel run (Majtnerová & Roušar, 2018). Evidently in a previous study Withania somnifera extracts significantly induce in apoptosis against Hepatocellular carcinoma cells (Ahmed et al, 2018 ). Another study reported that caspase activity was increase on a time dependent manner against HL- 60 cells (Malik, 2009 Figure 8. Caspase activity of decoction, negative control and positive control. Effect of WSD on MMP2, STAT3, JAK2, MMP9 ,Bcl-XL, Ccnd1, VEGF and p53 The effect of MMP2 and MMP9 in apoptosis and cell death MMP-2 has a role in promoting tumour angiogenesis, which is initially supported by interleukin 8 (IL-8) (Quintero-Fabián et al., 2019 ). In this study both MMP-2 and MMP-9 were down regulated with negative fold changes of -14.7 ± 0.9 and − 5.4 ± 1.1 respectively. This indicates that the WSD treatment effectively retards the angiogenesis and metastasis in the cancer. It was reported that Withania somnifera has great potential in down regulating MMP-2 and MMP-9, leading to the inhibition of cell migration in neuroblastoma (Kataria et al., 2013 ). The effect of Cyclin D1 in cell cycle arrest Cyclin D1 shows a negative fold change of -12.5 ± 0.7 where the expression is being downregulated. Considerably Withania somnifera has been reported in previous literature as an agent to reduce the Cyclin D1 level in human prostate cancer (Balakrishnan et al., 2017 ) cells and human neuroblastomas (Kataria et al., 2013 ). The effect of VEGF in reducing angiogenesis Withaferin A, the active component of Withania somnifera has been identified in a previous research study as a compound that reduces the expression of VEGF functioning as an anti-VEGF agent (Saha et al., 2013 ). In this study, VEGF has exhibited a negative fold change of 7.6, indicating the down-regulation of the expression. This indicates the effectiveness of the WSD in retarding angiogenesis and proliferation of the tumour. The effect of STAT3 and JAK2 in apoptosis According to the experimental results given in the Table 7 , the expression of both JAK2 and STAT3 were down regulated in 13.4 ± 1.4 and 10.7 ± 1.2 folds respectively which exhibited significantly higher fold changes, collectively this study demonstrated that the WSD decoction promotes apoptosis involving the JAK2/STAT3 gene expression pathway, leading to the death of the tumour cells. The effect of Withania somnifera on STAT3 and JAK2 pathways was described in related to renal carcinoma (Um et al., 2012 ) and breast cancers (J. Lee et al., 2010 ) in the literature. The effect of Bcl-XL in apoptosis As evident by, the differential expression of Bcl-XL dropped in around 14 folds (-14.4 ± 1.1), indicating the effectiveness of the decoction in promoting apoptosis. The effect of the Withania somnifera on reducing the expression level of Bcl-XL in human Neuroblastomas was reported in a previous study (Kataria et al., 2013 ). p53 the analysis demonstratep53 relative expression has increased significantly (11.7 ± 1.3 folds). This can be an overall effect generated from the DNA damage due to the upregulation of p53 and STAT3 down regulation. The upregulation of p53 promoted by Withania somnifera has also been previously reported (Munagala et al., 2011 ). Table 7 Names and short names of the genes with their fold change expressed as mean ± S.D of three independent experiments. Name of the gene Short name of the gene Fold change Matrix Metallopeptidase 2 MMP-2 -14.7 ± 0.9 Signal transducer and activator of transcription 3 STAT3 -10.7 ± 1.2 Janus kinase 2 JAK2 -13.4 ± 1.4 Matrix Metallopeptidase 9 MMP-9 -5.4 ± 1.1 B-cell lymphoma-extra large Bcl-XL -14.4 ± 1.1 Cyclin D1 Ccnd1 -12.5 ± 0.7 Vascular endothelial growth factor VEGF -7.6 ± 1.3 Tumor protein 53 p53 11.7 ± 1.3 Glyceraldehyde 3-phosphate dehydrogenase GAPDH -1.5 ± 0.1 4.0 Conclusions Poly-herbal decoction (PHD) is enriched with valuable anti-oxidative and anticancer properties which were differently and unevenly contributed by its individual components. Each component might be contributing in a balanced manner in such a way as to compensate any toxicity that might result from hyperactivities. As PHD is prepared by adding several herbs in a single mixture, each acts as a protective agent by reducing the effects of other components making it is tolerable and harmless to the normal cells and tissues. In summary, the poly-herbal decoction and all the individual plant decoctions that was used in this study have shown antioxidant and anticancer properties implying the medicinal value of traditional plant decoctions used in Sri Lanka. In addition, current study provides a scientific validation for the traditional use of this poly-herbal decoction in anti-cancer treatments. The findings of this study can be used as an initiation for performing more expanded cancer research using traditional plant decoctions towards a remedy for colorectal cancer. Abbreviations DPPH- 2,2-diphenyl-1-picrylhydrazyl FRAP- Ferric Reducing Antioxidant Power MTT- 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide RT-qPCR- Real-time quantitative Polymerase Chain Reaction LDH- Lactate dehydrogenase PVPP- Polyvinyl polypyrolidone MEM- Modified Eagle’s medium FBS- Fetal bovine serum Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Conflicts of Interest The authors declare that they have no conflict of interest Funding This project was funded by the University of Sri Jayewardenepura, Sri Lanka (Grant No: ASP/01/RE/SCI/2019/21). Author Contribution N. U and N. N wrote the manuscript. M.D.M.F, B. G. D. N. K. D, P.S and H.K reviewed and edited the manuscript. 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Each value is expressed as mean ± standard deviation (n=3)]\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/bd62463c71d3cc8cb6e24840.jpg"},{"id":94603316,"identity":"e5d0ee4c-2dbc-4754-9f56-6637f44737cd","added_by":"auto","created_at":"2025-10-28 20:09:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe cytotoxicity effects of polyherbal decoction and individual component decoctions. A) PHD, B) APD, C) TPD, D) TCD, E) WSD, F) WSD HEK293 cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/a321ebd09a8169e4f938fba1.jpg"},{"id":94612320,"identity":"4abb05d1-4f83-4f49-910c-2f0bdcad08ca","added_by":"auto","created_at":"2025-10-29 02:09:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity effects of WSD at 24 h, 48 h and 72 h against HCT 116 cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/00b0f4a9c17325ec8138c76a.jpg"},{"id":94612440,"identity":"7593ab10-541c-4828-80a9-4ca8b3fa2a7e","added_by":"auto","created_at":"2025-10-29 02:10:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose dependent curve of LDH leakage.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/96e004d7ec9f68bd421d5ad0.jpg"},{"id":94612462,"identity":"687f7282-d30f-4e14-8e59-eb37e0f26138","added_by":"auto","created_at":"2025-10-29 02:10:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":41849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColony formation of HCT 116 cells upon treatment and after 7 days. A) WSD treated cells, B) Positive control, C) Negative control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/2615ee9be87768970aca9ebe.jpg"},{"id":94612698,"identity":"ee687ac7-4dde-4d07-a0c3-4b11ec68c17b","added_by":"auto","created_at":"2025-10-29 02:11:28","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":54594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) Emits orange-red fluorescence upon Ethidium bromide intercalation with DNA in HCT 116 cells; indicating late apoptotic cells and dead cells, B) Fragmented nuclei C) Negative control (untreated cells) D) positive control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/077b55449b80cef2da6576da.jpg"},{"id":94612513,"identity":"67081dde-eccd-4e35-84a9-bf5e52fdc61b","added_by":"auto","created_at":"2025-10-29 02:10:42","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":41495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of genomic DNA fragmentation. DNA fragmentation was assessed on 2% agarose gel electrophoresis and ethidium bromide staining. Lane 1: Negative control (1), Lane 2: 100bp ladder, Lane 3:Positive control (1), Lane 4: Positive control (2), Lane 5: WSD treated cells (24 hrs)-(1), Lane 6: WSD treated cells (24 hrs)-(2), Lane 7: WSD treated cells (24 hrs)-(3), Lane 8: Negative control (2)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/95f7284a44456cb10d99fd22.jpg"},{"id":94612550,"identity":"168acf5a-fdec-452e-88e3-38d2034f0b2e","added_by":"auto","created_at":"2025-10-29 02:10:54","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":30397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaspase activity of decoction, negative control and positive control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/967057e67d156cc29ced1a35.jpg"},{"id":94603320,"identity":"25f8bc07-74d2-403a-a743-437073dc1bf1","added_by":"auto","created_at":"2025-10-28 20:09:13","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":25155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of fold change in MMP2, STAT3, JAK2, MMP9, Bcl-XL, Ccnd1, VEGF and p53.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/a04206cb1d065ad60bfd87b7.jpg"},{"id":104433516,"identity":"f9ffd2f1-ee62-480f-8ea9-725fef43060b","added_by":"auto","created_at":"2026-03-11 16:10:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2153544,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/60ca580f-3ca9-45e3-becd-29ae985d2659.pdf"},{"id":94603317,"identity":"b7807f77-9bd3-47dc-a714-81eca7e2d799","added_by":"auto","created_at":"2025-10-28 20:09:13","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":105691,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/6a66da3a92f36e86645201e7.jpg"},{"id":94603325,"identity":"bfb9d10b-3561-4a5b-821b-751d8f067929","added_by":"auto","created_at":"2025-10-28 20:09:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":699870,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/886e655a4cba887086e8a1d7.docx"},{"id":94612612,"identity":"34f06207-8bac-4b95-b051-476ed9f6dab5","added_by":"auto","created_at":"2025-10-29 02:11:12","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":53594,"visible":true,"origin":"","legend":"","description":"","filename":"GelphotoORI.png","url":"https://assets-eu.researchsquare.com/files/rs-7141234/v1/c2adda98f40664a3977ba291.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Vitro Anticancer Effects of a Traditional Sri Lankan Polyherbal Decoction and Its Individual Components Against Colorectal Cancer Cells","fulltext":[{"header":"1.0 Introduction","content":"\u003cp\u003eColorectal cancer is a significant global health concern globally. In 2020, approximately 1.9\u0026nbsp;million new colorectal cancer cases were diagnosed, accounting for 9.6% of all cancer incidences, making it the third most commonly occurring cancer worldwide (WHO, 2025). In the same year, colorectal cancer led to about 900,000 deaths, representing the second leading cause of cancer mortality globally (WHO, 2025).\u003c/p\u003e\u003cp\u003eChemotherapy and radiotherapy remain the currently practiced treatments for colorectal cancer. However, these therapies often result in significant adverse effects. While short-term side effects such as nausea, fatigue, and stomatitis typically subside within a few months, long term complications including infertility, cardiac dysfunction, and secondary malignancies like leukemia can persist for years (Nurgali et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Partridge et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Given these limitations, there is an urgent need to explore alternative therapeutic strategies that are both effective and safe. Natural products, particularly plant-based compounds, have gained considerable attention in cancer research due to their diverse bioactive properties, including immune modulation and enhanced survival benefits (Kumar et al., 2016; Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Jafarian et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSri Lanka has a long-standing tradition of herbal medicine, with historical records indicating the use of medicinal plant extracts and decoctions for over 3,000 years (Weragoda, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Many of these formulations are reputed to have anticancer properties (Jayasinghe et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Luo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Notably, a decoction derived from the bark of \u003cem\u003eAdenanthera pavonina\u003c/em\u003e and \u003cem\u003eThespesia populnea\u003c/em\u003e, the stem of \u003cem\u003eTinospora cordifolia\u003c/em\u003e, and the tuberous root of \u003cem\u003eWithania somnifera\u003c/em\u003e have been incorporated into Sri Lankan traditional medicine and have claimed to be effective in cancer therapy with particular emphasis against colorectal cancer (Kuruppu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever clinical studies or other forms of \u003cem\u003ein vivo\u003c/em\u003e or \u003cem\u003ein vitro\u003c/em\u003e efficacy studies has not been conducted for poly herbal decoction (PHD). Present study aims to evaluate the anticancer, cytotoxic and apoptosis inducing activity of the PHD and its individual components in colorectal cancer cells. Additionally, the study seeks to determine the individual contributions of each plant component to the overall bioactivity of the decoction as well as total phenolic, flavonoid, and tannin content and the antioxidant properties. By providing scientific validation for the traditional use of this formulation, this research contributes to the growing body of evidence supporting herbal medicine as a complementary approach in cancer therapy.\u003c/p\u003e"},{"header":"2.0 Methodology","content":"\u003cp\u003e\u003cb\u003eCollection and identification of plant materials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll plant species of \u003cem\u003eTinospora cordifolia\u003c/em\u003e (TC), \u003cem\u003eThespesia populnea\u003c/em\u003e(TP), \u003cem\u003eAdenanthera pavonina\u003c/em\u003e (AP) were collected from Kotte, Western Province, Sri Lanka. \u003cem\u003eWithania somnifera\u003c/em\u003e (WS) was purchased from a registered ayurvedic material vendor (Sridhara ayurvedic shop, Malabe, Western Province, Sri Lanka) in Sri Lanka. Each plant species was identified by a botanist at Bandaranaike Memorial Ayurvedic Research Institute (BMARI), Sri Lanka and voucher specimen were deposited at BMARI (voucher specimen numbers2093, 2094 and 2095 for AP, TP and TC respectively).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of plant decoctions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe aerial parts of TC, the bark of TP, the bark of AP and the rhizome of WS were freeze-dried and the poly-herbal decoction was prepared as of the methods given by the traditional medical practitioners. The individual decoctions were prepared by using 15 g of each freeze-dried aerial parts of TC, the bark of TP, the bark of AP and the rhizome of WS. In brief, the plant material was added with 1600 mL of deionized water and was heated till the volume reduced to 1/8th (200 mL) of the initial volume (Lindamulage and Soysa \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The decoction was centrifuged (5000 rpm, 15 min) and the supernatant was freeze-dried to obtain the dried powder dissolved in deionized water/phosphate-buffered saline (PBS) to obtain known concentrations of plant extracts. Hereafter, decoctions of TC, TP, AP and WS will be indicated as TCD, TPD, APD and WSD respectively. Poly-herbal decoction will be indicated as PHD.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstimation of Total phenolic, tannin and flavonoid contents\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eEstimation of Total Phenol Content (TPC)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTPC of the decoctions was determined by Falin-Ciocalteau method (Kaur and Kapoor \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Gallic acid was used as the standard and a standard curve was obtained at a concentration range of 5\u0026ndash;65 \u0026micro;g/mL of gallic acid. The TPC of the decoctions were calculated from the standard curve (Fernando et al, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). All determinants were carried out in triplicates.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEstimation of Total Flavonoids Content (TFC)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe aluminium chloride colorimetric method was carried out to determine the total flavonoid contents of the decoctions (Fernando and Soysa \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Quercetin was used as the standard and the standard curve was obtained at a concentration range of 10-1000 \u0026micro;g/mL of quercetin. The TFC of the decoctions were calculated from the standard curve. All determinants were carried out in triplicates.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEstimation of Tannin content (TAC)\u003c/em\u003e\u003c/p\u003e\u003cp\u003e The tannin content (TAC) of the plant decoctions was determined according to the following method described (Pulipati et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A volume of 500 \u0026micro;L of the decoction (20 mg/mL) was mixed with deionized water (500 \u0026micro;L) and 50 mg of polyvinyl polypyrolidone (PVPP) and it was vortexed and refrigerated (4 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e, for 15 min). The reaction mixture was subjected to vortex again and was centrifuged at 3000 g, for 10 min. A concentration series was prepared at 70-5000 \u0026micro;g/mL from the supernatant to estimate the tannin content. The rest of the reaction method was performed according to the phenolic content estimation. Tannic acid was used as the standard and a standard curve was obtained at a concentration range of 20\u0026ndash;73 \u0026micro;g/mL of tannic acid. The tannin content of the decoctions was calculated from the standard curve and all determinants were carried out in triplicates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of antioxidant properties\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eFree radical scavenging properties\u003c/em\u003e\u003c/p\u003e\u003cp\u003eDPPH radical scavenging assay (Soysa and Silva \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), Ferric reducing antioxidant power assay (FRAP Assay) (Jemli et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Hydroxyl radical scavenging assay (Halliwell et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, Poorna et al. 2013) were performed according to the standard methods given in the literature. All determinations were carried out in triplicate.\u003c/p\u003e\u003cp\u003e\u003cem\u003eProtein and lipid oxidation inhibition\u003c/em\u003e\u003c/p\u003e\u003cp\u003eInhibition of Protein oxidation was carried out using protein oxidation inhibition assay (Poorna et al. 2013; Martnez et al. 2001) and inhibition of lipid oxidation was performed using egg yolk lipids (Kizil et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Perera et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Dhar et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) to the standard methods reported in previous study.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell cultures\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHCT116 colorectal cancer cell line was used to determine the cytotoxicity of the targeted plant decoctions. HCT116 cell line was acquired from the Medical Research Institute, Colombo, Sri Lanka. It was cultured in Modified Eagle\u0026rsquo;s medium (MEM) supplemented with 10% fetal bovine serum (FBS), 3% L-glutamine, 7% Sodium bicarbonate, streptomycin (100 \u0026micro;g/mL), and penicillin (100 U/mL). Cells were incubated in humidified air at 37\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e. Exponentially growing (80% confluent) cells were passaged every 2\u0026ndash;3 days using 0.25% trypsin\u0026ndash;EDTA solution (Perera et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Razak et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vichai and Kirtikara \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Fernando et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxicity assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eDimethylthiazol-diphenyltetrazolium bromide (MTT) assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCytotoxicity was determined using MTT cell viability assay after being subjected to the 24 h treatment of the PHD and individual decoctions to HCT116 cells (Wageesha et al. 2017). Experiments were carried out in triplicates. WSD decoction was subjected to 24 h, 48 h, and 72 h after the initial screening.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of cell lysate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell plates were prepared and treated according to the previously described method (Fernando et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Each well was washed with PBS (100 \u0026micro;L, pH 7.4) three times. Thereafter, Triton-X-100 (100 \u0026micro;L, 0.1 v/v) was added to each well and subjected to sonication for 5 seconds. The cell lysates were then collected and centrifuged at 3000 rpm for 5 min to remove cell debris.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLDH leakage\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were treated with WSD decoction, assay was performed according to the manufacture\u0026rsquo;s description (LDH SCE mod. liquiUV, Human Diagnostics, Germany) for both the treated medium and cell lysate. The percentage LDH leakage was determined by the equation given below;\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Percentage\\:LDH\\:leakage\\:\\:=\\:\\frac{LDH\\:activity\\:in\\:the\\:supernatant}{Total\\:LDH\\:activity}\\)\u003c/span\u003e\u003c/span\u003e \u0026times;100 (Wageesha et al., 2017)\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony formation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTrypsinized cells were seeded in a culture dish and after 24 h cells were treated with the WSD as previously described. The colonies were stained with 6% glutaraldehyde and 0.5% crystal violet. The colonies were counted using stereomicroscope (Franken, 2006).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCaspase assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were seeded and treated with WSD as previously described, and the assay was performed according to the manufacturer\u0026rsquo;s description (Caspase 3 Activity Assay Kit, China (E-CK-A311).\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Caspase\\:activity\\:\\:=\\:\\frac{{Absorbance}_{sample}-{Absorbance}_{blank}}{{Absorbance}_{negative\\:control}-{Absorbance}_{blank}}\\)\u003c/span\u003e\u003c/span\u003e \u0026times;100\u003c/p\u003e\u003cp\u003e\u003cb\u003eEthidium bromide/acridine orange (EB/AO) assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDecoction-treated cells were stained with a 1:1 mixture of Ethidium bromide and acridine orange, 100 \u0026micro;g/mL, prepared in PBS and observed in UV under a fluorescent microscope (Wageesha et al., 2017). Morphological criteria were used to evaluate cell injury. Cells containing normal nuclear chromatin exhibit green nuclear staining. Cells containing fragmented nuclear chromatin due to apoptosis exhibit orange to red nuclear staining.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA fragmentation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTreated cells were trypsinized and were collected in microcentrifuge tubes (4\u0026times;10\u003csup\u003e5\u003c/sup\u003e per microcentrifuge tube). Cells were pelleted by centrifuging (2000 rpm, 5 min, 4 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃)\\)\u003c/span\u003e\u003c/span\u003e. TES lysis buffer (20 \u0026micro;L) was added, and the pellet was carefully resuspended. RNase was added (10 mg/mL, 10 \u0026micro;L) added and incubated for 2 hrs at 37 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e. Thereafter, Proteinase K (20 mg/mL, 10 \u0026micro;L) was then added and incubated at 50 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e overnight. The sample (10 \u0026micro;L) was mixed with 2 \u0026micro;L of DNA loading dye and was run in 1.5% agarose gel(Kasibhatla et al., 2006)(Wageesha et al., 2017).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene expression analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBased on the cytotoxicity, oxidative, and morphological changes results, a hypothetical gene expression pathway that might have expressed in driving cell death (apoptosis) was postulated. In that gene expression pathway, 9 genes were targeted (with the house keeping gene, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and primers were designed to analyze the relative gene expression during cell death.\u003c/p\u003e\u003cp\u003eSome primers were manually designed following the standard guidelines (Bustin \u0026amp; Huggett, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dieffenbach et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Either the RefSeq transcripts or all the transcript variants were considered in primer designing. A few primers were acquired from published literature. Primer sequence, alignment, position of annealing, nucleotide length, melting temperature and GC percentage of each primer are included in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRT-qPCR and Gene expression analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eRT-qPCR analysis of the genes was performed in Rotor Gene Q (Qiagen, Germany), following the given amplification parameters (40 cycles for Initial denaturation at 95 ℃ for 3 minutes, Denaturation at 95 ℃ for 30 seconds, Annealing at 60 ℃ for 30 seconds, Extension at 72 ℃ for 1.0-minute, Final extension at 72 ℃ for 1.0 minute, 4 ℃, \u0026infin;). Reaction mixtures were prepared as follow GoTaq\u0026reg; qPCR Master Mix, 2X; 10.0 \u0026micro;L (1X), Forward Primer (20X); 1.0 \u0026micro;L (250 nM), Reverse Primer (20X); 1.0 \u0026micro;L (250 nM), Nuclease-Free Water; 5.0 \u0026micro;L, cDNA template; 3.0 \u0026micro;L. Melting curves were obtained and the relative gene expression was evaluated. A small aliquot (10 \u0026micro;L) of each RT-qPCR product was loaded along with the loading buffer. The gel was run in ethidium bromide stained, 0.8% w/v agarose gel at 90 V. It was observed under UV after running for 15 minutes and 30 minutes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical data analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe data were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. For comparison, data were analyzed by regression and Pearson analysis. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers used in gene expression analysis\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePrimer sequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePrimer length\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAnnealing site\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMelting Temperature\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eGC content (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eProduct Length (cDNA)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMatrix metalloproteinase 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMMP2_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' TACAGGATCATTGGCTACACACC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e661\u0026ndash;683\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e63.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Dai et al., 2017)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMMP2_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GGTCACATCGCTCCAGACT 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e732\u0026ndash;750\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e63.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Dai et al., 2017)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMatrix metalloproteinase 9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMMP9_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' TTGACAGCGACAAGAAGTGG 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1155\u0026ndash;1174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e179\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Uehara et al., 2017)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMMP9_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GCCATTCACGTCGTCCTTAT 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1314\u0026ndash;1333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e62.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Uehara et al., 2017)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e18 S (house-keeping gene)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18S_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' CATTCGTATTGCGCCGCTAG 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e937\u0026ndash;956\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e63.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e135\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18S_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' CTACGACGGTATCTGATCGTC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1051\u0026ndash;1071\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e61.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e52.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eB-cell lymphoma-extra large\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBclxl_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GCGTAGACAAGGAGATGCAG 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e595\u0026ndash;614\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e62.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e136\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBclxl_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GCATTGTTCCCATAGAGTTCCAC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e708\u0026ndash;730\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e62.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Taghavi et al., 2019)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSignal transducer and activator of transcription 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSTAT3_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' AGCAGTTTCTTCAGAGCAGGT 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e755\u0026ndash;774\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e60.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSTAT3_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GCGTGATTCTTCCCACAGGC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e868\u0026ndash;888\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e60.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCyclin D1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCcnd1_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GATACCAGAAGGGAAAGCTTC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1141\u0026ndash;1161\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e60.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e189\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCcnd1_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' ATCTGTAGCACAACCCTCCTC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1309\u0026ndash;1329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e62.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e52.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVascular endothelial growth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVEGF_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' CTACCTCCACCATGCCAAGT 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1084\u0026ndash;1103\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e63.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e109\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Du et al., 2015)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVEGF_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GCAGTAGCTGCGCTGATAGA 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1173\u0026ndash;1192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e63.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Du et al., 2015)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP53_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' TATGAGCCGCCTGAGGTTGG 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1653\u0026ndash;1672\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e60.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(H\u0026uuml;bner et al., 2018)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP53_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' CGCACCTCAAAGCTGTTCCG 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1738\u0026ndash;1757\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e60.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(X. M. Li et al., 2020)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGlyceraldehyde 3-phosphate dehydrogenase (house-keeping gene)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGAPDH_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GGAGCGAGATCCCTCCAAAAT 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e554\u0026ndash;574\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e63.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e52.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e197\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Fu et al., 2019)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGAPDH_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' GGCTGTTGTCATACTTCTCATGG 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e728\u0026ndash;750\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e63.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(Fu et al., 2019)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eJanus Kinase 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJAK2_F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' TTGGAGCTTTGGAGTGGTTC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3784\u0026ndash;3803\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e62.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJAK2_R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5' ATCTGGGCATCCATCTGGTC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3942\u0026ndash;3961\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e63.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNewly designed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"3.0 Results and Discussion","content":"\u003cp\u003e\u003cb\u003eEstimation of total phenolic, flavonoid and tannin contents\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEstimated Total phenolic (TPC), total flavonoid (TFC) and total tannin contents (TAC) of the poly herbal decoction and individual plant decoctions are summarized in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. According to the results, APD and TPD have shown higher TFC and TAC contents. However, in TPC estimation, the highest value (49.01\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 mg GAE/g) was observed in PHD while TPD and WSD resulted in considerably lower values (1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg GAE/g, 0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mg GAE/g respectively). The estimation of TPC considers flavonoids and tannins, which encompasses a broad range of compounds including other phenol-type derivatives, this may be the reason for the deviations in TPC estimation (Muflihah, 2021).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTotal Phenolic Content, Total Flavonoid Content and Tannin Content of the PHD and individual plant decoctions; APD, TPD, TCD and WSD. The values are mean of three independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDecoctions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTPC (mg GAE/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTFC (mg QCE/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTAC (mg TAE/g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePHD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e49.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e24.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e44.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e64.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTPD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e74.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTCD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cb\u003eAntioxidant assays\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the present study, three different antioxidant assays are used to accurately confirm the antioxidant ability of each decoction.\u003c/p\u003e\u003cp\u003e\u003cem\u003eDPPH assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe present findings indicate that all decoctions induced a colorometric transition of DPPH confirming the antioxidant capacity. PHD exhibited antioxidant activity with an EC\u003csub\u003e50\u003c/sub\u003e value of 44.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 \u0026micro;g/mL, while TCD and WSD resulted with EC\u003csub\u003e50\u003c/sub\u003e value of 1025.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6 \u0026micro;g/mL 1011.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.9 \u0026micro;g/mL respectively. APD displayed significantly stronger antioxidant activity (EC\u003csub\u003e50\u003c/sub\u003e value of 15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026micro;g/mL) as given in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eHydroxyl radical scavenging assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAll the decoctions produced a characteristics pink chromogen upon thermal treatment, indicating the presences of phenolic compounds. PHD exhibited antioxidant activity with an EC\u003csub\u003e50\u003c/sub\u003e of 36.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;g/mL and TPD had the most potent antioxidant activity (EC\u003csub\u003e50\u003c/sub\u003e value of 13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;g/mL), while WSD resulted in weakest anti-oxidant activity with EC\u003csub\u003e50\u003c/sub\u003e 858.1\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3 \u0026micro;g/mL (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparative Antioxidant activities of PHD and individual plant decoctions. The values are mean of three independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDecoction/ Standard\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e values (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDPPH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHydroxyl radical scavenging assay\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePHD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTPD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTCD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1025.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e816.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1011.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e858.1\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAscorbic Acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e5.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eFerric ion reducing power assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe dose-response curves of ferric ion reducing power assay are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. TCD (200\u0026ndash;600 \u0026micro;g/mL of concentration range in between 0.300-1.000 of absorbance) and WSD (with 175\u0026ndash;600 \u0026micro;g/mL of concentration range in between 0.300\u0026ndash;0.600 of absorbance) were excluded in the figure.\u003c/p\u003e\u003cp\u003eResults from all three antioxidant assays, confirmed significant anti-oxidant activities in all tested decoctions with varying potency. APD and TPD demonstrated strongest antioxidant properties whereas TCD and WSD with the weakest antioxidant properties PHD exhibited intermediate anti-oxidant properties due to its composition containing individual components, PHD was prepared as a mixture including all the individual components together where it can be expected to have the highest antioxidant properties as a collective of all the herbal decoctions. However, this effect may be affected by the interactions and reactions of different components which may not undergo when they are in separate individual extracts and also no correlation was observed between antioxidant (DPPH) and TPC, TFC and TAC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOxidation assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eInhibition of Protein oxidation\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe maximum inhibitory activity (lowest EC\u003csub\u003e50\u003c/sub\u003e) was demonstrated in PHD at 223.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 \u0026micro;g/mL, while TCD exhibited a marginal inhibitory effect at EC\u003csub\u003e50\u003c/sub\u003e value of 511.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 \u0026micro;g/mL (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eInhibition of protein oxidation and lipid peroxidation shown by PHD and other individual plant decoctions; APD, TPD, TCD and WSD. The values are mean of three independent experiments (n\u0026thinsp;=\u0026thinsp;3)\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD)\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDecoction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e values (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProtein oxidation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLipid peroxidation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePHD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e223.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e383.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e343.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e206.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTPD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e260.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e207.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTCD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e511.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e523.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e295.0\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e156.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eInhibition of Lipid Peroxidation\u003c/em\u003e\u003c/p\u003e\u003cp\u003eLipid peroxidation was quantified spectrophotometrically by measuring the chromogenic transition in the reaction medium. WSD demonstrated the maximal inhibitory activity(EC\u003csub\u003e50\u003c/sub\u003e 156.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12.4 \u0026micro;g/mL), while APD (EC\u003csub\u003e50\u003c/sub\u003e 206.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 \u0026micro;g/mL) and TPD (EC\u003csub\u003e50\u003c/sub\u003e 207.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 \u0026micro;g/mL ) exhibited the minimal marginal inhibitory activity as given in Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. All decoctions exhibited ac considerable anti-oxidant activity against both protein oxidation a lipid oxidation. However, no significant correlation was observed between these protective effects, suggesting distinct mechanisms of action for each oxidative pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxicity assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMTT assay was employed in determining the cytotoxicity of the decoctions against the colorectal carcinoma. Level of cytotoxicity of the PHD and its individual decoctions against HCT 116 and HEK 293 cells were determined by MTT assay after 24 h of treatments (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the highest cytotoxic effect was observed with WSD (EC \u003csub\u003e50\u003c/sub\u003e value; 83.1 \u0026micro;g/mL), However, no significant e cytotoxic effect was detected in TCD decoction, while PHD (EC\u003csub\u003e50\u003c/sub\u003e 383.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 \u0026micro;g/mL) resulted in a comparatively higher EC\u003csub\u003e50\u003c/sub\u003e value than TPD (EC\u003csub\u003e50\u003c/sub\u003e 268.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 \u0026micro;g/mL\u003cb\u003e)\u003c/b\u003e the least active individual component (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The EC\u003csub\u003e50\u003c/sub\u003e values of the individual components resulted in a wide range from 83.1 \u0026micro;g/mL to 268.3 \u0026micro;g/mL.\u003c/p\u003e\u003cp\u003eA positive correlation was observed in PHD, APD, TPD and TCD decoctions, however a significant correlation was not observed in WSD, which demonstrated a strong cytotoxicity comparatively to other decoctions. While WSD exhibited potent anti-cancer activity, due to its low total phenolic, flavonoid and tannin content a significant correlation was not observed. Comparatively WSD exhibited highest anti-cancer activity against HCT 116 cells than individual components decoction and PHD. Based on this activity, WSD was selected for further studies.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLevel of cytotoxicity of the PHD and its individual decoctions against HCT 116 and HEK 293 cells by MTT assay after 24 h of treatments, expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three individual experiments (n\u0026thinsp;=\u0026thinsp;3). *n\u0026thinsp;=\u0026thinsp;2\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDecoctions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell line\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e values (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePHD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHCT 116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e383.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAPD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHCT 116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e191.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTPD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHCT 116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e268.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTCD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHCT 116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNot detected\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHCT 116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHEK 293\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e534.5\u0026thinsp;\u0026plusmn;\u0026thinsp;16.0*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e* The value is a mean of two independent experiments (n\u0026thinsp;=\u0026thinsp;2)\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn consistent to our study, A prior research reported the dose dependent and time dependent anti-cancer activity in \u003cem\u003eWithania somnifera\u003c/em\u003e crude extract against human malignant melanoma cells (Halder, 2015). Evidently a study performed with \u003cem\u003eWithania somnifera\u003c/em\u003e fruit extract resulted in cytotoxicity against HCT 116 cells with a LC\u003csub\u003e50\u003c/sub\u003e value of 410.2 \u0026micro;g/mL, and HepG2 cells with a LC\u003csub\u003e50\u003c/sub\u003e value of 164.7 \u0026micro;g/mL as the highest cytotoxicity demonstrated (Abutaha, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCytotoxicity effects of the WSD after 24, 48 and 72 h of treatments.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e values (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e81.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e48 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e72 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure SEQ Figure \\* ARABIC 3. At 24 hours, EC\u003csub\u003e50\u003c/sub\u003e of WSD was 81.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 \u0026micro;g/mL. When the incubation period was extended for 24 and 72 hours, the EC\u003csub\u003e50\u003c/sub\u003e values decreased to 17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;g/mL and 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;g/mL against HCT 116 cells respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLDH leakage\u003c/b\u003e\u003c/p\u003e\u003cp\u003eQuantification of membrane integrity damage was demonstrated upon WSD treatment within 24 h, a significant LDH leakage (59.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1%) was exhibited resulting in dose dependent cytotoxicity as demonstrated in Fig.\u0026nbsp;4. LDH leakage and increased level of LDH release were previously reported in \u003cem\u003eWithania somnifera\u003c/em\u003e extract-treated on androgen-independent prostate cancer 3 cells (PC3) exhibiting the anti-proliferative effect. (Balakrishnan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Similarly, in another study \u003cem\u003eWithania somnifera\u003c/em\u003e root powder was reported to have a significant suppression in lysosomal and cytoplasmic enzyme release in polymorphonuclear leucocytes cells (Rasool, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony forming assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs of the obtained data, the WSD decoction treatments have significantly reduced cell proliferation and differentiation into colonies (262\u0026thinsp;\u0026plusmn;\u0026thinsp;20). The negative control resulted in higher number of colonies after 7 days of the treatments (595\u0026thinsp;\u0026plusmn;\u0026thinsp;22), while the positive control resulted in 2\u0026thinsp;\u0026plusmn;\u0026thinsp;2 colonies, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eSumantran et al \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e reported that aqueous extract of \u003cem\u003eWithania somnifera\u003c/em\u003e roots had a dose dependent inhibition in colony formation with Chinese Hamster ovarian cell line. It also reported that \u003cem\u003eWithania somnifera\u003c/em\u003e root extract induce long term growth inhibition of CHO cells which was cell density dependent upon the drug treatment (Sumantran et al \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEthidium bromide/acridine orange (EB/AO) assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAcridine orange (AO) is a chromatin staining dye that emits green fluorescence upon intercalation and enters live cells with normal membrane potentials. Hence, it is important in live, healthy and proliferating cells. Ethidium bromide (EB) penetrates only the cells that have lost their membrane integrity and emits a fluorescence of red upon DNA intercalation. Further, it dominates over AO. This makes the live cells in green colour and apoptotic and dead cells in orange-red colour (Ribble et al., 2005).\u003c/p\u003e\u003cp\u003eNuclei stained with green colour indicate live cells, while greenish yellow shows early apoptotic cells. Condensed orange red nuclei demonstrate late apoptotic cells, whereas red colour indicates dead cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the EB/AO assay resulted in prominent apoptotic morphology in the HCT 116 cells upon treating with WSD for 24 hours. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, orange colour and red colour cells were observed, indicating the late apoptotic cells and dead cells respectively. When the cells were observed in high power, fragmented nuclei were observed as in 6B. The images 6C and 6D indicate the negative and positive control respectively. The morphological changes of the images indicate the ongoing apoptosis in the cells upon treatment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA fragmentation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e, HCT 116 cells resulted in DNA fragments upon treatment after 24 hours. According to the gel image visualization, faint bands of DNA fragments provide evidence of active apoptosis. As the negative control is free of such DNA fragments, it can be concluded that the WSD treatment has initiated apoptosis and DFF function, resulting DNA fragments in the HCT 116 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003c/div\u003e\u003cp\u003eDNA fragmentation is a main feature of apoptosis and thus is considered as a marker of apoptosis. During apoptosis, double-stranded DNA is cleaved at A and T-rich regions by the DNA fragmentation factor (DFF). The 40 kDa catalytic subunit (DFF40) provides the endonuclease activity of the DFF for DNA cleavage, while 45 kDa subunit (DFF45) provides the regulatory functions (Majtnerov\u0026aacute; \u0026amp; Roušar, 2018). At normal stages, DFF40 remains inactive, which is regulated by DFF45. Upon activation of Casapse3, the DFF40-DFF45 complex is cleaved. Active DFF40 is released, and hence, DNA fragmentation is started. It cleaves DNA in about 180 bp and its multiples (360 bp, 540 bp and 720 bp), resulting a ladder pattern upon agarose gel run (Majtnerov\u0026aacute; \u0026amp; Roušar, 2018).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEvidently in a previous study \u003cem\u003eWithania somnifera\u003c/em\u003e extracts significantly induce in apoptosis against Hepatocellular carcinoma cells (Ahmed et al, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Another study reported that caspase activity was increase on a time dependent manner against HL- 60 cells (Malik, 2009\u003cb\u003eFigure 8. Caspase activity of decoction, negative control and positive control.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of WSD on MMP2, STAT3, JAK2, MMP9 ,Bcl-XL, Ccnd1, VEGF and p53\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eThe effect of MMP2 and MMP9 in apoptosis and cell death\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMMP-2 has a role in promoting tumour angiogenesis, which is initially supported by interleukin 8 (IL-8) (Quintero-Fabi\u0026aacute;n et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study both MMP-2 and MMP-9 were down regulated with negative fold changes of -14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 and \u0026minus;\u0026thinsp;5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 respectively. This indicates that the WSD treatment effectively retards the angiogenesis and metastasis in the cancer. It was reported that \u003cem\u003eWithania somnifera\u003c/em\u003e has great potential in down regulating MMP-2 and MMP-9, leading to the inhibition of cell migration in neuroblastoma (Kataria et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eThe effect of Cyclin D1 in cell cycle arrest\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCyclin D1 shows a negative fold change of -12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 where the expression is being downregulated. Considerably \u003cem\u003eWithania somnifera\u003c/em\u003e has been reported in previous literature as an agent to reduce the Cyclin D1 level in human prostate cancer (Balakrishnan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) cells and human neuroblastomas (Kataria et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eThe effect of VEGF in reducing angiogenesis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWithaferin A, the active component of \u003cem\u003eWithania somnifera\u003c/em\u003e has been identified in a previous research study as a compound that reduces the expression of VEGF functioning as an anti-VEGF agent (Saha et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, VEGF has exhibited a negative fold change of 7.6, indicating the down-regulation of the expression. This indicates the effectiveness of the WSD in retarding angiogenesis and proliferation of the tumour.\u003c/p\u003e\u003cp\u003e\u003cem\u003eThe effect of STAT3 and JAK2 in apoptosis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAccording to the experimental results given in the Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the expression of both JAK2 and STAT3 were down regulated in 13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 and 10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 folds respectively which exhibited significantly higher fold changes, collectively this study demonstrated that the WSD decoction promotes apoptosis involving the JAK2/STAT3 gene expression pathway, leading to the death of the tumour cells. The effect of \u003cem\u003eWithania somnifera\u003c/em\u003e on STAT3 and JAK2 pathways was described in related to renal carcinoma (Um et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and breast cancers (J. Lee et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) in the literature.\u003c/p\u003e\u003cp\u003e\u003cem\u003eThe effect of Bcl-XL in apoptosis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAs evident by, the differential expression of Bcl-XL dropped in around 14 folds (-14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1), indicating the effectiveness of the decoction in promoting apoptosis. The effect of the \u003cem\u003eWithania somnifera\u003c/em\u003e on reducing the expression level of Bcl-XL in human Neuroblastomas was reported in a previous study (Kataria et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003ep53\u003c/em\u003e\u003c/p\u003e\u003cp\u003ethe analysis demonstratep53 relative expression has increased significantly (11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 folds). This can be an overall effect generated from the DNA damage due to the upregulation of p53 and STAT3 down regulation. The upregulation of p53 promoted by \u003cem\u003eWithania somnifera\u003c/em\u003e has also been previously reported (Munagala et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eNames and short names of the genes with their fold change expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D of three independent experiments.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName of the gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eShort name\u003c/p\u003e\u003cp\u003eof the gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFold change\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMatrix Metallopeptidase 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMMP-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSignal transducer and activator of transcription 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSTAT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJanus kinase 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJAK2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMatrix Metallopeptidase 9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMMP-9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB-cell lymphoma-extra large\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBcl-XL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCyclin D1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCcnd1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVascular endothelial growth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVEGF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor protein 53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.7 \u0026plusmn; 1.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyceraldehyde 3-phosphate dehydrogenase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-1.5 \u0026plusmn; 0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4.0 Conclusions","content":"\u003cp\u003ePoly-herbal decoction (PHD) is enriched with valuable anti-oxidative and anticancer properties which were differently and unevenly contributed by its individual components. Each component might be contributing in a balanced manner in such a way as to compensate any toxicity that might result from hyperactivities. As PHD is prepared by adding several herbs in a single mixture, each acts as a protective agent by reducing the effects of other components making it is tolerable and harmless to the normal cells and tissues. In summary, the poly-herbal decoction and all the individual plant decoctions that was used in this study have shown antioxidant and anticancer properties implying the medicinal value of traditional plant decoctions used in Sri Lanka. In addition, current study provides a scientific validation for the traditional use of this poly-herbal decoction in anti-cancer treatments. The findings of this study can be used as an initiation for performing more expanded cancer research using traditional plant decoctions towards a remedy for colorectal cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDPPH- 2,2-diphenyl-1-picrylhydrazyl\u003c/p\u003e\n\u003cp\u003eFRAP- Ferric Reducing Antioxidant Power\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMTT- 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide\u003c/p\u003e\n\u003cp\u003eRT-qPCR- Real-time quantitative Polymerase Chain Reaction\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLDH- Lactate dehydrogenase\u003c/p\u003e\n\u003cp\u003ePVPP- Polyvinyl polypyrolidone\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMEM- Modified Eagle’s medium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFBS- Fetal bovine serum\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflict of interest\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis project was funded by the University of Sri Jayewardenepura, Sri Lanka (Grant No: ASP/01/RE/SCI/2019/21).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN. U and N. N wrote the manuscript. M.D.M.F, B. G. D. N. K. D, P.S and H.K reviewed and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThe authors of this article are extremely grateful for the kind support of Dr. Nimal Jayathilake for the information provided in selecting and preparing herbal decoctions that are prescribed in Sri Lankan traditional cancer treatments.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe dataset supporting the conclusions of this article are included within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbutaha N. In vitro antiproliferative activity of partially purified Withania somnifera fruit extract on different cancer cell lines. J BUON. 2015;20(2):625\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmed W, Mofed D, Zekri AR, El-Sayed N, Rahouma M, Sabet S. 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Evidence-Based Complement Altern Med. 2013;2013(1):302426.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Poly herbal decoction, Withania somnifera, Anti-cancer, Anti-oxidant, Phytochemicals, Colorectal cancer","lastPublishedDoi":"10.21203/rs.3.rs-7141234/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7141234/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eColorectal cancer is globally the third highest prevalent and second highest mortality malignancy. Modern treatments were failed to reduce the cancer deaths to the expected level via producing exceptional side effects. On the contrary, traditional herbal medicine is gaining more attention in cancer remedies mainly due to its high efficacy and fewer side effects. The current study was aimed at investigating the anticancer and antioxidant properties of a poly-herbal decoction prescribed in Sri Lankan traditional cancer treatments. A polyherbal decoction (PHD) prepared using the \u003cem\u003eAdenanthera pavonina\u003c/em\u003e, \u003cem\u003eThespesia populnea\u003c/em\u003e, \u003cem\u003eTinospora cordifolia\u003c/em\u003e, and \u003cem\u003eWithania somnifera\u003c/em\u003e, and decoctions prepared from these four individual plants were used commonly in Sri Lankan traditional medicine for colorectal cancer treatments.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003einvestigated for their total polyphenolic content, anti-oxidant activity, in vitro anti-colon cancer and apoptosis inducing activity (in HCT116 cell line) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, Ferric Reducing Antioxidant Power (FRAP) assay, hydroxyl radical scavenging assay, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, caspase activity assay and Real-time quantitative Polymerase Chain Reaction (RT-qPCR) respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOut of the five decoctions, \u003cem\u003eW. somnifera\u003c/em\u003e decoction (WSD) exhibited strong anti-colon cancer activity (EC₅₀ = 83.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 \u0026micro;g/mL) while PHD and other decoctions exhibited limited anti-cancer activity (EC₅₀ \u0026gt;100 \u0026micro;g/mL). WSD suppressed colony formation and induced caspase dependent programmed cell death, and lactate dehydrogenase (LDH) leakage. Up regulation of tumor suppressor p53 gene expression and down regulation of expression of key oncogenic and anti-apoptotic genes MMP-2, MMP-9, STAT3, JAK2, Cyclin D1, VEGF, and Bcl-XL were observed.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOverall findings validates the traditional use of Poly Herbal Decoction and \u003cem\u003eW. somnifera\u003c/em\u003e decoction against colon cancer.\u003c/p\u003e","manuscriptTitle":"In Vitro Anticancer Effects of a Traditional Sri Lankan Polyherbal Decoction and Its Individual Components Against Colorectal Cancer Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-28 20:09:08","doi":"10.21203/rs.3.rs-7141234/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4bd8f3ba-ab99-4dcd-a874-8d9aa56f6bd3","owner":[],"postedDate":"October 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-11T16:10:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-28 20:09:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7141234","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7141234","identity":"rs-7141234","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}