Antiproliferation and Apoptosis of the Crude Extract of Andrographis

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1 Thai Journal of Physiological Sciences Vol. 21 No. 2 Oct Mar Original Article Antiproliferation and Apoptosis of the Crude Extract of Andrographis paniculata Nees, on Human Oropharyngeal Cancer Cells (KB) In Vitro ABSTRACT Andrographis paniculata Nees. has a surprisingly broad range of pharmacological effects for centuries and has not been associated with any major side effects. The Andrographis paniculata extract (APE) and its mains diterpenoid components have been found to be able to inhibit cancer cell proliferation, induce cellcycle arrest and promote apoptosis in different types of human cancer cell lines. However, there have been very limited studies on the anti-proliferative activities and the underlying mechanisms of APE in oropharyngeal cancer. The aim of this study is to evaluate the effects of APE on the molecular mechanisms for antiproliferative activity and induction of apoptosis in human oropharyngeal cancer cells (KB). The antiproliferative effect of APE against KB cancer cells was determined by colorimetric assay. The morphology of apoptotic nuclei was quantified using double fluorescent staining: DAPI and propidium iodide (PI). Cells were then visualized with fluorescence microscope. For each treatment group, nuclei were counted. Data were expressed as percentages of fragment nuclei in viable cells. The degree of fragmentation was analyzed using a 1.5% agarose gel electrophoresis followed by an SYBER gold staining. APE inhibited cell proliferation in a dose-dependent manner. Further, APE-induced cell death was associated with round cells, lost of cell-to-cell contract and fewer adherent cells when compared with control group. The IC 50 values for APE was 80 ± 3.7 µg/ml. Nuclear morphology in APE-treated cells exhibited chromatin condensation, and nuclear fragmentation as compared to control. Quantitative estimation of apoptotic nuclei in APE-treated cells dose 80 µg/ml for 48 hr. was 7.85 ± 0.9% (normal cell), ± 7.5% (viable cells with apoptotic nuclei) and 10.0 ± 1.5% (necrosis or late apoptotic nuclei). The oligonuclesomal DNA fragmentation in agarose gel was observed in a dose-dependent manner when cells were treated with AP extract 40, 80 and 180 µg/ml for 48 hr. These findings indicated that the APE exhibited the anti-proliferative effects via morphological changes typical of apoptosis including membrane blebbing, chromatin condensation, nuclear and DNA fragmentation. As apoptosis has become a therapeutic target in cancer research, the potential of APE may act as natural remedies combined with synthetic chemotherapeutic compounds that might improve efficacy and decrease side effects in oropharyngeal cancer. Key words: Andrographis paniculata Nees., KB cells, Antiproliferation, Apoptosis, DNA fragmentation Department of Medical Science, Faculty of Science, Burapha University, Chonburi Address for correspondence: Chantarawan Saengkhae, Department of Medical Science, Faculty of Science, Burapha University, Chonburi schantara@yahoo.com INTRODUCTION Since medicinal plants contain some good sources of bioactive compounds, recent research has also focused on natural products which possess anti-cancer properties. Such compounds are candidates for chemotherapeutic or chemopreventive agents against cancer which 40

2 Antiproliferation and Apoptosis of the Crude Extract of Andrographis paniculata Nees might decrease undesirable side effects from chemotherapies. There is always the possibility that the herbal medicine would be an effectively treat cancer 1. The oropharyngeal cancers are more common among men and in people over age 50. The risk increases with the amount of alcohol and tobacco that a person consumes. At present, the cancer treatment by chemotherapeutic agents, surgery and radiation have not been fully effective against the high incidence of developing a new cancer and unwanted side effects. Scientists continue to search for more effective cancer treatments and find better ways to reduce the side effects 1. The development of medicinal plants against oropharyngeal cancer remains one of the most challenging areas in cancer research. Andrographis paniculata Nees, commonly known as King of bitters, is an erect annual herb in the family Acanthaceae. It is a well known medicinal plant of Ayurveda as a popular remedy for the treatment of various disorders. The aerial parts (leaves and stems) of this herb were traditionally used for various medicinal purposes in India, China, Thailand and Southeast Asia. Extensive research has revealed that this herbal extract has a wide spectrum of pharmacological activities, including anti-diarrhea 2, anti-inflammatory 3, antibacterial 4, antiviral 5, immuno-stimulant 6,7, hepatoprotective 8-10, antiplatelet aggregation 11, anticancer 12,13. Andrographis paniculata extract (APE) has recently become popular as a safe natural folk remedy for drug in the prevention and treatment of uncomplicated common cold 14. The major bioactive constituents of APE are diterpene lactones including andrographolide, deoxyandrographolide and neoandrographolide 15. Andrographolide exerts its anti-inflammatory effects by inhibiting NF-kB 3. It has been well established that NF-kB is generally acting as a regulator of genes that keep the cell proliferating and protect the cell from conditions that cause it to die 16. In addition, some cancer cells secrete factors that cause NF-kB to become active. Therefore, the inhibitory effect of Andrographolide on NF-kB implies both anti-cancer and anti-inflammatory activity. Furthermore, the andrographolide and an ethanolic extract can stimulate both antigen-specific and nonspecific immune responses in mice suggesting its effective against a variety of infectious and cancer cell without any signs of toxicity 6. The diterpene andrographolide exhibited the immunomodualtory and anticancer activities in human colon cancer cells (HT-29) that can use for develop non-toxic anticancer molecules 7. Recently, anticancer activity of APE has been reported on different types of human cancer cell lines. Interestingly, the methanol extracts of APE and its isolated compounds exhibited growth inhibitory and differentiating activity on M1 (mouse myeloid leukaemia) cells 17. The ethanol extracts of APE and its mains diterpenoid components were significantly inhibited to human acute myeloid leukemic HL-60 cells by cell-cycle arrest at G0/ G1 phase and mitochondrial-mediated apoptosis 12. Andrographolide, the major diterpenoid of the Andrographis paniculata, induced apoptosis in human cancer cells via the receptor apoptotic pathway leads to mitochondria and then to downstream effector caspases 13. In addition, Andrographolide exerts direct anticancer activity on cancer cells by cell-cycle arrest at G0/G1 phase through induction of cell-cycle inhibitory protein p27 and decreased expression of cyclin-dependent kinase 4 (CDK4) 18. However, the effect of Andrographolide on apoptosis is controversial. For example, it induced apoptosis in various kinds of cancer cells 12,13,17,18 but it protects human umbilical vein endothelial cells (HUVECs) from growth factor deprivation-induced apoptosis via suppression of the mitochondrial and caspase dependent death pathway 19. So it needs to be further elucidated. The aim of this study is focus on the effects of APE on the anti-cancer activity as well as the possible mechanisms. A better understanding of the mechanisms of action of these anti-cancer candidates will facilitate development of novel nontoxic anti-cancer drugs derived from the potentially valuable medicinal plants. MATERIALS AND METHODS Chemicals The following chemicals were purchased from the following suppliers: propidium iodide (PI), 4'-6- Diamidino-2-phenylindole (DAPI) and SYBER Gold from invitrogen, Ltd. (Paisley, UK); dimethyl sulfoxide (DMSO) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) from Sigma Chemical Co. (St Louis, MO, USA); Cell culture media or materials were purchased from Gibco BRL (Gaithersburg, MD, USA) and InVitromex (Grevenbroich, Germany). 41

3 TJPS Vol. 21 No. 2 Oct Mar Plant materials and preparation of extract The aerial parts (stems and leaves) of Andrographis paniculata Nees. (Acanthaceae) were harvested from Dongbang District, Prajeanburi, Thailand in March, The raw materials were air dried. The grams of dried plant were extracted with ethyl acetate at room temperature. The extracts were concentrated by rotary evaporator to give grams of crude extract. The crude extract (APE) was dissolved in dimethylsulfoxide (DMSO) to obtain 1 mg/ml stock solution and then filtered through a 0.22 µm cellulose nitrate membrane and stored at -20 o C before use. Dilution was further dissolved in phosphate buffer saline (PBS). A voucher specimen No.5002 was deposited in Department of Chemistry, Burapha University. Cell culture and cell proliferation assay KB cell lines (human oropharyngeal epidermoid carcinoma) were obtained from National Cancer Institute of Thailand. Cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS), 1mM sodium pyruvate, and 100 U/ ml penicillin, and 100 µg/ml streptomycin and kept at 37 o C in a humidified atmosphere of 5% CO 2 and 95% air mixture. KB cells in logarithmic growth phase were collected. After digestion with trypsin-edta, cells/well were plated in flat-bottomed 96-well plates in 100-ml volumes and incubated for 24 hr. at 37 o C under a humidified 5% CO 2 and 95% air mixture to allow cell attachment. Cells were treated with 0.21% DMSO (vehicle control), Doxorubicin 0-10 µg/ml (positive control) and APE µg/ml (test sample) for 48 hr under the same conditions. At the end of 48 hr, 20 µl of 0.5% MTT in PBS were added to each well and plates were incubated at 37 o C for 3 hr. At the end of 3 hr, the supernatant was removed and replaced with 100 µl of DMSO. The metabolic reduction of soluble MTT by mitochondrial enzyme activity of viable cells into an insoluble colored formazan product was measured with a microplate spectrophotometer reader (Cecil Bioquest 2000 Series) at 570 nm. At least three separate experiments for each sample were used to determine the cell proliferation. Under these conditions, 0.21% DMSO was not toxic and cell survival in vehicle control was assumed 100%. The percentage of cell viability was calculated on the basis of the following formula: Absorbance at 570 nm of treated cells % Cell viability = 100 Absorbance at 570 nm of control cells Fluorescent microscopic analysis of nuclear fragment Cells cells/ml were cultured in polystylene flask (25 cm 2 ) and incubated for 24 hr (37 o C, 5% CO 2 and 95% air mixture). Then 80 µg/ml of APE was added and incubated for another 48 hr in the same condition. Doxorubicin (0.5 µg/ml) was used as a positive control. Cell grown in the presence of 0.21% DMSO but absence of APE was considered as a negative control. The morphology of the nuclei was quantified using fluorescence staining. Briefly, at designated time points, cells were collected and washed in PBS. The cells were then incubated with RNase A (20 mg/ml, 5 µl) in the dark for 30 minutes at room temperature. Harvested cells were washed three times with PBS, and then resuspended in PBS containing 5 µg/ml DAPI (used to identify nuclear fragmentation) and 5 µg/ml propidium iodide (to identify non-viable cells) for 30 min at 37 o C. The supernatant were discarded to remove unbound dye. The 10 µl of cells were mounted on the slide, covered with a coverslip and sealed the edges with nail polish. Condensed or fragmented nuclei in viable cells were then visualized through blue and green filter of fluorescence microscope (Olympus B 51) at 400 magnification. Normal nuclei can be identified by glowing bright and homogeny while apoptotic nuclei are condensed chromatin and fragmented morphology of nuclear bodies. For each treatment group, different nuclei were counted in random microscopic fields. Data were expressed as percentage of nuclei in different phases. At least three separate experiments for each sample were performed. DNA extraction and electrophoresis analysis The GF-1 Tissue DNA Extraction Kit (Vivantis) was used according to the manufacturer s instructions. After treatments, floating and adherent cells were washed with PBS and then lysed with digestion buffer containing proteinase K (20 mg/ml, 20 µl) at 60 o C overnight. RNaseA (20 mg/ml, 10 µl) was then added and incubated for 5 min at 37 o C. Genomic DNA was extracted with ice-cold absolute ethanol. DNA (400 ng) were 42

4 % cell viability Antiproliferation and Apoptosis of the Crude Extract of Andrographis paniculata Nees mixed with SYBER Gold (0.1 mg/ml, 1 µl) and loading buffer and loaded onto pre-solidified 1.5% agarose. The agarose gels were run at 150 V for 60 min in TBE buffer. Gels were observed and photographed under transilluminator (Clare Chemical Research). Data processing Data were expressed as mean ± standard error of the mean (S.E.M) of 3-4 different trials and analyzed with the software Microcal TM Origin 6. RESULTS To investigate the potential effects of APE on proliferation and survival of KB cells, the cells were exposed to µg/ml of APE for 48 h and cellular viability was analyzed by MTT assay. The APE markedly decreased viable cell numbers in a dose-dependent manner (Fig. 1). At 30 µg/ml of APE, cellular viability was reduced to ± 3.55% and at 250 µg/ml of APE the viability was reduced to less than 10%. The APE was less toxic than Doxorubicin in that the cell number decreased to less than 10% at concentrations 5 µg/ml. The IC50 values for APE and Doxorubicin on KB cells was 80 ± 3.7 µg/ml and 0.5 ± 0.02 µg/ml respectively. Marked morphological changes could be seen after 48 h treatment with both APE and Doxorubicin characterized by round cells, lost of cell-to-cell contract and fewer adherent cells when compared with control group. The apoptotic cell death is one of the mechanisms by which cell growth is suppressed. In order to gain more insight into cell-death pathways in KB cells treated with APE, morphology of apoptosis and the ordered DNA ladder detection were investigated. Both DAPI and PI staining are fluorescent nuclear dye that binds strongly to DNA. DAPI can pass through an intact cell membrane but PI is membrane impermeant that commonly used for identifying dead cells. KB cells treated with 0.21% DMSO produced rounded nuclei with homogenous DAPI staining and defined plasma membrane contours, but in cells treated with APE 80 µg/ml, there was an increase in the marked morphological changes of cellular apoptosis including membrane blebbing, condensation of chromatin, nuclear fragmentation and the formation of apoptotic bodies (Fig. 2). In vehicle control group, the quantitative estimation of nuclei was ± 4.2% (normal cells), 2.85 ± 0.7% (viable cells with apoptotic nuclei) and 2.85 ± 0.3% (necrosis or late apoptotic nuclei). When the cells were treated with APE 80 µg/ml for 48 h, the quantitative estimation of nuclei was 7.85 ± 0.9% (normal cell), ± 7.5% (viable cells 100 A 100 B APE, μg/ml Doxorubicin, (μg/ml) Fig. 1 Antiproliferative effects of APE (A) and Doxorubicin (B) to KB cells. Cells (2x105 cells/well) were exposed to APE ( µg/ml) or Doxorubicin (0-10 µg/ml) for 48 hr. Viable cell number was measured with MTT assay. Data were expressed as mean ± S.E.M of 4 replicates. 43

5 TJPS Vol. 21 No. 2 Oct Mar Fig. 2 Morphological features of nuclei of KB cells treated with DMSO (1-3), APE 80 µg/ml (4-6) and Doxorubicin 0.5 µg/ml (7-9) for 48 hr. Cells were observed by bright field morphology (1, 4, 7), DAPI staining (2, 5, 8) and PI staining (3, 6, 9). N: normal nuclei, C: chromatin condensation, F: nuclear fragmentation, L: late apoptosis and B: membrane blebbing. Table 1 Percentage of nuclear staining with DAPI/PI. KB cells were treated with APE (80 µg/ml) and Doxorubicin (0.5 µg/ml) for 48 hr. The percentage of cells with a normal or condensed or fragmented nucleus was estimated by counted directly with fluorescence microscope. The values are mean ± S.E.M of 2-3 replicates. % Normal cells % Apoptosis cells % Late apoptosis (Homogenous DAPI (Condensed or fragmented or necrotic cells staining) DAPI staining) (PI staining) 0.21% DMSO ± ± ± 0.3 APE 80 µg/ml 7.85 ± ± ± 1.5 Doxorubicin 0.5 µg/ml 9.09 ± ± ± 4.2 with apoptotic nuclei) and 10.0 ± 1.5% (necrosis or late apoptotic nuclei). In the treatment with Doxorubicin 0.5 µg/ml for 48 h, the quantitative estimation of nuclei was 9.09 ± 1.4% (normal cells), ± 2.1% (viable cells with apoptotic nuclei) and ± 4.2% (necrosis or late apoptotic nuclei) (Table 1). DNA fragmentation, a hallmark of apoptosis, was qualitatively demonstrated by incubating KB cells with different concentrations of APE. As shown in Fig. 3, oligonucleosomal DNA fragmentation was observed when cells were treated with 40, 80 and 180 µg/ml of APE for 48 hr. APE-induced apoptosis of KB cells exhibited a progressive increase in nonrandom fragmentation into a ladder with a concentration-dependent manner that is in good accordance with its growth suppressive effects. DISCUSSION The KB cell lines, derived from human epidermal carcinoma of the mouth, serve as a useful model system 44

6 Antiproliferation and Apoptosis of the Crude Extract of Andrographis paniculata Nees Fig. 3 A photograph of the SYBER Gold -stained gel, which is representative of three independent experiments, is shown. DNA fragmentation of KB cells exposed to APE and Doxorubicin for 48 hr. Cells were lysed and DNA was extracted and electrophoresed on 1.5% agarose gels and stained with SYBER GOLD for detection of DNA fragmentation. for studying anticancer activity. Andrographis paniculata has been used for centuries as an anti-inflammatory property that would otherwise cause cancer cells to stop proliferating, or to become more sensitive to the action of anti-cancer agents 3,16. From the results of cell viability studies with the MTT test, the APE obviously decreased viable cells in a dose-dependent manner and the IC50 value of APE was 80 ± 3.7 µg/ml at 48 hr incubation. This effect was not specific for KB cells because the methanol extract also inhibited the proliferation of human colon cancer (HT-29) cells. The IC 50 of crude extract in the same study was 10 µg/ml and IC 50 of its fractions ranged from µg/ml 7. In HL-60 cells, the IC 50 of ethanol extract was µg/ml while it was less effective in other cells 12. However, there are a number of important factors regarding the effect of AP extract on growth inhibition in cancer cells. It may also be constraint by the genetic background of the cell line under study. In addition, the growing region and seasonality play a role to the concentration of active ingredients existing in the extract. The dying cells in present study exhibit the morphological and biochemical features that characterize apoptosis, as shown by membrane blebbing, oligonucleosomal DNA fragmentation, chromatin condensation, and fragmentation of the cell into apoptotic bodies. The apoptotic nuclei were quantified using fluorescence double staining DAPI and PI that discriminate between viable and non-viable cells, and ideally between apoptosis and necrosis. When cells treated with APE 80 µg/ml for 48 hr, apoptotic nuclei in living cells can be identified by the condensed chromatin gathering at the periphery of the nuclear membrane or a marked reduction in nuclear fragment with an apoptotic manifestation of ± 7.5%. Quantitative apoptotic and late apoptotic nuclei of Doxorubicin-treated cells was ± 2.1% and ± 4.2% respectively. The mechanisms of cell death mediated by Doxorubicin utilize the apoptotic pathway through irreversible DNA damage with subsequent mitotic failure 20. Drastic decrease in apoptotic cells together with increase in late apoptotic cells was noticed in Doxorubicin-treated cells. Since Doxorubicin is more toxic than APE, it might also be that the apoptotic cells under the in vitro conditions cannot undergo rapid phagocytosis as in in vivo in the intact tissue 21. Extensive investigations into the molecular mechanisms underlying apoptotic cell death pointed to the presence of biochemical markers of apoptosis, that is, the fragmentation of nuclear DNA into oligonucleosomesized DNA fragments by an apoptotic nuclease. APEtreated cells presented with well-defined in the cleavage of genomic DNA in an oligonucleosomal laddering pattern upon agarose gel electrophoresis that is the hallmark of apoptosis. The apoptotic DNA fragmentation was in good agreement with the result obtained by cell proliferation assay and fluorescent microscopic analysis of nuclear fragment. However, further investigation of caspase activity which provides a direct link between endonucleases and apoptotic DNA fragmentation need to be further characterized. The process of tumor development and treatment resistance involve the cancer cells that obtain the capability to evade apoptosis by various pathways. The most cytotoxic anti-cancer drugs induce apoptosis and the ability of drug-induced apoptosis is associated with treatment sensitivity 22. The inhibiting cell proliferation and increasing apoptosis in cancer cells are effective strategy for preventing cancer growth. The present study did not identify the active components existing in the APE for its antiproliferative activity. Therefore, we do not know whether the induction of apoptosis by APE was due to single or combined effects of multiple agents contained in the extract. It has been previously reported that a major component of APE was diterpene lactone 45

7 TJPS Vol. 21 No. 2 Oct Mar such as andrographolide, deoxyandrographolide, dihydroandrographolide and neoandrographolide. Among these compounds, andrographolide was the highest degree of cytotoxicity on different types of human cancer cell lines 7. Andrographolide was also found to cause cell-cycle arrest at G0/G1 phase 18, induce apoptosis via extrinsic death receptor 13 and intrinsic mitochondria pathway 12 leading to downstream effecter caspases and apoptotic cell death. In addition to cytotoxic drug induce apoptosis, the most important targets of cancer therapy take account of the inhibition of selective angiogenesis and the activation of the immune system to target cancer cells. Treatment with andrographolide and ethanolic extract selectively inhibited tumor capillary sprouting without damaging the pre existing vasculature 23. Andrographolide and ethanolic extract stimulated both antigen specific and non-specific immune system and the extract was more potent than andrographolide, suggesting that other constituents also were immunostimulants 6. Similarly, the methanolic extract and purified diterpene andrographolides showed the immunostimulatory activity by enhance the IL-2 induction in human peripheral blood lymphocytes 7. The potent stimulator of the immune system makes it effective against a variety of infectious and cancer-causing agents. In another study, andrographolide and its extract were shown to produce a protective effect against liver toxicity. For example, the isolated andrographis diterpenes and the aqueous extract protected hepatocytes from damage induced by hexachlorocyclohexane 24, acetaminophen 25 and carbon tetrachloride or tertbutylhydroperoxide 26. It might induce hepatic drug metabolizing enzyme, cytochrome P In traditional medicine of Thailand, Chinese and India, Andrographis paniculata has long been perceived as safe. The acute and chronic toxicity of Andrographis paniculata in mice study that received oral extracts of at 15 g/kg body weight showed no evidence of organ damage and none of the mice died 28. Therefore, the dose used in the present study seems safe in terms of toxicity. Although Andrographis paniculata are much less toxic than most chemotherapeutic agents used to fight cancer, it may be seeing natural remedies combined with synthetic chemotherapeutic compounds that might improve efficacy and decrease side effects. The Andrographis paniculata is an inexpensive and easily obtained, so it would benefit many, especially people in developing countries where cancer is almost catastrophic. Additionally, the standardization of the active compounds in AP extracts need to perform further investigation. CONCLUSION In conclusion, the ethyl acetate extract of Andrographis paniculata exhibits an in vitro antiproliferative effect of KB cancer cells via apoptotic pathway. These results showed that APE was able to induce membrane blebbing, chromatin condensation and fragment of nuclear DNA into oligonucleosome-sized DNA fragments. Since very low toxic effects were noticeable, the APE may have potential as a chemopreventive agent. Additionally, the combination of APE with synthetic medications being used in therapies might improve efficacy and decrease side effects. ACKNOWLEDGEMENTS This work was partly supported by Department of Medical Science, Faculty of Science, Burapha University. We wish to thank Dr. Jongkolnee Jongaramruong for graciously providing the APE. REFERENCES 1. Kinghorn AD, Farnsworth NR, Soejarto DD, et al. Novel stragies for the discovery of plant derived anticancer agents. Pure Appl Chem 1999; 71(9): Gupta S, Choudhry MA, Yadava JNS, et al. Antidiarrhoeal activity of diterpenes of Andrographis paniculata (Kal-Megh) against Escherichia coli enterotoxin in in vivo models. Int J Crude Drug Res 1990; 28(4): Hidalgo MA, Romero A, Figueroa J, et al. Andrographolide interferes with binding of nuclear factor-kb to DNA in KL-60- derived neutrophilic cells. Br J Pharmacol 2005; 144: Ahmad I, Mehmood Z, Mohammad F. Screening of some Indian medicinal plants for their antimicrobial properties. J Ethnopharmacol 1998; 62: Calabrese C, Berman SH, Babish JG, et al. A phase I trial of andrographolide in HIV positive patients and normal volunteers. Phytother Res 2000; 14: Puri A, Saxena R, Saxena RP, et al. Immunostimulant agents from Andrographis paniculata. J Nat Prod 1993; 56: Kumar RA, Sridevi K, Kumar NV, et al. Anticancer and immunostimulatory compounds from Andrographis paniculata. J Ethnopharmacol 2004; 92:

8 Antiproliferation and Apoptosis of the Crude Extract of Andrographis paniculata Nees 8. Trivedi N, et al. Hepatoprotective and toxicological evaluation of Andrographis paniculata on severe liver damage. Indian J Pharmacol 2000; 32: Visen P, et al. Andrographolide protects rat hepatocytes against paracetamol-induced damage. J Ethnopharmacol 1993; 40: Kapil A, et al. Antihepatotoxic effects of major diterpenoid constituents of Andrographis paniculata. Biochem Pharmacol 1993; 46: Thisoda P, Rangkadilok N, Pholphana N, et al. Inhibitory effect of Andrographis paniculata extract and its active diterpenoids on platelet aggregation. Eur J Pharmacol 2006; 553: Cheung HY, Cheung SH, Li J, et al. Andrographolide isolated from Andrographis paniculata induces cell cycle arrest and mitochondrial-mediated apoptosis in human leukemia HL-60 cells. Planta Med 2005; 71: Zhou J, Zhang S, Ong CN, et al. Critical role of pro-apoptotic Bcl-2 family members in andrographolide-induced apoptosis in human cancer cells. Biochem Pharmacol 2006; 72: Roxas M, Jurenka J. Colds and influenza: A review of diagnosis and conventional botanical, and nutritional considerations. Altern Med Rev 2007; 12(1): Cheung HY, Cheung CS, Kong CK. Detection of bioactive diterpenoids from Andrographis paniculata by micellar electrokinetic chromatography. J Chromatogr 2001; 930: Micheau O, Lens S, Gaide O, et al. NF-kappaB signals induce the expression of c-flip. Mol Cell Biol 200; 21: Matsuda T, Kuroyanagi M, Sugiyama S, et al. Cell differentiation-inducing diterpenes from Andrographis paniculata Nees. Chem Pharm Bull 1994; 42: Rajagopal S, Kumar RA, Deevi DS, et al. Andrographolide, A potential cancer therapeutic agent isolated from Andrographis paniculata. J Exp Ther Oncol 2003; 3: Chen JH, Hsiao G, Lee AR, et al. Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol- 3-kinase/Akt pathway. Biochem Pharmacol 2004; 67: Bold RJ, Termuhlen PM & McConkey DJ. Apoptosis, cancer and cancer therapy. Surg Oncol 1997; 6(3): Grub S, Persohn E, Trommer W, et al. Mechanisms of cyclosporine-a induced apoptosis in rat hepatocytes primary cultures. Toxicol Appl Phramacol 2000; 163: Sellerss WR & Fisher DE. Apoptosis and cancer drug targeting. J Clin Invest 1999; 104(12): Sheeja K, Guruvayoorappan C & Kuttan G. Antiangiogenic activity of Andrographis paniculata extract and andrographolide. Int Immunopharmacol 2007; 7: Trivedi N, et al. Hepatoprotective and toxicological evaluation of Andrographis paniculata on severe liver damage. Indian J Pharmacol 2000; 32: Visen P, et al. Andrographolide protects rat hepatocytes against paracetamol-induced damage. J Ethnopharmacol 1993; 40: Kapil A, et al. Antihepatotoxic effects of major diterpenoid constituents of Andrographis paniculata. Biochem Pharmacol 1993; 46: Jarukamjorn K, Don-in K, Makejaruskul C, et al. Impact of Andrographis paniculata crude extract on mouse hepatic cytochrome P450 enzymes. J Ethnopharmacol 2006; 105: Sithisomwongse N, Phengchata J, Cheewapatana S, et al. Acute and chronic toxicity of Andrographis paniculata Nee. Thai J Pharm Sci 1989; 14(2):

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