Ethanolic Extract of Marsdenia condurango Ameliorates Benzo[a]pyrene-induced Lung Cancer of Rats -Condurango Ameliorates BaP-induced Lung Cancer in Rats-

Sourav Sikdar,Avinaba Mukherjee,Anisur Rahman Khuda-Bukhsh 대한약침학회 2015 Journal of pharmacopuncture Vol.18 No.2

The original article Ethanolic Extract of Marsdenia condurango Ameliorates Benzo[a]pyrene-induced Lung Cancer of Rats (J Pharmacopuncture 2014;17(2):7-17, DOI: http://dx.doi.org/10.3831/KPI.2014.17.011) by Sourav Sikdar, Avinaba Mukherjee, and, Anisur Rahman Khuda-Bukhsh, was published with an incorrect method in abstract due to a production error. The correct method in abstract are reported below. Methods: After one month of scheduled BaP feeding (50 mg/kg body-weight), lung cancer developed after four months. BaP-intoxicated rats were then treated with Condurango (0.06 mL) twice daily starting at the end of the four months for an additional one, two and three months, respectively. Effects of Condurango were evaluated by analyzing lung histology, reactive oxygen species (ROS) and antioxidant biomarkers, DNA-fragmentation, RT-PCR (Reverese Transcriptase- Polymerase Chain Reaction), ELISA (Enzyme linked immunosorbent assay) and western blot of several apoptotic signalling markers and comparing the results against those obtained for controls. We apologize to the authors and readers. Objectives: Condurango is widely used in various systems of complementary and alternative medicine (CAM) against oesophageal and stomach ailments including certain types of cancer. However, until now no systematic study has been conducted to verify its efficacy and dose with proper experimental support. Therefore, we examined if ethanolic extract of Condurango could ameliorate benzo[a]pyrene (BaP)-induced lung cancer in rats in vivo to validate its use as a traditional medicine. Methods: After one month of scheduled BaP feeding (50 mg/kg body-weight), lung cancer developed after four months. BaP-intoxicated rats were then treated with Condurango (0.06 mL) twice daily starting at the end of the four months for an additional one, two and three months, respectively. Effects of Condurango were evaluated by analyzing lung histology, reactive oxygen species (ROS) and antioxidant biomarkers, DNA-fragmentation, RT-PCR (Reverese Transcriptase- Polymerase Chain Reaction), ELISA (Enzyme linked immunosorbent assay) and western blot of several apoptotic signalling markers and comparing the results against those obtained for controls. Results: A histological study revealed gradual progress in lung tissue-repair activity in Condurango-fed cancer- bearing rats, showing gradual tissue recovery after three months of drug administration. Condurango has the capacity to generate ROS, which may contribute to a reduction in anti-oxidative activity and to an induction of oxidative stress-mediated cancer-cell death. Condurango-activated pro-apoptotic genes (Bax, caspase-3, caspase-9, p53, cytochrome-c, apaf-1, ICAD and PARP) and down-regulated antiapoptotic-Bcl-2 expression were noted both at mRNA and protein levels. Studies on caspase-3 activation and PARP cleavage by western blot analysis revealed that Condurango induced apoptosis through a caspase-3-dependent pathway. Conclusions: The anticancer efficacy of an ethanolic extract of Condurango for treating BaP-induced lung cancer in rats lends support for its use in various traditional systems of medicine.

Homeopathy - A Safe, Much Less Expensive, Non-Invasive, Viable Alternative for the Treatment of Patients Suffering from Loss of Lumbar Lordosis

Haque, Saiful,Das, Debarsi,Bhattacharya, Saugato,Sarkar, Tathagato,Khuda-Bukhsh, Anisur Rahman KOREAN PHARMACOPUNCTURE INSTITUTE 2016 Journal of pharmacopuncture Vol.19 No.4

Objectives: Loss of lumbar lordosis causing pain and curvature of the vertebral skeleton to one side is a relatively uncommon disease. To our knowledge, successful treatment of loss of lumbar lordosis with any potentized homeopathic drug diluted above Avogadro's limit (that is, above a potency of 12C) has not been documented so far. In this communication, we intend to document a relatively rare case of loss of lumbar lordosis with osteophytic lippings, disc desiccation, and protrusion, causing a narrowing of secondary spinal canal and a bilateral neural foramina, leading to vertebral column curvature with acute pain in an adolescent boy. Methods: The patient had undergone treatment with orthodox Western medicines, but did not get any relief from, or cure of, the ailment; finally, surgery was recommended. The patient's family brought the patient to the Khuda-Bukhsh Homeopathic Benevolent Foundation where a charitable clinic is run every Friday with the active participation of four qualified homeopathic doctors. A holistic method of homeopathic treatment was adopted by taking into consideration all symptoms and selecting the proper remedy by consulting the homeopathic repertory, mainly of Kent. Results: The symptoms were effectively treated with different potencies of a single homeopathic drug, Calcarea phos. X-ray and magnetic resonance imaging (MRI) supported recovery and a change in the skeletal curvature that was accompanied by removal of pain and other acute symptoms of the ailment. Conclusion: Homeopathy can be a safe, much less expensive, non-invasive, and viable alternative for the treatment of such cases.

Homeopathy - A Safe, Much Less Expensive, Non- Invasive, Viable Alternative for the Treatment of Patients Suffering from Loss of Lumbar Lordosis

Saiful Haque,Debarsi Das,Saugato Bhattacharya,Tathagato Sarkar,Anisur Rahman Khuda-Bukhsh 대한약침학회 2016 Journal of pharmacopuncture Vol.19 No.4

Objectives: Loss of lumbar lordosis causing pain and curvature of the vertebral skeleton to one side is a relatively uncommon disease. To our knowledge, successful treatment of loss of lumbar lordosis with any potentized homeopathic drug diluted above Avogadro’s limit (that is, above a potency of 12C) has not been documented so far. In this communication, we intend to document a relatively rare case of loss of lumbar lordosis with osteophytic lippings, disc desiccation, and protrusion, causing a narrowing of secondary spinal canal and a bilateral neural foramina, leading to vertebral column curvature with acute pain in an adolescent boy. Methods: The patient had undergone treatment with orthodox Western medicines, but did not get any relief from, or cure of, the ailment; finally, surgery was recommended. The patient’s family brought the patient to the Khuda-Bukhsh Homeopathic Benevolent Foundation where a charitable clinic is run every Friday with the active participation of four qualified homeopathic doctors. A holistic method of homeopathic treatment was adopted by taking into consideration all symptoms and selecting the proper remedy by consulting the homeopathic repertory, mainly of Kent. Results: The symptoms were effectively treated with different potencies of a single homeopathic drug, Calcarea phos. X-ray and magnetic resonance imaging (MRI) supported recovery and a change in the skeletal curvature that was accompanied by removal of pain and other acute symptoms of the ailment. Conclusion: Homeopathy can be a safe, much less expensive, non-invasive, and viable alternative for the treatment of such cases.

Homeopathic mother tincture of Conium initiates reactive oxygen species mediated DNA damage and makes HeLa cells prone to apoptosis

Anisur Rahman Khuda-Bukhsh,Kausik Bishayee,Avinaba Mukherjee,Avijit Paul 셀메드 세포교정의약학회 2012 TANG Vol.2 No.3

Adverse side-effects and lack of scientific validation of some chemotherapeutic agents prevent the use of many traditional medicines claimed to have anti-cancer effects. Ethanolic extract of Conium maculatum has long been used in traditional and alternative systems of medicine including homeopathy for the treatment of glandular enlargements, cancerous tumours or hard lumps of testicles, prostate, ovaries, breasts and/ or uterus, particularly in the breast. However, if and how it acts still remains scientifically unknown. This study aims to test if Conium extract (CE), used as mother tincture of Conium in homeopathy, has demonstrable anti-cancer potentials without having much cytotoxicity in normal cells. Cytotoxicity of the drug was tested by conducting MTT assay on both normal (peripheral blood mononuclear cells) and HeLa cells. We also evaluated DNA fragmentation and DNA damage by DAPI and diphenylamine assay. The LDH activity assay was done to evaluate the percentages of apoptosis and necrosis. ROS accumulation also was evaluated to pin-point the actual events of apoptosis. Administration of drug clearly demonstrated its anti-cancer potentials as evidenced by the DNA damage analysis. The ROS activity also increased in case of the CE treated cells. LDH data revealed that the mode of cell death was mainly apoptotic and not necrotic. CE appears to induce apoptosis of cancer cells through ROS mediated pathway, and has negligible cytotoxicity against normal cells.

Attenuation of p-dimethylaminoazobenzene initiated genotoxicity and cytotoxicity in mice by the combined treatment of a traditional homeopathic remedy Chelidonium Majus 200C and vitamin-C

Anisur Rahman Khuda-Bukhsh,Surjyo Jyoti Biswas,Susanta Roy Karmakar 셀메드 세포교정의약학회 2012 셀메드 (CellMed) Vol.2 No.4

The homeopathic remedy Chelidonium majus 200C (Chel-200) is traditionally used by homeopathic practitioners in liver ailments arising out of hepatotoxicity. The present investigation was aimed at examining whether vitamin C (L-ascorbic acid or AA), used in both traditional and orthodox medicines, can show better effects when used in combination with Chel-200, in favorably modifying the toxicological effects induced by the chronic feeding of p-dimethylaminoazobenzene (p-DAB, initiator) and phenobarbital (PB, promoter) in mice for 7 days through 120 days to induce hepatotoxicity and liver tumors. Mice were euthanized at 7, 15, 30, 60, 90, and 120 days of carcinogen feeding to assess various cytogenetical, biochemical and histological changes occurring in them. In a placebo controlled study, Chel-200 or the respective placebo (Alcohol-200C or Alc, “vehicle” of homeopathic drug), was orally administered to toxicant-fed mice. Sub-groups of the mice receiving Chel-200 were also fed either AA or an Alc placebo and their individual and conjoint effects were studied against the respective controls, to evaluate if the combination therapy of Chel-200 with AA can be of additional help in the amelioration of the toxicities generated by the toxicants. The combined feeding of Chel- 200 and AA appeared to reduce the cytotoxic and genotoxic effects significantly, when compared to either only the Chel-200 or AA fed group. A similar trend was also obtained in the results of scanning and transmission electron microscopic studies of the livers. Experiments in other mammalian models are warranted to confirm if these drugs in combination could be used in palliative care of human patients with liver diseases including cancer.

PLGA-Loaded Gold-Nanoparticles Precipitated with Quercetin Downregulate HDAC-Akt Activities Controlling Proliferation and Activate p53-ROS Crosstalk to Induce Apoptosis in Hepatocarcinoma Cells

Bishayee, Kausik,Khuda-Bukhsh, Anisur Rahman,Huh, Sung-Oh Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.6

Controlled release of medications remains the most convenient way to deliver drugs. In this study, we precipitated gold nanoparticles with quercetin. We loaded gold-quercetin into poly(DL-lactide-co-glycolide) nanoparticles (NQ) and tested the biological activity of NQ on HepG2 hepatocarcinoma cells to acquire the sustained release property. We determined by circular dichroism spectroscopy that NQ effectively caused conformational changes in DNA and modulated different proteins related to epigenetic modifications and c ell cycle control. The mitochondrial membrane potential (MMP), reactive oxygen species (ROS), cell cycle, apoptosis, DNA damage, and caspase 3 activity were analyzed by flow cytometry, and the expression profiles of different anti- and pro-apoptotic as well as epigenetic signals were studied by immunoblotting. A cytotoxicity assay indicated that NQ preferentially killed cancer cells, compared to normal cells. NQ interacted with HepG2 cell DNA and reduced histone deacetylases to control cell proliferation and arrest the cell cycle at the sub-G stage. Activities of cell cycle-related proteins, such as $p21^{WAF}$, cdk1, and pAkt, were modulated. NQ induced apoptosis in HepG2 cells by activating p53-ROS crosstalk and induces epigenetic modifications leading to inhibited proliferation and cell cycle arrest.

Quercetin Down-regulates IL-6/STAT-3 Signals to Induce Mitochondrial-mediated Apoptosis in a Non-small-cell Lung-cancer Cell Line, A549

Mukherjee, Avinaba,Khuda-Bukhsh, Anisur Rahman KOREAN PHARMACOPUNCTURE INSTITUTE 2015 Journal of pharmacopuncture Vol.18 No.1

Objectives: Quercetin, a flavonoid compound, has been reported to induce apoptosis in cancer cells, but its anti-inflammatory effects, which are also closely linked with apoptosis, if any, on non-small-cell lung cancer (NSCLC) have not so far been critically examined. In this study, we tried to determine if quercetin had any demonstrable anti-inflammatory potential, which also could significantly contribute to inducing apoptosis in a NSCLC cell line, A549. Methods: In this context, several assays, including cytotoxicity, flow cytometry and fluorimetry, were done. Gene expression was analyzed by using a western blot analysis. Results: Results revealed that quercetin could induce apoptosis in A549 cells through mitochondrial depolarization by causing an imbalance in B-cell lymphoma 2/Bcl2 Antagonist X (Bcl2/Bax) ratio and by down-regulating the interleukine-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway. An analysis of the data revealed that quercetin could block nuclear factor kappa-light-chain-enhancer of activated B cells (NF-${\kappa}B$) activity at early hours, which might cause a down-regulation of the IL-6 titer, and the IL-6 expression, in turn, could inhibit p-STAT3 expression. Down-regulation of both the STAT3 and the NF-${\kappa}B$ expressions might, therefore, cause down-regulation of Bcl2 activity because both are major upstream effectors of Bcl2. Alteration in Bcl2 responses might result in an imbalance in the Bcl2/Bax ratio, which could ultimately bring about mitochondria mediated apoptosis in A549 cells. Conclusion: Overall, the finding of this study indicates that a quercetin induced anti-inflammatory pathway in A549 cells appeared to make a significant contribution towards induction of apoptosis in NSCLC and, thus, may have a therapeutic use such as a strong apoptosis inducer in cancer cells.

Two Homeopathic Remedies Used Intermittently Provide Additional Protective Effects Against Hepatotoxicity Induced by Carcinogens in Mice

Nandini Bhattacharjee,Anisur Rahman Khuda-Bukhsh 사단법인약침학회 2012 Journal of Acupuncture & Meridian Studies Vol.5 No.4

The purpose of the study was to evaluate whether potentized cholesterinum (Chol) intermittently used with another homeopathic remedy, Natrum Sulphuricum (Nat Sulph) can provide additional benefits in combating hepatotoxicity generated by chronic feeding of carcinogens, p-dimethylaminoazobenzene (p-DAB), and phenobarbital (PB). Mice were categorized into subgroups: normal untreated (Gr-1); normal + alcohol “vehicle” (Alc) (Gr-2), 0.06% p-DAB +0.05% PB (Gr-3), p-DAB+PB+Alc (Gr-4), p-DAB+PB+Nat Sulph-30 (Gr-5), p-DAB+PB+Chol-200 (Gr-6), p-DAB+PB+Nat Sulph-30+Chol-200 (Gr-7), p-DAB+PB+Nat Sulph-200 (Gr-8), and DAB+PB+Nat Sulph-200+Chol-200 (Gr-9). Hepatotoxicity was assessed through biomarkers like aspartate and alanine aminotransferases (AST and ALT), acid and alkaline phosphatases (AcP and AlkP), reduced glutathione content (GSH), glucose 6-phosphate dehydrogenase (G6PD), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), and analysis of lipid peroxidation (LPO) at 30, 60, 90, and 120 days and antioxidant biomarkers like superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) were assayed. Electron microscopic studies (scanning and transmission) and gelatin zymography for matrix metalloproteinases were conducted in liver. The feeding of the homeopathic drugs showed intervention in regard to the increased activities of AST, ALT, AcP, AlkP, GGT, LDH, and LPO and decreased activities of G6PD, SOD, CAT, GR, and GSH noted in the intoxicated mice, more appreciable in Groups 7 and 9. Thus, combined therapy provided additional antihepatotoxic and anticancer effects.

Evidence of an Epigenetic Modification in Cell-cycle Arrest Caused by the Use of Ultra-highly-diluted Gonolobus Condurango Extract

Kausik Bishayee,Anisur Rahman Khuda-Bukhsh,Sourav Sikdar 대한약침학회 2013 Journal of pharmacopuncture Vol.16 No.4

Objectives: Whether the ultra-highly-diluted remedies used in homeopathy can effectively bring about modulations of gene expressions through acetylation/deacetylation of histones has not been explored. Therefore, in this study, we pointedly checked if the homeopathically-diluted anti-cancer remedy Condurango 30C (ethanolic extract of Gonolobus condurango diluted 10-60 times) was capable of arresting the cell cycles in cervical cancer cells HeLa by triggering an epigenetic modification through modulation of the activity of the key enzyme histone deacetylase 2 vis-a-vis the succussed alcohol (placebo) control. Methods: We checked the activity of different signal proteins (like p21WAF, p53, Akt, STAT3) related to deacetylation, cell growth and differentiation by western blotting and analyzed cell-cycle arrest, if any, by fluorescence activated cell sorting. After viability assays had been performed with Condurango 30C and with a placebo, the activities of histone de-acetylase (HDAC) enzymes 1 and 2 were measured colorimetrically. Results: While Condurango 30C induced cytotoxicity in HeLa cells in vitro and reduced HDAC2 activity quite strikingly, it apparently did not alter the HDAC1 enzyme; the placebo had no or negligible cytotoxicity against HeLa cells and could not alter either the HDAC 1 or 2 activity. Data on p21WAF, p53, Akt, and STAT3 activities and a cell-cycle analysis revealed a reduction in DNA synthesis and G1-phase cell-cycle arrest when Condurango 30C was used at a 2% dose. Conclusion: Condurango 30C appeared to trigger key epigenetic events of gene modulation in effectively combating cancer cells, which the placebo was unable to do.

Ethanolic Extract of Marsdenia condurango Ameliorates Benzo[a]pyrene-induced Lung Cancer of Rats

Sourav Sikdar,Anisur Rahman Khuda-Bukhsh,Avinaba Mukherjee 대한약침학회 2014 Journal of pharmacopuncture Vol.17 No.2

Objectives: Condurango is widely used in various systems of complementary and alternative medicines (CAM) against oesophageal and stomach ailments including certain types of cancer. However, until now no systematic study has been conducted to verify its efficacy and dose with proper experimental support. Therefore, we examined if ethanolic extract of Condurango could ameliorate benzo[a]pyrene (BaP)-induced lung cancer in rats, in vivo to validate its use as traditional medicine. Methods: Fifteen male and 15 female SD rats were treated with 0.28 mg/kg of SBV (high-dosage group) and the same numbers of male and female SD rats were treated with 0.2 mL/kg of normal saline (control group) for 13 weeks. We selected five male and five female SD rats from the high-dosage group and the same numbers of male and female SD rats from the control group, and we observed these rats for four weeks. We conducted body-weight measurements, ophthalmic examinations, urinalyses and hematology, biochemistry, histology tests. Results: A histological study revealed gradual progress in lung tissue-repair activity in Condurango-fed cancer-bearing rats, showing gradual tissue recovery after three months of drug administration. Condurango has the capacity to generate ROS, which may contribute to a reduction in anti-oxidative activity and to an induction of oxidative stress-mediated cancer cell-death. Condurango-activated pro-apoptotic genes (Bax, caspase-3, caspase-9, p53, cytochrome-c, apaf-1, ICAD and PARP) and down-regulated antiapoptotic-Bcl-2 expression were noted both at mRNA and protein levels. Studies on caspase-3 activation and PARP cleavage by western blot analysis revealed that Condurango induced apoptosis through a caspase-3-dependent pathway. Conclusion: The anticancer efficacy of an ethanolic extract of Condurango for treating BaP-induced lung cancer in rats lends support for its use in various traditional systems of medicine.