NAMPT-targeting PROTAC promotes antitumor immunity via suppressing myeloid-derived suppressor cell expansion

Nicotinamide phosphoribosyl transferase (NAMPT) is considered as a promising target for cancer therapy given its critical engagement in cancer metabolism and inflammation. However, therapeutic benefit of NAMPT enzymatic inhibitors appears very limited, likely due to the failure to intervene non-enzymatic functions of NAMPT. Herein, we show that NAMPT dampens antitumor immunity by promoting the expansion of tumor infiltrating myeloid derived suppressive cells (MDSCs) via a mechanism independent of its enzymatic activity. Using proteolysis-targeting chimera (PROTAC) technology, PROTAC A7 is identified as a potent and selective degrader of NAMPT, which degrades intracellular NAMPT (iNAMPT) via the ubiquitin-proteasome system, and in turn decreases the secretion of extracellular NAMPT (eNAMPT), the major player of the non-enzymatic activity of NAMPT. In vivo, PROTAC A7 efficiently degrades NAMPT, inhibits tumor infiltrating MDSCs, and boosts antitumor efficacy. Of note, the anticancer activity of PROTAC A7 is superior to NAMPT enzymatic inhibitors that fail to achieve the same impact on MDSCs. Together, our findings uncover the new role of enzymatically-independent function of NAMPT in remodeling the immunosuppressive tumor microenvironment, and reports the first NAMPT PROTAC A7 that is able to block the pro-tumor function of both iNAMPT and eNAMPT, pointing out a new direction for the development of NAMPT-targeted therapies.

Reconstitution of biosynthetic pathway for mushroom-derived cyathane diterpenes in yeast and generation of new "non-natural" analogues

Mushroom-derived cyathane-type diterpenes possess unusual chemical skeleton and diverse bioactivities. To efficiently supply bioactive cyathanes for deep studies and explore their structural diversity,

Sulfite as the substrate of C-sulfonate metabolism of α, β-unsaturated carbonyl containing andrographolide: analysis of sulfite in rats' intestinal tract and the reaction kinetics of andrographolide with sulfite / 中国天然药物

One-sixth of the currently known natural products contain α, β-unsaturated carbonyl groups. Our previous studies reported a rare C-sulfonate metabolic pathway. Sulfonate groups were linked to the β-carbon of α, β-unsaturated carbonyl-based natural compounds through this pathway. However, the mechanism of this type of metabolism is still not fully understood, especially whether it is formed through enzyme-mediated biotransformation or direct sulfite addition. In this work, the enzyme-mediated and non-enzymatic pathways were studied. First, the sulfite content in rat intestine was determined by LC-MS/MS. The results showed that the amount of sulfite in rat intestinal contents was from 41.5 to 383 μg·g

Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione:Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions / 药物分析学报

Unsymmetrical bisacridines (UAs) are a novel potent class of antitumor-active therapeutics.A significant route of phase II drug metabolism is conjugation with glutathione (GSH),which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes.The aim of this work was to investigate the GSH-mediated metabolic pathway of a representative UA,C-2028.GSH-supplemented incubations of C-2028 with rat,but not with human,liver cytosol led to the formation of a single GSH-related metabolite.Interestingly,it was also revealed with rat liver microsomes.Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450 (CYP450) inhibitor 1-aminobenzotriazole.Therefore,the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate.In turn,incubations of C-2028 and GSH with human recombinant glutathione S-transferase(GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form,respectively.These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule.Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction.Both GSH S-conju-gates were characterized by combined liquid chromatography/tandem mass spectrometry.Mechanisms for their formation were proposed.The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important.This may affect the patient's drug clearance due to GST activity,loss of GSH,or the interactions with GSH-conjugated drugs.Moreover,GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

Protective effects of carnosic acid against aging in a premature cellular senescence model and in a D-galactose induced mouse model / 药学学报

This study aimed to investigate the effect and possible mechanism of carnosic acid (CA) on delaying aging. The effects of CA on senescence-related β-galactosidase (SA-β-Gal) activity and expressions of p53, p21 and p16 were evaluated by an oxidative challenge induced premature 2BS cell senescence model. Meanwhile, the animal experiment was approved by the Ethics Committee of Zhejiang Hospital. Male C57 BL/6J mice were injected with 100 mg·kg-1·d-1 D-galactose (D-gal) for 8 weeks to establish an aging model in vivo, and CA at 5 and 10 mg·kg-1·d-1 were given ig administration at the same time. Morris water maze test was used to test the spatial memory ability. Then the serum and tissue samples were collected for the detections of malondialdehyde (MDA), total superoxide dismutase (T-SOD), interleukin-6 (IL-6), tumor necrosis factor α (TNFα) and advanced glycation end products (AGEs) as well as the protein expression of p53, p21 and p16 in hippocampus of brain. The results showed that H2O2 induced increment of SA-β-Gal activity (95%) was prevented by CA treatment (35%) and the enhanced protein expressions of p53, p21 and p16 in H2O2 exposed 2BS cells were alleviated by CA treatment, suggesting a potent protective role of CA against premature senescence induced by oxidative challenge. For in vivo study, D-gal induced declined spatial memory ability was partly reversed by CA administration. Besides, the serum and cerebral levels of MDA, IL-6, TNFα and AGEs were attenuated by CA treatment when compared to those in model mice. And the protein expressions of p53, p21 and p16 in mice hippocampus were suppressed by CA in D-gal treated mice. Taken together, our results showed that CA protects premature senescence induced by oxidative stress and D-gal, which is related to its antioxidative, antiinflammatory roles and inhibition on non-enzymatic glycosylation.

Non-enzymatic covalent modifications: a new link between metabolism and epigenetics

Epigenetic modifications, including those on DNA and histones, have been shown to regulate cellular metabolism by controlling expression of enzymes involved in the corresponding metabolic pathways. In turn, metabolic flux influences epigenetic regulation by affecting the biosynthetic balance of enzyme cofactors or donors for certain chromatin modifications. Recently, non-enzymatic covalent modifications (NECMs) by chemically reactive metabolites have been reported to manipulate chromatin architecture and gene transcription through multiple mechanisms. Here, we summarize these recent advances in the identification and characterization of NECMs on nucleic acids, histones, and transcription factors, providing an additional mechanistic link between metabolism and epigenetics.

Non-enzymatic covalent modifications: a new link between metabolism and epigenetics

Epigenetic modifications, including those on DNA and histones, have been shown to regulate cellular metabolism by controlling expression of enzymes involved in the corresponding metabolic pathways. In turn, metabolic flux influences epigenetic regulation by affecting the biosynthetic balance of enzyme cofactors or donors for certain chromatin modifications. Recently, non-enzymatic covalent modifications (NECMs) by chemically reactive metabolites have been reported to manipulate chromatin architecture and gene transcription through multiple mechanisms. Here, we summarize these recent advances in the identification and characterization of NECMs on nucleic acids, histones, and transcription factors, providing an additional mechanistic link between metabolism and epigenetics.

Screening for active components of Sophorae Flos on inhibiting AGEs formation based on non-enzymatic glycation reaction / 中国中药杂志

Sugar-poison caused blood-heat is the pathological basis of many complications of diabetes. Advanced glycation end products( AGEs) are considered as the potential glycotoxic factor that can cause blood-heat. Sophorae Flos hold the effect of removing pathogenic heat from blood. In this study,chromatographic non-enzymatic glycation reaction system of bovine serum albumin( BSA)/methylglyoxal( MGO) and Sophorae Flos was established to identify active components in Sophorae Flos inhibiting AGEs formation. The HPLC was used to analyze chromatograms before and after the incubation of Sophorae Flos and methylglyoxal. Changes of chromatographic peaks of eight compounds was found. It is speculated that this change may be due to new substance produced by the reaction of active components in Sophorae Flos and methylglyoxal,and these active components may be flavonoid component rutin. Further investigation for the effects of rutin and MGO reaction( 1 ∶ 1,1 ∶ 3,3 ∶ 1) for 6 days on the formation of AGEs was performed. The results showed that the inhibition activity of rutin on AGEs production was most obvious when the reaction ratio was 1 ∶3,and the most inhibition was in 24 h and stabilized after 3 d. The product of the reaction of rutin with MGO was identified by LC-ESI-MS/MS,which indicated that the newly formed seven substances were the mono-and di-MGO adducts of rutin. This study showed that rutin is the active component on Sophorae Flos for removing pathogenic heat from blood by forming new compounds to inhibit the formation of sugar poison products,which provides reference for rational application of Sophorae Flos.

Pathological Overview and Antioxidant Status of Thyme on H2O2-induced Spleen Tissues Damage

Objectives: To study the preventive effect of Thymus algeriensis essential oil (TAS) against hydrogen peroxide (H2O2)-induced spleen toxicity in rats. Materials and Methods: Rats were treated with Hydrophobic fractions of Thymus algeriensis (180 mg/kg body weight, n=6), H2O2 (0.1, 1 mmol/L body weight, n=6) and the exposure to both drugs orally for 15 days. Histological examination was performed and the levels of biochemical parameters and lipid peroxides were determined. Results: In spleen tissue protein, catalase, superoxide dismutase, and glutathione (GST, GPx and GSH) levels were increased significantly (P<0.05) in the essential oil pretreated rats when compared to H2O2. TAS decreased the intracellular malondialdehyde (MDA) levels in spleen tissues. Vascular congestion was seen in spleen of high dose H2O2-treated rats and normal architecture of tissues was observed in other groups. Conclusion: The biochemical parameters and histopathology examination support the cytoprotective effect of Thyme which could be attributed to terpenes.

Inhibition of puerarin on formation of advanced glycation end products in vivo and in vitro / 中草药

Objective: To study the influence of puerarin on advanced glycation end products (AGEs) formation in vivo and in vitro. Methods: C57BL/6 mice were fed with high fat diet and injected with streptozotocin (40 mg/kg) to establish diabetic model. After modeling, mice were randomly divided into blank control group, model group, aminoguanidine group (100 mg/kg), low-, mid-, and high-dose puerarin (50, 100, and 200 mg/kg) groups. After treatment of eight weeks, the levels of fasting blood-glucose (FBG), oral glucose tolerance test (OGTT), glycated serum protein (GSP), and AGEs in serum were detected. To establish non-enzymatic glycation reaction model in vitro, glucose and bovine serum albumin (BSA) were incubated with puerarin at different concent rations respectively for 144 h. The productivity of non-enzymatic advanced glycation end products was detected by aspectrophotometer. Results: Puerarin can significantly lower the level of FBG and oral glucose tolerance in diabetic mice, and inhibit the productivity of GSP and AGEs in serum. Besides, puerarin also significantly inhibits the productivity of AGEs in vitro in a concentration-dependent manner. Conclusion: Puerarin possesses significant inhibitory effect on the productivity of AGEs in vivo and in vitro.

A Non-Enzymatic Glucose Sensor Based on Polydopamine/Cu Microparticles Self-assembled Multilayer Films / 分析化学

A highly stable multilayer film modified glassy carbon electrode (GCE) containing polydopamine (PDA) and Cu microparticles was fabricated by layer-by-layer self-assembly technique. The fabrication process was based on the self-polymerization of dopamine and electroless deposition of Cu microparticles on PDA coating. The fabrication process of multilayer films was characterized by UV-Vis spectra. The electrochemical performance of GCE / (PDA/ Cu) n modified electrode for glucose detection was investigated by cyclic voltammetry and amperometric current-time curve under alkaline conditions. At detection potential of 0. 35 V, the GCE / ( PDA/ Cu) 4 modified electrode presented a linear range of 0. 5 - 9. 0 mmol/ L with a detection limit of 5. 8 μmol/ L (S / N=3). Moreover, the sensitivity of the modified electrode was tunable by controlling the number of bilayers of the multilayer films. The modified electrode showed highly selective, stable and fast amperometric sensing of glucose. It was applied to determine glucose in human blood serum with satisfactory results.

Evaluation of in vitro enzymatic and non-enzymatic antioxidant properites of leaf extract from Alpinia Purpurata (Vieill.) K. Schum / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the enzymatic and non-enzymatic antioxidants of leaf extract from Alpinia purpurata.</p><p><b>METHODS</b>One gram of fresh leaf of Alpinia purpurata was grinded in 2 mL of 50% ethanol and centrifuged at 10,000×g at 4°C for 10 min. The supernatant obtained was used within 4 h for various enzymatic antioxidants assays like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), ascorbate oxidase, peroxidase, polyphenol oxidase (PPO) and non-enzymatic antioxidants such as vitamin C, total reduced glutathione (TRG) and lipid peroxidation (LPO).</p><p><b>RESULTS</b>The leaf extract of Alpinia purpurata possess antioxidants like vitamin C 472.92±6.80 μg/mg protein, GST 372.11±5.70 μmol of 1-chloro 2,4 dinitrobenzene (CDNB)-reduced glutathione (GSH) conjugate formed/min/mg protein, GPx 281.69±6.43 μg of glutathione oxidized/min/mg protein, peroxidases 173.12±9.40 μmol/g tissue, TRG 75.27±3.55 μg/mg protein, SOD 58.03±2.11 U/mg protein, CAT 46.70±2.35 μmol of H2O2 consumed/min/mg protein in high amount whereas ascorbate oxidase 17.41±2.46 U/g tissue, LPO 2.71±0.14 nmol/L of malondialdehyde formed/min/mg protein and PPO 1.14±0.11 μmol/g tissue in moderate amount.</p><p><b>CONCLUSION</b>Alpinia purpurata has the potential to scavenge the free radicals and protect against oxidative stress causing diseases. In future, Alpinia purpurata may serve as a good pharmacotherapeutic agent.</p>

Inhibitory effects of extracts from Moutan Cortex on formation of advanced glycation end-products (AGEs) in vitro and specificity screening of its potential active components / 中国中药杂志

In this study, bovine serum albumin (BSA)/methylglyoxal (MGO) non-enzymatic glycosylation reaction system was used for the evaluation of the inhibitory effects of Moutan Cortex extracts on the formation of AGEs. The HPLC-LC-ESI-MS/MS technology was adopted to test and indentify active components in Moutan Cortex against AGEs formation. The different concentrations of extracts (crude herb concentration 50, 100, 150, 200, 250 g•L⁻¹) from Moutan Cortexwas determined by fluorospectrophotometry, indicating an activity against AGEs formation in different concentrations of extracts, the inhibition ratio were (36.2±5.3)%, (43.5±6.2)%, (55.4±7.8)%, (68.6±6.7)%, (70.4±8.2)%, respectively after 6-day reaction in a dose dependent manner. Besides, the forming speed of AGEs tended to be steady after 24 h reaction. The HPLC technology was used to analyze chromatograms before and after the incubation of Moutan Cortex and methylglyoxal, identify changes in five chromatographic peaks and show decrease or increase in chromatographic peaks. These substances were trigalloyl glucose, tetragalloyl glucose, galloylpaeoniflorin, hexagalloyl glucose and benzoylpaeoniflorin after LC-ESI-MS/MS identification. Extracts from Moutan Cortex showed the remarkable inhibitory effects against formation of AGEs in BSA/glucose system. Furthermore, these potential active components might be associated with the efficacy of Moutan Cortex on treatment of diabetic nephropathy, which enriches basic studies for Moutan Cortex and provides ideas and reference basis for subsequent studies.

Chemical constituents from the stems of Gymnema sylvestre / 中国天然药物

AIM@#To study the chemical constituents of stems of Gymnema sylvestre (Retz.) Schult.@*METHODS@#Chromatographic techniques using silica gel, C18 reversed phase silica gel, and prep-HPLC were used. The structures were elucidated on the basis of MS and spectroscopic analysis (1D and 2D NMR), as well as chemical methods.@*RESULTS@#Seven compounds were isolated and their structures were elucidated as conduritol A (1), stigmasterol (2), lupeol (3), stigmasterol-3-O-β-D-glucoside (4), the sodium salt of 22α-hydroxy-longispinogenin-3-O-β-D-glucopyranosyl-(1→3)-β-D-glu-curono-pyranosyl-28-O-α-L-rhamnopyranoside (5), oleanolic acid-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (6), and the sodium salt of 22α-hydroxy-longispinogenin 3-O-β-D-glucuronopyranosyl-28-O-α-L-rhamnopyranoside (7). The inhibition activities of compounds 1, 5-7 on non-enzymatic glycation of protein in vitro were evaluated.@*CONCLUSION@#Compound 7 is a new triterpenoid saponin. It was shown that compounds 1, 5-7 have weak inhibition activities for non-enzymatic glycation of protein in vitro.

Expression of advanced glycation end-products in photoaging skin / 中华医学美学美容杂志

Objective To detect the expression of advanced glycation end-products (AGEs) in the sun exposure and non-exposure skin,to observe the elastic protein in the same location,and to explore the relationship between non-enzymatic glycation reactions and,photo-aging skin morphological changes.Methods In the exposure and non-exposure specimens from 30 patients,elastic fibers were stained with Gomori staining,and immunohistochemistry for AGEs was performed.Results AGEs expressed clearly positive in all elastic fibers of degeneration markedly (+ + above) of exposure skin,and appeared line-like in markedly(+ + above)elastic fibers.While in all non-exposure skin,AGEs expressed negative.In the exposure skin with middle or old age,compared with non-exposure skin,AGEs expressed more and elastic fibers were hyperplasia with thickening,curling and irregular distribution.The skin parts of AGEs expressed accorded with where elastic fibers degenerated.Conclusions Ultraviolet radiation can induce denaturation of elastic fibers and non-enzymatic glycation may contribute to the mechanism of skin photo-aging.

Estado redox materno en el tercer trimestre de la gestación y su relación con el flujo útero-placentario / Maternal redox state in the third trimester of pregnancy and its relationship with uteroplacental flow

Introducción: durante la gestación el óxido nítrico liberado por las células endoteliales de las arterias uterinas y la vasculatura umbilical promueve la vasodilatación y facilitan el flujo sanguíneo al feto. Los factores prooxidantes pueden, por el contrario, causar disfunción endotelial y comprometer el flujo útero-placentario. Objetivo: determinar la relación entre el estado redox materno en el tercer trimestre de la gestación y el flujo útero-placentario. Métodos: se realizó un estudio observacional analítico, que incluyó a 65 gestantes de las áreas de salud del municipio Bayamo, en el período comprendido desde diciembre de 2011 hasta abril de 2012. Se midió el índice de pulsatilidad promedio de las arterias uterinas y de la arteria umbilical por flujometría doppler, así como, biomarcadores del estado redox en sangre materna: se midió el Potencial Reductor Férrico del suero, la concentración sérica del malondialdehído más el 4-hidroxinonenal, de la vitamina C, la albúmina, el ácido úrico y la de glutatión reducido eritrocitario. Resultados: la mayoría de los índices de pulsatilidad promedio de las arterias uterinas y el de la umbilical se encontraron entre el percentil 95 y 50 para la edad gestacional. Los valores del malondialdehído más el 4-hidroxinonenal como indicadores de daño oxidativo fueron bajos, mientras que se detectaron altos valores de la vitamina C, del glutatión reducido y de la actividad de la enzima superóxido dismutasa. Las concentraciones de la vitamina C se asociaron de manera directa y significativa con el índice de pulsatilidad promedio de las arterias uterinas. Conclusiones: se concluye que el comportamiento de los biomarcadores del estado redox se corresponde con un adecuado estado antioxidante y con el estado del flujo útero-placentario, no obstante, a que solo las concentraciones de la vitamina C se asociaron con este.

Introduction: during gestational stage, nitric oxide released by placenta endothelial cells, uterine arteries, and umbilical vasculature promote vasodilatation and facilitate blood flow to the fetus. Pro-oxidant factors may, however, cause endothelial dysfunction and compromise the uterus-placentary flow. Objective: to determine the relationship between maternal redox status in the 3rd. trimester of pregnancy and uterus-placental flow. Methods: a prospective observational study was performed; it included 65 pregnant women from Bayamo municipality from December 2011 to April 2012. The average pulsatility index of the uterine arteries and umbilical artery using Doppler flowmetry, as well as redox status biomarkers in maternal blood were measured: Ferric reducing potencial, Serum malondialdehyde plus 4-hydroxynonenal, vitamin C, albumin, uric acid, and erythrocyte reduced glutathione concentration were measured. Total extracellular superoxide dismutase and catalase activity were also measured. Results: most of the average pulsatility index of the uterine and cord arteries was found between the 95 and 50 percentile for gestational age. Malondialdehyde values plus 4-hydroxynonenal were low, whereas high values were detected from vitamin C, reduced glutathione and the superoxide dismutase enzyme activity. Vitamin C concentrations were directly and significantly associated with mean pulsatility index of the uterine arteries. Conclusions: the behavior of redox status biomarkers corresponds to an adequate antioxidant status and the state of, however, only the concentrations of vitamin C were associated with uterus-placental flow.

Screening for anti-diabetic effective fraction in leaves of Olea europaea / 中草药

Objective: To screen the anti-diabetic effective fraction in the leaves of Olea europaea. Methods: Different fractions (Frs. A-F) were prepared using reflux extraction and separation with macroporous resins. α-Amylase system, non-enzymatic glycation system, and alloxan-induced diabetic models of mice were used to evaluate the anti-diabetic activities of the different fractions, respectively. Results: Frs. C-F fractions showed the significant inhibitory activities against α-amylase system and non-enzymatic glycation system. Especially, fraction D showed the best anti-diabetic activity compared with the other fractions, which also displayed a good dose-effect relationship. The anti-diabetic activities of Frs. C and D were both obvious and Fr. D showed more significance. Conclusion: The leaves of O. europaea show the potential anti-diabetic activity and the most effective fraction is ascribed to Fr. D.

Aldehyde seaweed polysaccharide composites serve as artificial liver carriers / 中国组织工程研究

10.3969/j.issn.2095-4344.2013.25.014

Effect of acute exposure of N, N-Dimethylformamide, an industrial solvent on lipid peroxidation and antioxidants in liver and kidney of rats.

N, N-Dimethylformamide (DMF), an industrial solvent widely used throughout the world is a known toxic compound. Here, we studied the effects of acute exposure of DMF on liver and kidney in rats. Rats were treated intraperitoneally with a single dose of DMF (1.5 g/kg) for 24 and 48 h. Hepatic and nephrotoxicity was confirmed based on the significant increase in the serum levels of aspartate amino transferase, alanine amino transferase, alkaline phosphatase, g-glutamyl transferase, urea, creatinine and electrolytes. Oxidative stress was assessed by determining the levels of lipid peroxidation (LPO) and antioxidants in liver and kidney. The LPO levels were elevated in both the tissues upon DMF exposure, whereas the activities of enzymatic antioxidants SOD, CAT and Gpx and non-enzymatic antioxidants (glutathione and vitamin C) were declined. The hepatic- and nephrotoxicity was further confirmed by the increasing incidence of inflammation in the histopathological studies. The findings indicate that acute exposure of DMF results in oxidative stress, antioxidant deficiency, attenuating liver and kidney marker enzymes, resulting in tissue inflammation and damage.

In Vitro Evaluation of the Anticancer Effect of Methanolic Extract of Alstonia scholaris Leaves on Mammary Carcinoma.

Breast cancer is the leading cause of cancer associated death among women worldwide. Current cancer treatments include chemotherapy, radiotherapy, and surgery. However, since these conventional methods often have undesirable side effects,new focus towards the use of plant extract to treating cancer with eliminating the side effects. The objective of present study is to asses the anticancer effect of leaves of Alstonia scholaris, using the cytosolic marker enzymes like Aspartate transaminase (AST),Acid phosphatase(ACP) , Alkaline phosphatase(ALP), Lactate dehydrogenase (LDH), Gamma - glutamyl transferase(γ– GT), and 5'-nuclcotidase(5'-NT) in vitro over breast cancer tissue. These are key enzymes in the metabolic pathways and these are the target for the drugs used in chemotherapy. An elevated level of enzyme concentration signals the presence of malignancy .The effect of leaves of Alstonia scholaris is also assessed by studying the effect of non-enzymatic antioxidants like vitamin A, E, C in vitro over breast cancer tissue. The present study revealed the leaves of methanolic extracts of Alstonia scholaris on cancer cells/tumor cells in vitro has been justified by its cytotoxic effect and anti proliferative effect.