1. in such features as cell antigenicity, invasiveness,

1.   Introduction:Cancer being a catastrophe of the frame of higher multicellularorganisms consequential from the abnormal growth of the cells attainsmodifications in the statement of copious amendments, prompting dysregulationof the conventional cell programming concerned with the cell division and cellseparation. This upshot in an unevenness of cell replication and cell passingthat stimulates the development of a tumor cell population. The characters thatportray a lopsided growth of a malignant tumor are the competence to strikelocally, to sweep to neighboring lymph nodes, and to metastasize far off organsin the body. Clinically, growth seems, by all accounts, to be an extensiveassortment of diseases with various phenotypic attributes. As a cancerousgrowth ensues, genetic drift in the cell population forms cell heterogeneity insuch features as cell antigenicity, invasiveness, metastatic potential, rate ofcell proliferation, differentiation state, and response to chemotherapeuticagents. At the molecular level, all cancers have plentiful things in mutual,which implies that the ultimate biochemical lesions steering to malignanttransformation and progression can be fabricated by a common but not analogouspattern of alterations of gene readout. In general, malignant cancers causenoteworthy morbidity and will be lethal to the host if not treated.

Omissionsto them give the marks to be concealed, laidback cancers that may remainclinically undetectable (or in situ), permits the host to have a standard lifeexpectancy. There are over 100 diverse categories of cancer, and eachis classified by the type of cell that is initially affected, and these includebreast cancer, cervical cancer, skin cancer, leukemia, lung cancer, prostatecancer, and so on. Chronic degenerative diseases likecancer have touched rampant proportions and are regarded as a serious medicalcondition and therefore, the treatments of these diseases are of clinicalimportance (WHO, 2005). In consequence, the medicinal plants play a pivotalrole and are considered as the basis for health preservation and careworldwide. There havebeen researches done on the anti-cancerous properties of plants and recognizedfor centuries.

 The National Cancer Institute (NCI) has screenedapproximately 35,000 plant species for conceivable anticancer activities. Amongthem, about 3,000 plant species have established reproducible anticanceractivity. Various studieshave concentrated on the anticarcinogenic properties of plants, to name a few, Abrus precatorius, Albizza lebbeck Alstoniascholaries Anacardium occidentale hepatoma, Asparagus racemosa, Boswelliaserrata, Ethyrina suberosa, Euphorbia hirta, Gynandropis pentaphylla, Nigellasativa, Peaderia foetida, Picrorrhiza kurroa, Withania somnifera, Annonamuricata. Due to the therapeutic ability of the species Annona muricata ofthe family Annonaceae, wide range of studies has been made over the lastdecade.

The bioactivity as well as toxicity of this species has diverted theattention towards itself.  2.   Annona muricata: Descriptionand ethnomedicinal uses:Annona muricata, commonly called Soursop or Graviola, isa tropical plant species renowned for its edible fruit which has selectedrestorative benefits, yet in addition particular toxicological impacts. This plantis a species of the genus Annona, of the Annonaceae family, order Magnolialesand Division Magnoliophyta.

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The genus Annona comprises over 70 species among which A. muricata is the mostextensively grown. The Soursop tree grows up to 5– 10 m tall and 15– 83 cm inbreadth with low branches. It has a tendency to blossom and fruit a largesegment of the year, however there are more characterized seasons relying uponthe altitude.

It is scattered in the tropical areas. The flowers of the plant are showy and areyellow-green colored. The fruit here is an ovoid berry, dull greenin shading. Each fruit may comprise 55–170 black seeds when fresh and theytransform to light brown when dry. The flesh is white and creamy withdistinctive aroma and flavor.

Figure 1: A)Annona muricata, fruit tree    B) Leaves     C) Fruit, an ovoid berry    D) Flower  Traditionalmedicinal benefits ofleaves, bark, fruit and seed of A.muricata have been the emphasis of myriad medicinalroutines. The most significantly used preparation in traditional medicine isthe decoction of bark, root, seed or leaf and applications are wide-ranging.The ingestion of leaves decoction is used as analgesic and also it is used tocope with discomfort accompanying colds, flu and asthma; and to treat cutaneous(external) and internal parasites. The use of leaves to treat malaria is verysubstantial.

The fruit is not only cherished as food, but the juice is used asgalactogogue to treat diarrhea, heart and liver diseases, and againstintestinal parasites. Customary therapeutic qualities of A. muricata have been recognized intropical locales to ponder different afflictions, for example, fever, torment,respiratory and skin disease, bacterial infections, hypertension, aggravation,diabetes and cancer.

There have been a number of reports on theethno-medical uses of A.muricata leavesincluding treatments for hypertension, diabetes and cancer. Mostparts of the A. muricata tree, similar to that of the other Annona species,namely A. squamosa and A. reticulata arewidely used as local and habitual medicines contrary to a large number of humaninflamations, infections and disorders. The pulp of the fruit is used as naturalmedicine for joint pains, dysentery, neuralgia, diarrhea, rheumatism, fever,malaria, parasites, skin rushes and worms, and also is eaten to increase mother’s milk after parturition.

The leavesare used to control cystitis, , body aches and diabetes. Additionally, theadministration of the leaf’s decoction is thought to display anti-rheumatic andneuralgic effects. In addition, the cooked leaves are topically used to treatabscesses and rheumatism that was reported by de Sousa OV andcolleagues.(9) The crushedseeds are thought to have anthelmintic activities antaagonistic to external andinternal worms. In tropical Africa, the plant is employed as an astringentpesticide, and insecticide; along this it is used to treat coughs, pain andskin diseases. In India, the fruit and flower are used as remedies againstcatarrh, while the root-bark and leaves are well known to have an antiphlogistic and anthelmintic activities which was worked uponby Adewole SO, Ojewole J.

(2)InMalaysia, the crude extract of A. muricata and A.squamosa is used as a syrup on the head to buffer from fainting. In SouthAmerica and tropical Africa, including Nigeria, leaves of A.muricata areimplemented as an ethnomedicine opposed to tumors and cancer. Moreover, thesmooth muscle relaxant, hypotensive hypoglycemic, anti-inflammatory, sedative,and antispasmodic effects accredit to the leaves, barks and roots of A.muricata.

Moreover along with the ethnomedicinal uses, the fruits arewidely employed for the preparation of beverages, ice creams,and syrups (3, 21, 23, 26) . 3.   Phytoconstituents: Thereare more than 200 chemical compounds that have been documented and isolatedfrom this plant; the foremost being the alkaloids, phenols, flavonoids andacetogenins. Based on the in vitro studies, extracts and phytochemicals of A. muricata have been sorted out asanti-microbial, anti-inflammatory, anti-protozoan, antioxidant, insecticide,larvicide, and cytotoxic totumor cells. Studies on the extracts and isolated compounds of A.

muricata showed contraceptive,antitumor, anti-ulceric, wound healing, hepato-protective, anxiolytic,anti-stress, anti-inflammatory, anti-icteric and hypoglycemic activities.Furthermore, there have been clinical studies carried out in order to boost thehypoglycemic activity of the ethanolic extracts of A. muricata leaves. Mechanisms of action of a few pharmacologicalactivities have been explicated, such as cytotoxic, antioxidant, antimicrobial,anti-nociception and hypotensive activities.

Nonetheless, some phytochemicalcompounds isolated from A. muricatahave shown a neurotoxic effect in vitro and in vivo. Thus, these crude extractsand isolated compounds requires further studies to define the magnitude of theeffects, optimal dosage, long-term safety, and potential side effects. (32)Constant examinations on diverse partsof the A. muricata have shown the occurrence of varieties of phytoconstituents and compounds, including flavonol triglycosides (FTGs) alkaloids(ALKs), phenolics (PLs), megastigmanes (MGs), cyclopeptides (CPs) and essentialoils. The existence of various minerals such as Ca, Na, Fe, K, Cu and Mg implythat regular intake of the A.

muricata fruit can help furnish essential nutrients to thehuman body. However, Annona species, including A. muricata, have beenshown to be a vital source of annonaceous acetogenin compounds (AGEs). Almostall the parts inclusive of the fruits, leaves, stems androots of this plant are known to be rich in flavonoids, isoquinoline alkaloidsand annonaceous acetogenins. (23,25,26,28,35,42) Acetogenins are a unique category of C-35/C37 secondarymetabolites obtained from long chain (C-32/C34) fatty acids in the polyketidepathway. They are basically illustrated by combining fatty acids with  2-propanol unit at C-2 that gives a methyl-substituted?, ?-unsaturated ?-lactone. Starting with the discovery of uvaricin from Uvariaaccuminata in 1982, there have been several acetogenins that areidentified. About 500 have been reported from numerous parts of the plants inthe Annonaceae family.

Because of the exceptional structures and broad spectrumof biological activities, AGEs have drawn significant scientific interest oflate. The active annonaceous acetogenins have shown to be successful ininducing death in cancer cells that are resistant to even chemotherapeuticdrugs. Besides their remarkable anti proliferative efficacy, these annonaceousacetogenins have been endorsed to debilitating side effects such asneurotoxicity suggesting that these components can easily traverse theblood–brain barrier and are known to cause atypical Parkinson’s disease, thusrestricting their development as new drug entities. Various biologicalactivities have been reported for AGEs, including antimalarial, anti-parasiticand pesticidal activities. However, the physiological activities of AGEs areinitially characterized by the toxicity against cancer cells and inhibitions ofthe mitochondrial complex I. (11)4.    Anticancer activity:There are plentiful reports that signify anti-proliferative effects ofvarious extracts of the plant and isolated AGEs against various cancer celllines.

As mentioned earlier cancer is categorized based on the primary tissueit occurs in. The antitumor effects of A.muricata against various cancer cell lines are described here.Breast Adenocarcinoma: Anoncogene, the epidermal growth factor receptor (EGFR) that is quite often overexpressedin breast cancer (BC) and is linked with poor prognosis and drug resistance. Studieshave revealed that there is a selective inhibition of breast cancer cells viaEGFR down regulation by the extract of Graviola. Thus, EGFR is a rationaltarget for BC therapy development.

In addition, xenografts mouse model studies experimentshave showed that the fruit extract reduces the growth of BC cells. The Fruit extractspecifically suppressed the growth of EGFR-overexpressing human BC (MDA-MB-468)cells but did not in non-tumorigenic human breast epithelial cells (MCF-10A). These reports bolster theevidence that Graviola has selective anti-growth effects between cancer andnon-cancer cells (8). In a report by Yu-Min Koaand colleagues it was shown that Graviola favors apoptosis in ER-relatedpathways. In addition, it was also noted that Graviola had subsided MCF-7 tumorgrowth while hindering ER-cyclin D1 and Bcl-2 protein expressions in nude mice(44). A promisingantitumor effect was reported in an in vivo studyon 7,12-dimethylbenzene anthracene (DMBA)-induced cell proliferation in thebreast tissues of mice. Oral administration of the Graviola leaves supposedly haveprotective effects towards the development of breast carcinogenesis was shownby DMBA as it induced a protective effect against DNA damage. Lung Cancer: The A549 cells (adenocarcinomichuman alveolar basal epithelial cells) when treated with aqueousextracts of A.

muricata showed elevated levels of ROS, reduction of MMP (matrixmetalloproteinases) through the upheaval of expression of Bax and decreasedexpression of Bcl-2. These events led to the release of cytochrome c release tothe cytosol. The released cyt-c activatedcaspase-9 and caspase-3, thus causing apoptosis. Concomitantly, there was acell cycle arrest at G0G1 phase.

The incubation of A549 cells with superoxidedismutase and catalase predominantly suppressed the cytotoxicity that was inducedby the extract. This highlighted that the intracellular ROS plays an importantrole in cell death. Anonna muricata also has had impacts on Lewis lung carcinoma (LLC)tumor cell lines that were examined both invivo as well as in vitro wasshown in a study by Zhao GX1 et,. al. Thisvalidated that Graviola had antitumor activity by limiting the natural growthof the lung tumors (45). NADH oxidase inhibition in cancer cell lines, downregulation of the P-glycoprotein pump via ATP depletion and Cell cycle arrestat S-phase progression has shown to be affected by Graviola through itsanti-cancerous and cytotoxic mechanisms, which has been shown by additionalresearch.

 Liver Carcinoma:The aqueous extracts of Annona muricata was tested for theactivation of caspases in Huh-7 human liver cancer cells. Huh-7 cells whentreated with the extracts, both caspase-9 and caspase-3 activities in the cellswere uplifted. This suggested that the extract had induced apoptosis by activatingthe mitochondrial mediated intrinsic pathway.

The protein analysis (westernblot) of caspases demonstrated that the expressions of the cleaved caspases wasdose dependent. Considering these results, it was concluded that the extract ofA.muricata leaves have a goodpotential to play a vital role as cancer chemotherapeutic agents. Furthermore, thereports by ____________indicate the reduction in the viability of hepatocellular carcinoma cell linespossibly through G0/G1 orS phase arrest or via induction of sub-G0/G1 DNA fragmentation.

This mechanismremains yet to be confirmed. Pancreatic Cancer (PC): Pancreatic tumor aggressiveness is alliedwith a heightened metabolic activity and glucose concentration of malignanttumors. Also a hypoxic environment has been proven tobe necessary for the oncogenic and metabolic transformation and the survival ofthe pancreatic cancer cells. In particular, it is believed that resistanceto drugs is induced by hypoxia by the activation of PI3K, Akt, NF-?B and MAPKpathways 51.  The cells after being treated with the Graviolaextract, showed low viability as the phosphorylation of the molecules washindered by the extract. Accordingly, the major transcription factor that isactivated under hypoxic conditions, HIF-1a expression was investigated. The reportsstated that a product of the extract had blocked glucose transporters (GLUT1and GLUT4), NF-?B, HIF-1a and otherglycolytic enzymes like LDHA, which reduced the uptake of glucose molecules andthe production of ATP by the PC cells. This overall reduction caused the celldeath.

In accord with the ATP reduction, the extract also increased the ROSlevels within the cells that eventually led to necrosis. 52–54. Necrotic agentsby itself being less significant in the treatment of cancer as they induce alocal inflammation. But the inflammation thus caused leads to the activation ofthe innate immune system to initiate anti-tumor activities. 52. This effect alongwith the progression of PC cells was evaluated in the KrasG12DPdx1- Cre mice (55,56).

The mice with adeveloping pancreatic intraepithelial neoplastic (PanIN) lesions was fed withthe plant extract. The cells were analyzed and it was concluded that thecytotoxic effects were particular to the tumor cells. Thus the administrationof Graviola caused glycolytic inhibition was seen in the PC cells that led tothe depletion of the tumor. Prostate CancerThe anti-proliferative effects of the waterextract of leaves of A.

muricata was examined in vivo against the benignprostatic hyperplasia (BPH-1) cell line and the rat prostates were observed. Normalhistology of all the other testes was observed. There was a significantreduction in the size of the seminal vesicles of the test groups and displayeda remarkable atrophy with a raised cellularity and zero secretion in theacinii. The apoptotic characteristics of the glandular epithelium was typicallyseen (pycknotic nuclei and nuclear material towards the periphery). Also, the Baxprotein was up regulated, while Bcl-2 was suppressed.

Thus it was concludedthat Annona muricatahas anti-proliferative effects on BPH-1 cells and deduces the prostate size,possibly through apoptosis (5). The effect of Graviola extract againstthe prostate cancer cell lines has also been expounded in vitro. Experimentshave been performed to show that Graviola initiates necrosis in PC-3 cellsthrough the inhibition of cellular metabolism and tumor mobility. Furtherevaluation depicted the downregulation of the expression of the hypoxia-relatedfactors and glycolytic factors following treatment in PC cells with Graviola(41).

The inhibition ofprostate cancer proliferation, viability and clonogenic colonies have been provedas an outcome of the Graviola leaf extract (GLE) pharmacokinetics andabsorption kinetics (43). by Yang C et, .al Colon Cancer:In a study performed by Jaramillo MC et al., the mechanism of action of ethylacetate extract of A.

muricata leaves against colon cancercells (HT-29 and HCT-116) and lung cancer cells (A549) has been illustrated. Theleaf extract was proficient to induce apoptosis in colon and lung cancer cellsthrough the mitochondrial-mediated pathway. This anti-proliferative effect wasalongside with cell cycle arrest in the G1 phase (20).

However,the migration and invasion of colon cancer cells were profoundly inhibited bythe leaf extract. The in vivo chemo preventive potential of the ethyl acetateextract of the A. muricata leaves against azoxymethane-inducedcolonic aberrant crypt foci (ACF) in rats was validated by Moghadamtousi andcolleagues.

Oral dosage that was administered for 60days caused a significantreduction of ACF formation in rats when tested by methylene blue staining ofthe colorectal specimens. PCNA and Bcl2 proteins were down regulated whereasBax protein was up regulated after the administration of the extract. This was depictedin the immunohistochemistry analysis where theywere compared with cancer control group. In addition the levels of enzymaticantioxidants showed an increase and a suppression was seen in malondialdehydelevel of the colon tissue homogenates. This suggested the restraint of lipidperoxidation. An AGE annomuricin E was found to inhibit the growth of HT-29 cells.The cytotoxic effect of annomuricin E was enhanced by the G1 cell cycle arrest.Annomuricin activated the mitochondrial events comprising the dissipation ofthe mitochondrial membrane potential and caused the leakage of cytochrome cfrom the mitochondria.

Followed by this, annomuricin E activated caspase 3/7and caspase 9 responsible for the apoptosis. Furthermore, Moghadamtousi and colleagues examined thatethyl acetate extract of Annona muricata leaves (EEAM) exerted astriking cytotoxic effects on HCT-116 cells as determined by MTT and LDHassays. Flow cytometric analysis illucidated the cell cycle arrest at G1 phaseand also the externalization of phosphatidylserine acting as an indicator ofthe induction of apoptosis.

EEAM treatment activated excessive accumulation ofROS followed by disruption of MMP, cytochrome c leakage andactivation of the initiator and executioner caspases in both colon cancer cells.These processes subsequently steer toattenuation of mitochondrial membrane potential (MMP) and cytochrome c release.Release of cytochrome c activates apotosome and the intrinsic caspase cascadethat triggers execution of apoptosis through DNA fragmentation. Immunofluorescence analysis portrayed theup-regulation of Bax and down-regulation of Bcl-2 proteins while treated withEEAM.

Furthermore, EEAM conspicuously blocked the migration and invasion ofHT-29 and HCT-116 cells. (29,30,31).  Thus, these findings verify the usage of A. muricata leaves inethnomedicine against cancer and emphasize annomuricin E as one of the contributingcompounds in the anticancer activity of A.

muricata leaves.Ovarian Cancer:Apart from its various medicinal properties, acetogeninsin Annona muricata showed potent anti-ovarian cancer activity which selectivelyattack cancerous cells without harming healthy cells and preventing metastasis.Apoptosis of granulosa cells of the ovary has been initiated by this acetogeninin many organs of cancerous cells of humans. The plant had proved to be aneffective anti-tumor and anti-cancer medicinal plant, and thus represents asource for new antiovarian cancer drug discovery. In vitro cytotoxicity testswere performed using MTT assay as described by Xu-jie and Chu 11. In vivoPilot Experiment An in vivo pilot experiment was performed with thirty Swissalbino mice consisting of all females (average weights 18-25g). In order tomimic advanced ovarian cancer, the mice were injected intraperitoneally (i.

p.)with 1 x 104 OV7-96020764-CDNA-(20uL) cell line into the abdominal cavity toform ascites. Five groups of mice were examined: four control mice (notreatment), twelve mice treated with crude and pure compound from A. muricataleaf and four mice treated with doxorubicin (10 mg) after ascites had formed.Cells of ascites of two mice were frozen and stored for future experiments. Tostudy the reduction of swollen abdomen, 5 mg/kg doxorubicin (Rubex) and theisolated compound at a final concentration of 20 mg/kg were administered i.p.

 Leukemia: Investigationshave also been done to check the ability of ethanolic extracts of Annona muricata leaves for its cytotoxicity potential and capacity ofinducing apoptosis in K562 cancer cells, a chronic myelogenous cell line. The activity of Caspase-3was remarkably enhanced during the apoptosis stimulated by the extract at a lowquantity. Terminal deoxynucleotidyl transferase-mediated dUTP nick-endlabelling (TUNEL) assay results verified the process of apoptosis. The Caspase-3activity and TUNEL assay reports endorsed that the ethanolic extract of A. muricata leaves induced apoptosis inK562 cell lines. Ezirim, AU and colleagues thus concluded in their paper that Annona muricata could be considered as a natural source for thepreparation of pro apoptotic drugs. Also in the reports by, Constant Anatole Pieme and others, A.

muricata had exhibited anti proliferative effects on HL-60 cells(Human promyelocytic leukemia cell line) by promoting the lossof cell viability, morphological differences, loss in membrane mitochondrialpotential and G0/G1 phase cell arrest. Their conclusions confirmed the efficacyof A. muricata as an agent ofchemotherapeutic and cytostatic activity in HL-60 cells (7).Cervical Cancer:In a study by A N Artanti et,al Our result demonstratedthat polyketide derivatives from Annonamuricata have growth inhibitory and cytotoxic effect on cervical cancercell line.

The acetogenins from Annonamuricata L. leaves performed potent cytotoxic effect on HeLa cells. Decreasingcell viability may be because of either cell death or cell cycle arrest. Themechanism of cell cycle distribution is also associated with some of celuller proteinespecially p53 protein. p53 is a tumor suppresor protein. In this study, theyobserved that AGEs from Annona muricatatreatment increased p53 level in nucleus. Therefore, AGE isolation may be amandableas viral inhibitor agent and as competitor of vaccine to prevent thedevelopment of cervical cancer.

In conclusion, extracts of Annona muricata L.leaves indicate has potential to be developed as a co-chemotherapeutic agent onHeLa cell lines, it can exhibit potential abbility with p53 stabilization.Further molecular target detection to investigate its cellular pathway needs tobe conducted.