Pharmacogn Rev. 2013 Jan-Jun; 7(13): 7380.doi:10.4103/0973-7847.112853PMCID:PMC3731883Phytochemistry and medicinal properties ofPhaleria macrocarpa(Scheff.) Boerl. extractsRabia Altaf,Mohammad Zaini Bin Asmawi,Aidiahmad Dewa,1Amirin Sadikun,2andMuhammad Ihtisham UmarDepartment of Pharmacology, School of Pharmaceutical Sciences, University Sains Malaysia, Minden, Pulau Pinang, Malaysia1Department of Physiology, School of Pharmaceutical Sciences, University Sains Malaysia, Minden, Pulau Pinang, Malaysia2Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, University Sains Malaysia, Minden, Pulau Pinang, MalaysiaAddress for correspondence:Dr. Rabia Altaf, Department of Pharmacology, School of Pharmaceutical Sciences, University Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia. E-mail:moc.liamg@mahsithi.aibarReceived 2012 Dec 5; Revised 2013 Feb 3; Accepted 2013 Jun 1.Copyright: Pharmacognosy ReviewsThis is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractPhaleria macrocarpa, commonly known as Mahkota dewa is a medicinal plant that is indigenous to Indonesia and Malaysia. Extracts ofP. macrocarpahave been used since years in traditional medicine that are evaluated scientifically as well. The extracts are reported for a number of valuable medicinal properties such as anti-cancer, anti-diabetic, anti-hyperlipidemic, anti-inflammatory, anti-bacterial, anti-fungal, anti-oxidant and vasorelaxant effect. The constituents isolated from different parts ofP. macrocarpainclude Phalerin, gallic acid, Icaricide C, magniferin, mahkoside A, dodecanoic acid, palmitic acid, des-acetylflavicordin-A, flavicordin-A, flavicordin-D, flavicordin-A glucoside, ethyl stearate, lignans, alkaloids andsaponins. The present review is an up-to-date summary of occurrence, botanical description, ethnopharmacology, bioactivity and toxicological studies related toP. macrocarpa.Keywords:Gallic acid, God's crown, magniferin, Mahkota dewa, phalerin, thymelaceaeOCCURRENCE, BOTANICAL DESCRIPTION AND ETHNOPHARMACOLOGYPhaleria macrocarpa, commonly known as God's crown, Mahkota dewa or Pau is an Indonesian plant of family Thymelaceae that grows in tropical areas of Papua island. It is a complete tree, including stem, leaves, flowers and fruits [Figure 1]. Its height ranges from 1 m to 18 m with 1 m long straight root exuding sap, brownish green bark and white wood. It grows 10-1,200 m above sea level with a productive age that ranges from 10 to 20 years. The leaves are green and tapering with length and width ranging from 7 cm to 10 cm and 3-5 cm respectively. The flowers make a compound of 2-4, with color from green to maroon. Pit is round, white and poisonous[1] and fruit is of eclipse shape with a diameter of 3 cm. Fruits are green when un-ripened and become red on ripening.[2] Seeds exist as 1-2 seeds per fruit and are brown, ovoid and anatropous. Although the herb is being used in both un-processed and processed form, however, the former can be poisonous and toxic.[3]P. macrocarpais being considered generally as a treatment of life style diseases.[4] Extracts ofP. macrocarpaare reported for a number of pharmacological activities, including anti-tumor, anti-hyperglycemia, anti-inflammation, anti-diarrhoeal, vasodilator, anti-oxidant, anti-viral, anti-bacterial and anti-fungal effect. Its stem is used to treat bone cancer; egg shells of seeds are used in treating breast cancer, cervix cancer, lung diseases, liver and heart diseases while leaves contain constituents that treat impotence, blood diseases, allergies, diabetes mellitus and tumors.[5] In this review article, we have reviewed medicinal properties ofP. macrocarpaextracts and chemical constituents that are isolated from its extracts to update current knowledge regarding this valuable plant and to provide guidelines for its future implications in the medical field.PHYTOCHEMICAL STUDIESQualitative phytochemical studiesA variety of chemical constituents have been found in different parts ofP. macrocarpain varying concentrations. These include mahkoside A, dodecanoic acid, palmitic acid, des-acetyl flavicordin-A, flavicordin-A, flavicordin-D, flavicordin-A glucoside, ethyl stearate, lignans and sucrose.[4] Yang and co-workers isolated mahkoside A (4,4 dihydroxy-2-methoxybenzophenone-6-O--D-glucopyranoside) for the first time from the pit ofP. macrocarpaalong with 6 known compounds including magniferin (a C-glucosylxantone), kaempferol-3-o--D-glucoside, dodecanoic acid, palmitic acid, ethyl stearate, and sucrose.[6] Lignans isolated from different parts ofP. macrocarpa, when subjected to chiral column analysis, have been found to include pinoresinol (79 4% [] -enantiomer excess), lariciresinol (55 6% [] -enantiomer excess) and matairesinol (pure [+] -enantiomers).[7] The bark and fruits are rich in saponins, alkaloids, polyphenolics, phenols, flavanoids, lignans and tannins.[8,9,10] Isolated constituents of fruit include Icariside C3, magniferin, and gallic acid.[1,11,12,13] Phalerin was first isolated from leaves ofP. macrocarpaas a benzophenone glycoside (3,4,5, trihydroxy-4-methoxy-benzophenone-3-O--D-glucoside) by Hartatiet al.[14] The same compound was isolated from fruits later by Oshimiet al.,[11] however the proposed structure (2,4,6, trihydroxy-4-methoxy-benzophenone-3-O--D-glucoside) was slightly different from the PRevious report. The pericarp of fruit contains kaempferol, myricetin, naringin and rutin.[2] Naringin and quercitin are found in mesocarp and seeds[2] while phorboesters, des-acetyl flavicordin-A and 29-norcucurbitacin derivatives have been isolated from seeds.[15] The chemical structures of the vital constituents isolated fromP. macrocarpaare shown inFigure 2.P. macrocarpaextracts are reported to contain alkaloids and saponins as well.[4] There is, however, a need of investigating the extracts thoroughly for the identification of these alkaloids and saponins and to relate them with the reported biological properties ofP. macrocarpaextracts.Quantification of isolated phytoconstituentsThe extracts ofP. macrocarpaare not yet standardized fully to give a detailed percent quantity of its isolated constituents. However, the concentration of few of its constituents in a particular fraction ofP. macrocarpaextracts are reported. For instance, methanol extracts of fruits ofP. macrocarpa(when serially extracted in petroleum ether, chloroform, methanol and water) is reported to have phalerin content up to 9.52%.[16] The crude paste (32 g) obtained from 3.2 kg ofP. macrocarpapit was found to consist of 9.1% Mahkoside A, 0.21% kaempferol and 0.1% magniferin, whereas 60% of this crude paste was consisted of sucrose.[6] More recently, Kimet al. has advised an extraction method that has as much as 2.1% yield of magniferin by using pressurized hot water (subcritical water) as solvent.[17] This high yield of magniferin was found to be dependent strongly on extraction temperature and weakly on pressure. The yield of magniferin with subcritical water was found to be lesser than that with methanol (2.5%) but higher than that with water (1.8%).The total phenolic content of mesocarp, pericarp and seeds was found to be 60.5 0.17, 59.2 0.04 and 47.7 1.04 mg galic acid equivalent/gram of dry weight (GAE/gDW) respectively.[18] The total flavonoid content of pericarp was found to be maximum (161.3 1.58 mg RE (rutin equivalent)/g DW) when compared to that of mesocarp and seeds (131.7 1.66 and 35.9 2.47 mg RE/g DW respectively).[18]A detailed phytochemical study ofP. macrocarpaextracts is imperative to get a more accurate estimation of its constituents. There is an utmost need of establishing chemical methods for the isolation of the already identified pharmacologically active constituents such as magniferin, phalerin, icariside and gallic acid with maximum possible yield.BIOACTIVITYAnticancer activityLeaves and fruits ofP. macrocarpaare being used since years in treatment of different types of cancer[9] especially against breast cancer[5,19] and brain tumor.[20]P. macrocarpasupplementation with adriamycin cyclophosphamide (AC) is reported for its synergistic effect in reducing tumor growth in breast cells by inducing apoptosis, but also for its protective effect on liver and kidney damage caused by AC.[21] Two of its constituents, phalerin and gallic acid, are proven to pay a major contribution to its cytotoxic properties.[1,22]Methanolic semipolar extract ofP. macrocarpa(designated as DLBS1425) was proved to manifest its anticancer activity in breast cancer cells by acting as an anti-proliferative, anti-angiogenic and apoptotic inducer.[23] This extract contained 20.26% phalerin and it induced apoptosis by DNA fragmentation, caspase-9 activation and by regulation of B-cell lymphoma 2 protein (Bcl-2) and Bcl-2 associated X proteins (BAX) at mRNA level. Bcl-2 are anti-apoptotic proteins encoded by Bcl-2 genes that act as check points in apoptosis regulation, while BAX are pro-apoptotic proteins of Bcl-2 gene family. Phalerin and gallic acid up-regulate BAX protein in a dose dependent manner while down-regulate the expression of Bcl-2 mRNA,[22,24] which causes sustained elevation of calcium levels in mitochondria [Figure 3]. This induces opening of voltage dependent anion channels and cause release of a small amount of (Cytochrome-c, an inter-membrane space protein) from mitochondria. Cyt-c interacts with inositol triphosphate receptor on endoplasmic reticulum causing the release of endoplasmic reticulum-calcium, enhancing overall level of calcium ions, which further triggers massive release of cyt-c. This cyt-c binds to WD-40 domain of C-terminus of apoptotic protease activating factor-1 (APAF-1) and ceases auto-inhibition of this domain while other two domains of APAF-1 as caspase activation and recruitment domain (CARD) and Nucleotide binding adaptor shared by APAF-1, R and CED-4 (NB-ARC) adenosine tri phosphatase domain (ATPase domain) remains in autoinhibited state. Cyt-c binding to APAF-1 triggers stable binding of ATP/dATP at nucleotide binding site of APAF-1. In the presence of seven cyt-c molecules and seven APAF-1 proteins, oligomerisation of APAF-1 into wheel-like heptagonal structure occurs and causes activation of apoptosome. The apoptosome causes cleavage and recruitment of inactive procaspase-9 (APAF-3) to activated caspase-9 molecules. Caspases are cysteine-aspartic-proteases consisting of initiator caspases as 2, 8, 9, 10 and effector caspases as 3, 6 and 7. The activated initiator caspase-9 further activates effector caspases, which triggers cascade of caspases 3, 6, 7. This activates DNAase to cause DNA fragmentation thus inducing apoptosis.[23]DLBS1425 also suppressed cyclo-oxygenase 2 mRNA expression and vascular endothelial growth factor-C (VGEF-C) mRNA expression. It caused a reduction in vascular permeability which further reduced endothelial cell proliferation and migration, resulting into anti-angiogenesis. Protein kinase-C was also inhibited by this extract causing down-regulation of transcriptional factors as activator protein-1, contributing further to its anti-proliferative activity.[23] Gallic acid along with acting on BAX and Bcl-2 genes, also acts on PI3K/Akt/mammalian target of rapamycin (mTOR) pathway.[20] In addition to these two pro-apoptotic mechanisms, gallic acid is also reported to increase reactive oxygen species in cancer cell linesin vitro.[25]Phosphatidyl-inositol-3-kinase (PI3K) activates Akt (protein kinase-B) that regulates cell survival. This further activates mTOR, which regulates cell growth and proliferation. In cancerous cells, this pathway becomes overactive and decreases apoptosis, which allows proliferation. Gallic acid exerts its anti-proliferative effect by down-regulating survival of Akt/mTOR.[24] Gallic acid also down-regulates phosphorylation of Ras/mitogen activated protein kinase pathway, thus suppressing A disintegrin and Mettalloproteinase (ADAM)-metallopeptidase domain 17 (tumor necrosis factor alpha converting enzyme) therefore, decreasing cell invasion and proliferation.[26] Gallic acid has been found to interfere with interaction of C-X-C chemokine receptor type-4 and C-X-C chemokine ligand-12 which inhibits PI3K activation. This inhibition further inhibits phosphorylation of Akt thus down-regulating VGEF at both protein and mRNA level, combating progression of angiogenesis and tumor.[27,28] Gallic acid has been found to inhibit growth of Hela cells and human umbilical vein endothelial cells (HUVEC) cellsin vitrowith median inhibitory concentration (IC50) of 80 M and 400 M for Hela and HUVEC cells respectively.[25]Ethanolic leaf extracts ofP. macrocarpais reported for its ability to increase expression of NKG2D (type-II integral membrane protein) and CD-122 (a subunit of interleukin-2), the surface molecules which enhances the activity of killer triggering receptors on natural killer cells (NKC) of spleen thus enhancing their killing activity. It increases interferon-gamma production, which is a glycoprotein that activates immune cells, macrophages and NKC, ultimately increasing recognition of infection or tumor by up-regulating T-lymphocytes.[29,30]Antihyperglycemic activityHyperglycemia is a condition in which excessive amounts of glucose circulates in blood plasma. The extracts ofP. macrocarpafruits have been found to lower the post-prandial hyperglycemia.[31,32] Highest activity is being shown by n-butanol extract of young and ripened fruits followed by ethyl acetate extract and then methanol extract. The -glucosidase enzyme is present in the brush border of the small intestine, which breaks down oligosaccharides, trisaccharides and starch to glucose and other monosaccharides. The inhibition of -glucosidase reduces rate of carbohydrates digestion, thus reducing the breakdown of carbohydrates into glucose. Glucose absorption, therefore, reduces and blood glucose level decreases contributing to hypoglycemic effect.P. macrocarpafruit competitively inhibits pancreatic -amylase and membrane bound intestinal hydrolase as isomaltase, maltase and sucrose by inhibiting -glucosidase. This delays glucose absorption and lowers post prandial hyperglycemia in short-term effect, while reduces HbA1c (glycated hemoglobin) in long term effect.[31] Methanol extracts of pericarp is recently reported to decrease blood glucose on 12thday in diabetic rats by 56.25% and 58.33% when compared with diabetic control and pre-treatment value respectively.[33] A further fractionation of n-butanol fraction of this pericarp methanol extract has revealed the presence of phalerin in the most active sub-fraction up to 22.50%.[16]We have already mentioned about the presence of rutin in mesocarp and pericarp ofP. macrocarpafruits. Rutin is reported to have a significant anti-diabetic effect in rats,[34] however, the anti-diabetic effect ofP. macrpcarpafruit has not been linked with the presence of rutin so far.Antihyperlipidemic activityThe increased body mass index causes increased level of total cholesterol and low-density lipoproteins (LDL) while reduces the levels of high-density lipoproteins. Increased cholesterol level in the body is termed as hypercholesterolemia. Imbalance in cholesterol hemostasis can create certain health problems as arteriosclerosis and heart diseases. The fruit ofP. macrocarpacontains many active compounds as alkaloids, saponins and polyphenols, one of them is gallic acid that is found to regulate cholesterol hemostasis. Gallic acid decreases the cholesterol level in the body by up-regulating LDL-R (low-density lipoprotein receptors) and pro-protein convertase subtilisin/kexin type-9 (PCSK9) via sterol regulatory element binding protein transcription factor (SREBP-2-TF) up-regulation.[8] High-levels of cholesterol in cells inhibit transcription of LDL-R and PCSK-9, which decreases intake of plasma cholesterol into cells. Gallic acid present in fruit ofP. macrocarpaincreases the number of LDL-R, which enhances binding of LDL particles in the blood to LDL-R. This further triggers LDL-R mediated endocytosis causing internalization into the peripheral cells decreasing circulating LDL-level, thus, reducing cholesterol level in cells. Gallic acid also up-regulates PCSK-9-mRNA expression. PCSK-9 binds to EGF-A (epidermal growth factor like repeat A) domain of cell surface LDL-R. Receptor-mediated endocytosis is initiated. PCSK-9-LDLR complex is formed and goes inside the cell and routed to lysosomes and degraded. This causes regulation of cell surface LDLR and thus decreases cholesterol levels.[35] SREBPs activates expression of 30 genes involved in synthesis of cholesterol, fatty acids and triglycerides.[36] SREBP up-regulates expression of 3-hydroxyl-3-methyl gentaryl-coenzyme A reductase. If cellular cholesterol level is high, SREBP exists as a membrane bound precursor and SREBP-cleavage activating protein (SCAP) remains inactivated, but if cellular cholesterol level decreases, SCAP becomes activated and escorts SREBP to golgi bodies. Proteolytic cleavage of SREBP occurs by site-1-protease and site-2-protease, which activates transcription factor of SREBP named as nuclear SREBP that translocates into nucleus. Inside the nucleus, SREBP binds to sterol regulatory elements activating genes involved in lipid hemostasis, thus controlling cholesterol balance.[8,37,38]Antibacterial and anti-fungal activityLeaves and seeds ofP. macrocarpaare found to have profound antibacterial activity.[39] Flavanoids, saponins, polyphenols and tannins present in the fruit highly inhibit gram positive bacteria as compared to gram-negative bacteria[40] due to the outer permeability barrier in gram-negative bacteria. These bacteria includeBacillus cereus, Bacillus subtilis, Enterobacter aerogenes, Eschericia coli, Klebsiella pneumonia, Micrococcus luteus, Pseudomonas aeroginosa, Staphylococcus aureus. It exhibits its antimicrobial activity by different mechanisms as inhibiting nucleic acid synthesis, energy metabolism or cytoplasmic membrane function.[41] Kaempferols are found to inhibitS. aureus, Enterococcus faecalis, Escherichia coliandP. aeroginosa. The methanol extracts of fruit ofP. macrocarpais found to have good activity againstP. aeroginosaand strong activity againstE. coli,[42]B. cereusandStreptococcus aureuswith the diameter of the inhibitory zone (DIZ) as 15-18 mm. Ethyl acetate extract has shown good activity againstE. coli, Klebsiella pneumoniaeandStreptococcus ubelliswhile strong activity againstP. aeroginosa, Streptococcus aureusandB. cereuswith DIZ as 15-27 mm. The hexane and chloroform extract have found lowest activity with DIZ less than 10 mm.[41] Phorbolesters inP. macrocarpaseeds inhibit growth of certain fungi asAspergillus niger, Fusarium oxysporum, Ganoderma lucidum, andMucor indicus.[2]Anti-inflammatory activityP. macrocarpais found to have potent anti-inflammatory activity[2] due to its contents, including terpenoids, saponins, tannins, flavanoids and phenols such as rutin and cathecol. During the process of inflammation, lipopolysaccharides of invading bacteria (for instance) bind to the tool like receptors (TLRs) on the dendritic cells, macrophages or antigen presenting cells (APCs). The cytoplasmic domains of TLRs on surface of APCs change and they cause activation of inactive protein kinase in the cytoplasm as PI3-kinase or Akt. This activation brings about cascade of changes that involve gene transcription factors including nuclear factor kappa-B (NF-kB). Tail of NF-kB have nuclear localization signals (NLS) that remains inactive as long as it remains attached with inhibitory kappa-B (IkB). Cascade of changes causes phosphorylation of IkB, which gets separated from NLS resulting into its activation. Activated NLS finds a way into the nucleus where it induces transcription of pro-inflammatory genes and production of COX1, COX2, leukotrienes and cytokines starts, which form prostaglandins and TNF- from arachidonic acid. These act on endothelial cells of post-capillary venules and stimulate synthesis of adhesion molecules as intracellular adhesive molecules and vascular adhesive molecules. Lymphocytes are attached with these adhesion molecules, invade the surrounding tissue and cause inflammation.Semipolar methanolic extract of fruit ofP. macrocarpaDLBS1425 (containing 20.26% phalerin) is reported to inhibit inflammation.[23] It suppresses COX2-mRNA expression even in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This inhibition of COX2-mRNA causes a decrease in prostaglandin E2 (PGE2) synthesis thus inhibiting inflammation. More recently, phalerin is reported to have mild inhibitory effect on xanthine oxidase and lipo-oxygenase (34.83 4.64 and 23.47 9.43% inhibition respectively). However its inhibitory effect on Hyaluronidase was found to be non-significant (1.34 0.57% inhibition).[43]P. macrocarpais also found to treat primary dysmenorrhea in which the production of leukotrienes and prostaglandins F2- and E2 is enhanced at a large level and may cause increased uterine smooth muscles tone and contractions causing abdominal cramps.[10] The pericarp and mesocarp extract of fruit have shown moderate anti-inflammatory activity while seed extract has shown weak activity.[2] Cytokines by binding with macrophages up-regulate nitric oxide synthase enzyme which forms NO (nitric oxide) from l-arginine. NO plays an important role in inflammation.[44] Extracts ofP. macrocarpainhibit NO production in a dose dependent manner thus inhibiting inflammation.Antioxidant activityFree radicals or reactive oxygen species have deleterious effects on human body, foodstuffs and fats, which developed an urge to find antioxidant substances from natural sources, which either delay or inhibit oxidation.[45] The antioxidant activity of an extract is associated with its free radical scavenging activity. Different types of assays are developed to determine the antioxidant properties of plant extracts as ferric thiocyanate assay, thiobarbituric acid assay, ferric reducing antioxidant power assay and DPPH (2,2-diphenyl-1-picryl-hydroxyl) assay.[9,41] DPPH, discovered 50 years ago, is a violet colored free radical used to determine antioxidant properties of plant extracts and is a strong indicator for measuring antioxidant capacity in human plasma too.[46] Reactive oxygen creates oxidative stress in the body and along with other damages can also mediate alloxan-induced liver damage.[47]Fruit and leaves ofP. macrocarpaare found to possess flavanoids and phenolics[41] which make it a potent antioxidant. The constituents present in mesocarp, pericarp and seed extract ofP. macrocarpaare responsible for antioxidant activity such as gallic acid[24] and 6-dihydroxy-4-methoxybenzophenone-2-o--D-glucoside which shows activity on DPPH.[48] Fruit extracts ofP. macrocarpahas been found to increase the level of SOD (superoxide dismutase),[47,48] which are enzymes of three types, SOD1, 2 and 3, that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide thus acting as an antioxidant. Pericarp extract has found to possess highest activity as an antioxidant while seed extract the lowest.[9]Vasorelaxant activityFor centuries, the leaves and fruit ofP. macrocarpahave been used to counter a number of diseases, including vascular problems and high blood pressure.[1,8] Dried flesh fruit powder[19] and egg shells of seeds[5] have been empirically considered potent cure of hypertension and heart diseases.[2] Epidemiological studies have shown that its two major constituents have effect on cardiovascular system, the kaempferol, a flavanoid that reduces the risk of cardiovascular diseases[49] and icariside, that is a moderate vasorelaxant reducing hypertension.[11] Icariside which is being isolated from chloroform extracts ofP. macrocarpafruit enhances vasorelaxant responses of isoproterenol and inhibits noradrenaline induced contractions contributing to the increase of second messengers as cyclic adenosine monophosphate and cyclic guanosine mono phosphate, phosphodiesterase inhibition and adenylatecyclase activation.[11]TOXICOLOGICAL STUDIESIn spite of a number of medicinal properties claimed in traditional medicine for the extracts ofP. macrocarpa, there are equally known poisonous tendencies of the extracts as well. However, we have not found a considerable supporting literature to evaluate the nature of the possible side effects. Even scientifically, there is a lack of credible and detailed report about the toxicity ofP. macrocarpaextracts. The only available literature consists of some preliminary toxicity reports. In tradition medicine, eating of unprocessed ripened fruits ofP. macrocarpais believed to cause oral ulcers,[3] however, neither the possible mechanism of this toxicity has been evaluated scientifically, nor the responsible constituents ofP. macrocarpafruits that are responsible for this effect are identified and quantified so far. Consumption ofP. macrocarpaat a dose higher that 27 mg/kg is reported to show embryo-fetotoxicity in female mice.[50] Butanol extracts of ripened fruits, when given to mice at doses of 0, 42.5, 85 and 170 mg/kg intraperitoneally, is reported to cause mild necrosis of proximal convoluted tubules in mice kidney at a dose higher than 85 mg/kg.[51] Ethanol extracts ofP. macrocarpa, when given to Javanese Quail in doses of 50, 100 and 200 mg/kg for two months are reported to cause mild hepatic hypertrophy and an increase in serum glutamate pyruvate transaminase activity at a dose of 100 mg/kg.[52] Like fruits, seeds ofP. macrocarpaare also reported for their toxicity. Des-acetylfevicordin-A and its derivatives isolated from seeds ofP. macrocarpaare reported to exert toxicity in brine shrimp (Artemiasalina) with a median lethal dose (LD50) of 3 ppm for des-acetylfevicordin-A and from 5 ppm to 12 ppm for its derivatives.[15] The available literature until today is, however, not enough to evaluate the toxic profile of different extracts of this invaluable medicinal plant. This lack of toxicity data creates doubt about the success of employingP. macrocarpaextracts in treating different ailments.CONCLUSIONThe scientific research has suggested a significant biological potential ofP. macrocarpaextracts. The phytoconstituents and the bioactivities associated with these constituents as presented in this short but concise review [Figure 4] is strongly believed to be helpful for those researchers who are already working, or planning to start evaluating a particular biological aspect of this precious herb. There is an utmost need to evaluate toxicity of its fruit, seeds and other extracts with a special focus on the estimation of LD50of the extracts and the identification of the responsible constituents. Anti-cancer effect of fruit extracts is already well-established, but the activity report is based only on thein-vitroassay. There is still no report of the evaluation of this otherwise tremendousin vitrocytotoxic effect ofP. macrocarpaextracts in anin-vivotumor model, insisting further clinical trials first in animals and then in human. Biologically active extracts of the plant can be further exploited in the future for the pharmaceutical and neutraceutical industry as well.FootnotesSource of Support:The authors thank Universiti Sains Malaysia for providing financial support and fellowship: [USM.IPS/JWT/1/19(JLD7)]Conflict of Interest:None declared.REFERENCES1.Sufi A. Mataram: Heinrich-Heine-University Dusseldorf; 2007. Lignans inPhaleria macrocarpa(Scheff.) 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Phalerin, a new benzophenoic glucoside isolated from the methanolic extract of Mahkota Dewa (Phaleria macrocarpa) leaves.Majalah Farmasi Indones.2005;16:517.15.Kurnia D, Akiyama K, Hayashi H. 29-Norcucurbitacin derivatives isolated from the Indonesian medicinal plant,Phaleria macrocarpa(Scheff.) Boerl.Biosci Biotechnol Biochem.2008;72:61820.[PubMed: 18256498]16.Ali RB, Atangwho IJ, Kaur N, Abraika OS, Ahmad M, Mahmud R, et al. Bioassay-guided antidiabetic study ofPhaleria macrocarpafruit extract.Molecules.2012;17:49865002.[PubMed: 22547320]17.Kim WJ, Veriansyah B, Lee YW, Kim J, Kim JD. Extraction of mangiferin from Mahkota Dewa (Phaleria macrocarpa) using subcritical water.J Indus Eng Chem.2010;16:42530.18.Hendra R, Ahmad S, Oskoueian E, Sukari A, Shukor MY. Antioxidant, anti-inflammatory and cytotoxicity ofPhaleria macrocarpa(Boerl.) Scheff Fruit.BMC Complement Altern Med.2011;11:110.[PMCID: PMC3354343][PubMed: 22070850]19.Astuti E, Raharjo TJ, Eviane D. Yogyakarta Indonesia: Proceeding of International Conference On Chemical Sciences; 2007. Cytotoxicity ofPhaleria macrocarpa(Scheff) boerl fruit flesh and seed extract of ethanol and its effect against p53 and Bcl-2 genes expression of normal cell 2007; pp. 14.20.Lu Y, Jiang F, Jiang H, Wu K, Zheng X, Cai Y, et al. Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells.Eur J Pharmacol.2010;641:1027.[PMCID: PMC3003697][PubMed: 20553913]21.Riwanto I, Budijitno S, Dharmana E, Handojo D, Prasetyo SA, Eko A, et al. Effect ofphaleria macrocarpasupplementation on apoptosis and tumor growth of C3H mice with breast cancer under treatment with adriamycin-cyclophosphamide.Int Surg.2011;96:16470.[PubMed: 22026311]22.Agung BS, Faried A, Arifin MZ, Wiriadisastra K, Ohta T. Herbal Medicine Isolation,Phaleria macrocarpafor primary glioblastoma multiforme cells.Ann Epidemiol.2008;18:70841.23.Tjandrawinata RR, Arifin PF, Tandrasasmita OM, Rahmi D, Aripin A. DLBS1425, aPhaleria macrocarpa(Scheff.) Boerl. extract confers anti proliferative and proapoptosis effects via eicosanoid pathway.J Exp Ther Oncol.2010;8:187201.[PubMed: 20734918]24.Faried A, Kurnia D, Faried LS, Usman N, Miyazaki T, Kato H, et al. Anticancer effects of gallic acid isolated from Indonesian herbal medicine,Phaleria macrocarpa(Scheff.) Boerl, on human cancer cell lines.Int J Oncol.2007;30:60513.[PubMed: 17273761]25.You BR, Moon HJ, Han YH, Park WH. Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis.Food Chem Toxicol.2010;48:133440.[PubMed: 20197077]26.Sutthanont N, Choochote W, Tuetun B, Junkum A, Jitpakdi A, Chaithong U, et al. Chemical composition and larvicidal activity of edible plant-derived essential oils against the pyrethroid-susceptible and -resistant strains of Aedes aegypti (Diptera: Culicidae)J Vector Ecol.2010;35:10615.[PubMed: 20618656]27.Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 Pathway in Cancer.Clin Cancer Res.2010:292731.[PubMed: 20484021]28.Liang Z, Brooks J, Willard M, Liang K, Yoon Y, Kang S, et al. CXCR4/CXCL12 axis promotes VEGF-mediated tumor angiogenesis through Akt signaling pathway.Biochem Biophys Res Commun.2007;359:71622.[PMCID: PMC1986788][PubMed: 17559806]29.Muhammad GM, Sofia MH, Sismindari The effect of mahkota dewa (Phaleria macrocarpa(Scheff) Boerl) leaf etanolic extract on splenic NK 1.1 cells activity.Periodic Medical Sci.200830.OCallaghan CA. NKG2D, Molecule Pages.200931.Sugiwati SK, Leonardus BS, Bintang M. -Glucosidase inhibitory activity and hypoglycemic effect ofPhaleria macrocarpafruit pericarp extracts by oral administration to rats.J App Sci.6:23126.32.Triastuti A, Paltiel HJ, Choi JW.Phaleria macrocarpasuppress nephropathy by increasing renal antioxidant enzyme activity in alloxan-induced diabetic rats.Nat Prod Sci.2009;15:16772.33.Rabyah BA, Item JA, Navneet K, Elsnoussi AH, Ali MJ, Asmawi MZ, Mahmud R. Hypoglycemic and anti-hyperglycemic study ofPhaleria macrocarpafruits pericarp.J Med Plant Res.2012;6:198290.34.Nagasawa T, Tabata N, Ito Y, Nishizawa N. Suppression of early and advanced glycation by dietary water-soluble rutin derivative in diabetic rats.Int Congr Ser.2002;1245:4035.35.Steinberg D, Witztum JL. Inhibition of PCSK9: A powerful weapon for achieving ideal LDL cholesterol levels.Proc Natl Acad Sci U S A.2009;106:95467.[PMCID: PMC2701045][PubMed: 19506257]36.Eberl D, Hegarty B, Bossard P, Ferr P, Foufelle F. SREBP transcription factors: Master regulators of lipid homeostasis.Biochimie.2004;86:83948.[PubMed: 15589694]37.Wong J, Quinn CM, Brown AJ. SREBP-2 positively regulates transcription of the cholesterol efflux gene, ABCA1, by generating oxysterol ligands for LXR.Biochem J.2006;400:48591.[PMCID: PMC1698594][PubMed: 16901265]38.Zeng L, Liao H, Liu Y, Lee TS, Zhu M, Wang X, et al. Sterol-responsive element-binding protein (SREBP) 2 down-regulates ATP-binding cassette transporter A1 in vascular endothelial cells: A novel role of SREBP in regulating cholesterol metabolism.J Biol Chem.2004;279:488017.[PubMed: 15358760]39.Shodikin A.Microbiology.Surabaya: Faculty of Medicine, University of Airlangga; 2009. Antimicrobial activity of etanol extract of Mahkota Dewa (Phaleria macrocarpa) fruits and leaves against pseudomonas aeruginosa by agar dilution and scanning electron microscopy.40.Tri WA, Eko S, Ismail MA, Shafiur MR. Effect of alloe vera (Alloe vera) and crown of god fruit (Phaleria macrocarpa) on sensory, chemical, and microbiological attributes of Indian mackerel (Restrelliger neglectus) during ice sto.Int Food Res J.2012;1:11925.41.Yosie A, Effendy MAW, Sifzizul TMT, Habsah M. Antibacterial, radical-scavenging activities and cytotoxicity properties ofPhaleria Macrocarpa(Scheff.) Boerl leaves in HEPG2 cell lines.Int J Pharmac Sci Res.2011;2:17006.42.Poeloengan M, Komala I. Screening for antibacterial properties of some plants and chemical antibiotic against two isolates ofescherichia colifrom diarrhea calves in Indonesia.The 1stInternational Seminar on Animal Industry Bogor Indonesia.2009:4.43.Fariza IN, Fadzureena J, Zunoliza A, Chuah AL, Pin KY, Adawiah I. Anti-inflammatory activity of the major compound from methanol extract ofPhaleria macrocarpaleaves.J App Sci.2012:11951198.44.Wu CC. Nitric Oxide and Inflammation.Current medicinal chemistry- antiinflammatory and antiallergy agents: Bentham Science Publishers;2004;3:21722.45.Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin.J Sci Toxicol.2004;26:2119.46.Ionita P. Is DPPH stable free radical a good scavenger for oxygen active species?Chem Pap.2005;1147.Triastuti A, Paltiel HJ, Choi JW.Phaleria macrocarpasuppresses oxidative stress in alloxan-induced diabetic rats by enhancing hepatic antioxidant enzyme activity.Nat Prod Sci.2009;15:3743.48.Martirosyan DM. Vol. 4. Texas USA: Food Science Publishers; 2009. Functional food for chronic diseases- Obesity, Diabetes, Cardiavascular disorders and AIDS; pp. 1409.49.Caldern-Montao JM, Burgos-Morn E, Prez-Guerrero C, Lpez-Lzaro M. A review on the dietary flavonoid kaempferol.Mini Rev Med Chem.2011;11:298344.[PubMed: 21428901]50.Haryono AS. NW. toxic effects of Phaleria (Phaleria macrocarpa) in mice (Mus musculus) swiss webster.J Biotika.2008;5:428.51.Ahmad S. Effect of butanol extract of maturated mahkota dewa (Phaleria macrocarpa) fruit on kidney tissue of Mice (Mus musculus)Biodiversitas.2006;7:27881.52.Armenia EF, Widya RM, Rusdi DJ, Netty M. Anti-Atherosclerotic effect and liver toxicity of ethanolic extract ofPhaleria macrocarpa(Scheff. Boerl) fruit on Japanese Quail.Asian Symposium on Medicinal Plants, Spices and other natural product XII (ASOMP), Padang, Indonesia, 13-18 November 2006Figures and TablesFigure 1

Botanical description ofPhaleria macrocarpashowing a typical (a) small flower bud, (b) green tapering leaves, (c) un-ripened green fruit (c), and (d) fully grown red fruitFigure 2

Chemical structures of constituents isolated fromPhaleria macrocarpaextracts: (a) fevicordin-A, (b) fevicordin-D, (c) magniferin, (d) kaempferol, (e) myricetin, (f) naringin, (g) gallic acid, (h) rutin, (i) quercitin, (j) lariciresinol, (k) pinoresinol, (l) matairesinol. The structures are drawn by using ChemBio Draw 12.0Figure 3

Mechanism of pro-apoptotic effect of gallic acid and phalerin isolated fromPhaleria macrocarpaextracts. Bcl-2: B-cell lymphoma 2 protein, BAX: Bcl-2 associated X proteins, Cyt-c: Cytochrome C, VDAC: Voltage dependent anion channels, PI3/Akt: Phosphoinositide 3/protein kinase B pathwayFigure 4

Phytoconstituents isolated fromPhaleria macrocarpawith their respective biological activity

Methods & Cell Culture Protocols Concentrating Cells: A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture. Counting Cells in a Hemacytometer: How to count and calculate the number of cells from a stock flask or culture dish. Cryopreservation of Mammalian Cells

Dissociation of Cells from Culture Vessels - Cell Dissociation Buffers

Dissociation of Cells from Culture Vessels - Other Reagents

Growth Factor Supplementation for Specific Cells: Reference Chart

Guidelines for Maintaining Cultured Cells: Learn what is subculture and when to do it,review media recommendationsfor common cell lines and how to dissociate cells as well as the benefits of using TrypLE as an alternative to trypsin. Media Preparation from Powder and Concentrates

Preparing Salts Solutions from Powder Concentrates Subculturing Adherent Cells: A general procedure for subculturing adherent mammalian cells in culture Subculturing Suspension Cells: A general procedure for subculturing mammalian and insect cells in suspension culture. Freezing Cells: A general procedure to properly cryopreserving cell lines. Thawing Frozen Cells: A general procedure for properly thawing cell lines. Trypan Blue Exclusion:A rapid assay to accurately determine the viability of your cells. Use of Antibiotics and Antimycotics

Useful Numbers for Cell Culture Red Blood Cell Lysis Using ACK Lysing Buffer

Cryopreservation of Mammalian CellsMammalian cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous cell lines, and to avoid aging and transformation in finite cell lines. Before cryopreservation, cells should be characterized and checked for contamination. There are several common media used to freeze cells.

For serum-containing medium, the constituents may be as follows: complete medium containing 10% glycerol complete medium containing 10% DMSO (dimethylsulfoxide) 50% cell-conditioned medium with 50% fresh medium with 10% glycerol or 10% DMSOCryopreservation media generally consists of a base medium, cryopreservative, and a protein source. The crypreservative and protein protect the cells from the stress of the freeze-thaw process. A serum-free medium has generally low or no protein; but one can still use it as a base for a cryopreservative medium in the following formulations:

For serum-free medium, some of the common media constituents may be: 50% cell-conditioned serum free medium with 50% fresh serum-free medium containing 7.5% DMSO fresh serum-free medium containing 7.5% DMSO and 10% cell culture grade DMSO.Protocol: Suspension Cultures1. Count the number of viable cells to be cryopreserved. Cells should be in log phase. Centrifuge the cells at ~200 to 400 x g for 5 min to pellet cells. Using a pipette, remove the supernate down to the smallest volume without disturbing the cells.2. Resuspend cells in freezing medium to a concentration of 1 x 107to 5 x 107cells/ml for serum containing medium, or 0.5 x 107to 1 x 107cells/ml for serum-free medium.3. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4C refrigerator, and start the freezing procedure within 5 minutes.4. Cells are frozen slowly at 1C /min. This can be done by programmable coolers or by placing vials in an insulated box placed in a -70C to -90C freezer, then transferring to liquid nitrogen storage.Protocol: Adherent Cultures1. Detach cells from the substrate with dissociation agents. Detach as gently as possible to minimize damage to the cells.2. Resuspend the detached cells in a complete growth medium and establish the viable cell count.3. Centrifuge at ~200 x g for 5 min to pellet cells. Using a pipette, withdraw the supernate down to the smallest volume without disturbing the cells.4. Resuspend cells in freezing medium to a concentration of 5 x 106to 1 x 107cells/ml.5. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4C refrigerator, and start the freezing procedure within 5 min.6. Cells are frozen slowly at 1C /min. This can be done by programmable coolers or by placing vials in an insulated box placed in a -70C to -90C freezer, then transferring to liquid nitrogen storage.Reference:1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 220, Alan R. Liss,Inc., New York.

To thaw cryopreserved cells,see this protocolDissociation of Cells from Culture Vessels with Enzyme-free Cell Dissociation BuffersThe following is a general procedure to rapidly remove various cell lines from the substratum while maintaining cellular integrity. This procedure is not meant to be universally applicable for all cell lines. The optimal conditions and concentrations employed for individual systems should be determined empirically. Cell viability should be routinely monitored at the time of subculturing. Cell viability should be greater than 90%.1. Warm all reagents to 37C prior to use.2. Remove growth medium from cells.3. Thoroughly rinse cell monolayers with 5 ml Ca++- and Mg++-free PBS per T75 flask or 100 mm dish. Gently rock the flask (or dish), allowing the solution to bathe the cells for 30 to 60 seconds at room temperature. Aspirate the rinse solution and discard.4. Repeat step 3.5. Add approximately 5 ml of Cell Dissociation Buffer per T75 flask or 100 mm dish and gently bathe cells by rocking at room temperature for 1 to 2 minutes. You may check for dissociation under the microscope. Aspirate solution and discard.6. Firmly tap flask or dish against palm of hand to dislodge cells. If cells do not detach quickly, allow to sit at room temperature for another 2 to 5 minutes and tap flask against palm of hand again. This may be repeated for cells that are more strongly adherent (with 5 more mls of dissociation buffer). After the cells are visibly detached add at least 5 ml of complete growth medium. Resuspend cells in growth medium.Reference:1.Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.Ordering InformationTop of FormCatalog #NameSizeList Price (SGD)Qty

13151014Cell Dissociation Buffer, enzyme-free, PBS100 mLContact Us

13150016Cell Dissociation Buffer, enzyme-free, Hank's Balanced Salt Solution100 mLContact Us

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Dissociation of Cells from Culture Vessels Using Other ReagentsThe following is a general procedure to rapidly remove various cell lines from the substratum while maintaining cellular integrity. This procedure is not meant to be universally applicable for all cell lines. The optimal conditions and concentrations employed for individual systems should be determined empirically. Cell viability should be routinely monitored at the time of subculturing. Cell viability should be greater than 90%.1. Remove and discard spent cell culture media.2. Wash cells using a balanced salt solution without calcium and magnesium or wash with EDTA. Add wash solution to the side of the flask opposite the cells. Rinse the cell sheet by rocking the flask for 1 to 2 min and discard wash solution.3. Add the dissociation solution of choice at 2 to 3 ml/25 cm2to the side of the flask opposite the cells. Be certain that the dissociation solution covers the cell sheet. Incubate the flasks at 37C. Rock the flasks gently. Generally, cells are dissociated in 5 to 15 min. The actual time needed to dissociate cells will vary according to cell line. Monitor the process carefully to avoid cell damage. In addition to rocking gently, flasks of cell lines that are characteristically difficult to remove from the substratum may be tapped to expedite removal.4. When the cells are completely detached, stand the flask in the upright position to allow the cells to drain to the bottom of the flask. Add complete media to the flask. Disperse the cells by pipetting repeatedly over the surface of the monolayer. Count and subculture the cells.Reference:1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.Ordering InformationTop of FormCatalog #NameSizeList Price (SGD)Qty

14190144DPBS, no calcium, no magnesium500 mLContact Us

15400054Trypsin-EDTA (0.5%), no phenol red100 mLContact Us

15090046Trypsin (2.5%), no phenol red100 mLContact Us

12604013TrypLE Express Enzyme (1X), no phenol red100 mLContact Us

Cell LineCell TypeGrowth or Attachment FactorSuggested Conc. (ng/ml)MediumSerum Suppl.

MCF 7Human mammary carcinomaEGF(a)100DMEM2% FBS

HFHuman foreskin fibroblastEGF(a)2DMEM10% FBS

--Primary hepatocyteEGF(a)10DMEM10% FBS

WBRat hepatic eptheliumEGF(a)10Richter's MEM10% FBS

HeLaHuman cervical carcinomaEGF(a)2.5 x104DMEM10% FBS

A431Epidermal carcinomaEGF(a)2.5 x 104DMEM10% FBS

--Primary human fibroblastEGF(a)1x103MEM Earle's

10% FBS

MKN-7Human adenocarcinomaEGF(a)50DMEM8% FBS

BALB/c 3T3Mouse fibroblastEGF(a)50DMEM/F-12--

TM-4Mouse testes epitheliumEGF(a)3DMEM/F-12--

NRK-49FRat fibroblastEGF(a)50DMEM/F-12--

GH3Rat neuroblastomabFGF1F-12--

BHK-21Baby hamster kidneybFGF3DMEM/F-125% Calf

Growth Factor Supplementation for Specific CellsGrowth and attachment factors, natural and recombinant, are used in conjunction with other essential nutrients and hormones to maximize biological stimulation. The optimal conditions and concentrations of growth factor for particular cell types must be empirically determined. The following table provides a summary of conditions for growth and attachment factor supplementation reported to give maximal stimulation for various cell lines. It can be used to establish initial concentrations prior to optimization.What is Subculture?Subculturing, also referred to aspassaging, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.Characteristic Growth Pattern of Cultured CellsThe growth of cells in culture proceeds from thelag phasefollowing seeding to thelog phase, where the cells proliferate exponentially. When the cells in adherent cultures occupy all the available substrate and have no room left for expansion, or when the cells in suspension cultures exceed the capacity of the medium to support further growth, cell proliferation is greatly reduced or ceases entirely (see Figure 4.1 below). To keep them at an optimal density for continued growth and to stimulate further proliferation, the culture has to divided and fresh medium supplied.Figure 4.1:Characteristic Growth Pattern of Cultured Cells. The semi-logarithmic plot shows the cell density versus the time spent in culture. Cells in culture usually proliferate following a standard growth pattern. The first phase of growth after the culture is seeded is the lag phase, which is a period of slow growth when the cells are adapting to the culture environment and preparing for fast growth. The lag phase is followed by the log phase (i.e., logarithmic phase), a period where the cells proliferate exponentially and consume the nutrients in the growth medium. When all the growth medium is spent (i.e., one or more of the nutrients is depleted) or when the cells occupy all of the available substrate, the cells enter the stationary phase (i.e., plateau phase), where the proliferation is greatly reduced or ceases entirely.

When to Subculture?The criteria for determining the need for subculture are similar in adherent and suspension cultures; however, there are some differences between mammalian and insect cell lines.Cell Density Mammalian cells: Adherent cultures should be passaged when they are in the log phase, before they reach confluence. Normal cells stop growing when they reach confluence (contact inhibition), and it takes them longer to recover when reseeded. Transformed cells can continue proliferating even after they reach confluence, but they usually deteriorate after about two doublings. Similarly, cells in suspension should be passaged when they are in log-phase growth before they reach confluency. When they reach confluency, cells in suspension clump together and the medium appears turbid when the culture flask is swirled. Insect cells:Insect cells should be subcultured when they are in the log phase, before they reach confluency. While tightly adherent insect cells can be passaged at confluency, which allows for easier detachment from the culture vessel, insect cells that are repeatedly passaged at densities past confluency display decreased doubling times, decreased viabilities, and a decreased ability to attach. On the other hand, passaging insect cells in adherent culture before they reach confluency requires more mechanical force to dislodge them from the monolayer. When repeatedly subcultured before confluency, these cells also display decreased doubling times and decreased viabilities, and are considered unhealthy.Exhaustion of Medium Mammalian cells:A drop in the pH of the growth medium usually indicates a build up of lactic acid, which is a by-product of cellular metabolism. Lactic acid can be toxic to the cells, and the decreased pH can be sub-optimal for cell growth. The rate of change of pH is generally dependent on the cell concentration in that cultures at a high cell concentration exhaust medium faster than cells lower concentrations. You should subculture your cells if you observe a rapid drop in pH (>0.1 0.2 pH units) with an increase in cell concentration. Insect cells:Insect cells are cultured in growth media that are usually more acidic that those used for mammalian cells. For example, TNM-FH and Graces medium used for culturing Sf9 cells has a pH of 6.2. Unlike mammalian cell cultures, the pH rises gradually as the insect cells grow, but usually does not exceed pH 6.4. However, as with mammalian cells, the pH of the growth medium will start falling when insect cells reach higher densities.Subculture SchedulePassaging your cells according to a strict schedule ensures reproducible behavior and allows you to monitor their health status. Vary the seeding density of your cultures until you achieve consistent growth rate and yield appropriate for your cell type from a given seeding density. Deviations from the growth patterns thus established usually indicate that the culture is unhealthy (e.g., deterioration, contamination) or a component of your culture system is not functioning properly (e.g., temperature is not optimal, culture medium too old). We strongly recommend that you keep a detailedcell culture log, listing the feeding and subculture schedules, types of media used, the dissociation procedure followed, split ratios, morphological observations, seeding concentrations, yields, and any anti-biotic use.It is best to perform experiments and other non-routine procedures (e.g., changing type of media) according to your subculture schedule. If your experimental schedule does not fit the routine subculture schedule, make sure that you do not passage your cells while they are still in the lag period or when they have reached confluency and ceased growing.Media Recommendations for Common Cell LinesMany continuous mammalian cell lines can be maintained on a relatively simple medium such as MEM supplemented with serum, and a culture grown in MEM can probably be just as easily grown in DMEM or Medium 199. However, when a specialized function is expressed, a more complex medium may be required. Information for selecting the appropriate medium for a given cell type is usually available in published literature, and may also be obtained from the source of the cells or cell banks.If there is no information available on the appropriate medium for your cell type, choose the growth medium and serum empirically or test several different media for best results. In general, a good place to start is MEM for adherent cells and RPMI-1640 for suspension cells.Insect cells are cultured in growth media that are usually more acidic than those used for mammalian cells such as TNM-FH and Graces medium.The conditions below can be used as a guide when setting up a new mammalian cell culture: Recommended Media for Common Cell TypesDissociating Adherent CellsThe first step in subculturing adherent cells is to detach them from the surface of the culture vessel by enzymatic or mechanical means. The table below lists the various cell dissociation procedures.ProcedureDissociation AgentApplications

Shake-offGentle shaking or rocking of culture vessel, or vigorous pipetting.Loosely adherent cells, mitotic cells

ScrapingCell scraperCell lines sensitive to proteases; may damage some cells

Enzymatic dissociationTrypsinStrongly adherent cells

Enzymatic dissociationTrypsin + CollagenaseHigh density cultures, cultures that have formed multiple layers, especially fibroblasts

Enzymatic dissociationDispaseDetaching epidermal cells as confluent, intact sheets from the surface of culture dishes without dissociating the cells

Enzymatic dissociationTrypLE dissociation enzymeStrongly adherent cells; direct substitute for trypsin; applications that require animal origin-free reagents

TrypLE Dissociation EnzymesTrypLE Express and TrypLE Selectare microbially produced cell dissociation enzymes with similar kinetics and cleavage specificities to trypsin. Although TrypLE enzymes can directly substitute for trypsin in dissociation procedures without a need for protocol changes, we recommend that you initially optimize the incubation time for dissociation. Because TrypLE enzymes are recombinant fungal trypsin-like proteases, they are ideal for applications that require animal origin-free reagents. The table below compares TrypLE Express and TrypLE Select to trypsin.TrypLE Express and TrypLE SelectTrypsin

Completely free of animal- and human derived componentsPorcine- or bovine-derived

Stable at room temperature for at least six monthsNot stable at room temperature

Does not require inactivationRequires inactivation with serum or other inhibitors

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