Plants provide us with many biologically active compounds that have possible curative consequence on a myriad of diseases. Leea indica ( Burm. f. ) Merrill ( Leeaceace ) is a traditional Chinese medicinal works widely used for the intervention of assorted unwellnesss including ini¬‚ammations, diabetes, tegument diseases and malignant neoplastic disease. Previous in vitro cytotoxicity consequences have verified its anti-cancer activity against Ca Ski cervical malignant neoplastic disease cell lines.
To insulate the active components from the ethyl ethanoate fraction obtained from the leaf infusion of Leea indica via cytotoxicity guided attack. The consequence of the compounds on the ordinance of cell proliferation, cell rhythm and programmed cell death in Ca Ski cells was evaluated.
The compounds were characterized by 1D-NMR ( 1H NMR, 13C NMR and DEPT-135 ) , 2D-NMR ( COSY, HMQC and HMBC ) and LC-MS methods. The cytotoxic and anti-proliferative effects were investigated utilizing 3- ( 4,5-dimethylthiazol-2-yl ) -2,5-diphenyltetrazolium bromide ( MTT ) and trypan bluish exclusion ( TBE ) assays. The cell rhythm distribution and programmed cell death initiation were analyzed by flow cytometry. Quantitative real-time PCR ( Q-PCR ) was used to mensurate the look of proliferative cell atomic antigen ( PCNA ) .
Two cycloartane triterpenoid glycosides, mollic acid arabinoside ( MAA ) and mollic acid xyloside ( MAX ) were isolated for the first clip from Leea indica. These compounds were showed to hold anti-cancer activity for the first clip. They demonstrated strong dose-dependent cytotoxic effects on Ca Ski cells with high selectivity over the normal MRC-5 cells. The more powerful compound, MAA caused a lessening in the viability and proliferation of Ca Ski cells as assessed by MTT and TBE checks. The cytostatic ( anti-proliferative ) and cytocidal ( apoptotic ) effects of MAA were reflected by S-phase apprehension, decreased of PCNA look and initiation of bomber G1cells.
The MAA isolated from Leea indica showed possible anti-cancer activity, which is cytotoxic, anti-proliferative, and apoptotic on Ca Ski cervical malignant neoplastic disease cells. Therefore, this survey provides the scientific grounds for the ethno-medicinal usage of Leea indica as anticancer works and may pave the manner for future development as a malignant neoplastic disease chemotherapeutic agent.
Leea indica ( Burm. f. ) Merrill is a leafy works belonging to the Leeaceae household, which is grown in South East Asia, China and India. The foliages, roots and whole works of L. indica are widely used for many medicative values. The foliages are used to handle diabetes and the unction prepared from roasted foliages is used to relief symptoms of dizziness ( Chatterjee and Prakashi, 1994 ; Prajapati et al. , 2003 ; Pullaiah and Naidu, 2003 ) . The foliages paste is locally used to alleviate itchiness, tenderness, eczema, leprosy, sprain and bone break ( Rahman et al. , 2007 ; Yusuf et al. , 2007 ) . On the other manus, the root is claimed to hold sudatory, anti-diarrhoeal, anti-dysenteric, anti-spasmodic effects and are frequently used to handle cardiac and skin diseases ( Chatterjee and Prakashi, 1994 ) . All portion of the works is used to handle concern, organic structure strivings and tegument ailments ( Burkill, 1966 ; Lattif et al. , 1984 ) .
In position of the traditional usage of L. indica for assorted medicative intents, some phytochemical and biological surveies have been conducted. The earliest phytochemical survey on the foliages of L. indica reported the isolation of ?-tocopherol, ?-amyrin and ?-sitosteryl-?-D-glucopyranoside ( Saha et al. , 2005 ) . This was followed by the designation of 11 hydrocarbons, phthalic acid, palmitic acid, 1-eicosanol, solanesol, farnesol, phthalic acid esters, Gallic acid, lupeol and ursolic acid ( Srinivasan et al. , 2008 ) . Meanwhile, the indispensable oil extracted from the flower of L. indica was found to incorporate di-isobutylphthalate, di-n-butylphthalate, n-butylisobutylphthalate and butylisohexylphthalate and monobutyl carbonotrithioate ( ( Srinivasan et al. , 2009 ) . Anti-microbial activity has been reported in the foliage, root and indispensable oil from the flower of L. indica ( Srinivasan et al. , 2009 ; Srinivasan et al. , 2010 ) . Furthermore, the foliage was found to hold hypo-glycemic and anti-strychnine activities ( Dhar et al. , 1968 ) while the root was found demoing phosphodiesterase repressive activity ( Temkitthawon et al. , 2008 ) . L. indica was besides possessed singular anti-oxidant and anti-inflammatory activities, as shown by strong DPPH free extremist scavenging and strong azotic oxide repressive effects ( Saha et al. , 2004 ) . Recently, the high anti-oxidant activity of L. indica was besides reported by other bookmans ( Li and Liu, 2009 ; Krishnaiah et al. , 2010 ) .
In Leeaceae household, L. guineense and L. macrophylla were ethno-medicinally used to handle malignant neoplastic disease disease ( Graham et al. , 2000 ; Choudhary et al. , 2008 ) . The leaf decoction of L. indica is consumed by adult female during gestation and bringing, or for birth control ( Bourdy and Walter, 1992 ) . The foliages are besides used to handle obstetric diseases and organic structure hurting ( Srithi et al. , 2009 ) . It is besides an ingredient in the readying to handle leukorrhea, enteric malignant neoplastic disease and uterus malignant neoplastic disease ( Saralamp, 1997 ) . Furthermore, the dried foliages are used as a tea drink to forestall malignant neoplastic disease and alleviate cancer-related symptoms. In our old cytotoxicity showing, the petroleum ethanol infusion and fractions ( ethyl ethanoate, hexane and H2O ) were found to suppress the growing of Ca Ski cervical malignant neoplastic disease cell line ( Wong and Kadir, 2011 ) . This farther provides the grounds for the usage of L. indica as folkloric intervention of malignant neoplastic disease.
To the best of our cognition, the anti-cancer potency of L. indica has non yet been studied extensively. In the present survey, we report the farther advancement in an on-going research on L. indica whereby the active fraction ( ethyl ethanoate ) was subjected to bioassay-guided attack in order to insulate the active chemical components and farther measure its cytotoxic, anti-proliferative and apoptotic consequence on Ca Ski cells
From the old study ( Wong and Kadir, 2011 ) , the fresh foliages of L. indica were collected, authenticated, extracted and fractionated. A voucher specimen ( 47365 ) was deposited at the herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
The active ethyl ethanoate fraction ( 50g ) was dissolved in MeOH and loaded onto Diaion HP-20 SS ( Supelco, Bellefonte ) column, eluted utilizing a gradient dissolver system of 40 % MeOH and 60 % H2O with 10 % MeOH increase. Thin bed chromatography ( TLC ) analysis were performed on pre-coated silicon oxide gel 60 F254 home bases ( 0.2mm midst, Merck ) and musca volitanss were detected by UV light after spraying with 10 % H2SO4 followed by heating. Based on the TLC profiles, a sum of nine combined fractions ( designated F1-F9 ) were pooled together. MTT check was performed on each fraction. The active F8 was farther subjected to silica gel ( 200-400 mesh, Merck ) column chromatography. The nomadic stage was consisted of CHCl3: MeOH: H2O ( C: Meter: H, v/v ) . The initial solvent composing was 100 % C, and so it was changed to C: M ( 9.5: 0.5 ) , followed by C: Meter: H ( 9: 1: 0.1 ) , C: Meter: H ( 8.5: 1.5: 0.1 ) , C: Meter: H ( 8: 2: 0.2 ) , C: Meter: H ( 7: 3: 0.5 ) , C: Meter: H ( 6.5: 3.5: 0.5 ) , C: Meter: H ( 6: 4: 1 ) and eventually to 100 % M. A sum of six fractions ( F81-F86 ) were obtained. The active F83 was farther fractionated once more on silicon oxide gel 60 column utilizing C: Meter: H system. The initial dissolver was 100 % C, and so it was changed to C: Meter: H ( 9: 1: 0.1 ) , followed by C: Meter: H ( 8.5: 1.5: 0.1 ) , C: Meter: H ( 8: 2: 0.2 ) , C: Meter: H ( 7: 3: 0.5 ) , and eventually to 100 % M. Another six fractions ( F831-F836 ) were obtained. The active F835 was further purified by prep-TLC ( silica gel 60 F254 glass home bases, size 20cm ten 20cm, Merck ) utilizing C: Meter: H ( 7: 3: 0.5 ) as dissolver system and yielded compounds 1 ( 55.9mg ) and 2 ( 26.6mg ) .
Structure elucidation of the compounds was determined by spectral techniques. The compounds were dissolved in pyridine-d5 solution. The 1H, 13C and DEPT-135 NMR informations were recorded on a Bruker DRX 300 spectrometer, while the 2D-NMR spectra ( COSY, HMQC and HMBC ) were obtained by JEOL ECA 400. LC-MS analysis was performed utilizing LTQ Orbitrap mass spectrometer ( Thermo Fisher Scientific ) fitted with an electrospray interface.
The human cervical epidermoid carcinoma cell line ( Ca Ski, ATCC figure CRL-1550 ) and human fibroblast cell line ( MRC-5, ATCC figure CCL-171 ) were purchased from the American Type Culture Collection ( ATCC, USA ) . Ca Ski cells were maintained in RPMI 1640 Medium ( Sigma ) and MRC-5 cells in EMEM ( Eagle Minimum Essential Medium ) ( Sigma ) . The media were supplemented with 10 % ( v/v ) heat-inactivated foetal bovine serum ( PAA Lab, Austria ) , 100µg/ml streptomycin and 100unit/ml penicillin ( PAA Lab, Austria ) and 50µg/ml amphotericin B ( PAA Lab, Austria ) . The media were filter sterilized utilizing a 0.22µm filter membrane ( Minisart, Sartorius stedim ) . The cells were cultured in 5 % CO2 brooder at 37 & A ; deg ; C in a humidified ambiance.
MTT check was modified from Mossmann and used to measure the cytotoxic effects of each fraction and compounds. MTT check is widely used to measure the viability and/or the metabolic province of the cells based on mitochondrial respiratory activity ( Mossmann, 1983 ) . A sum of 5×103 Ca Ski or MRC-5 cells were seeded into 96-well home bases and allowed to adhere for 24h. After 24h, the cells were treated with the fractions or compounds in the concluding concentrations runing from 3.125 to 100?g/ml. Control cells were treated with vehicle DMSO to acquire the concluding concentration of 0.5 % v/v. The cells were so incubated for 72h. After 72h exposure period, MTT ( 5mg/ml ) was added and further incubated for 4h at 37 & A ; deg ; C. The medium was so aspirated and the formazan crystals generated from the feasible cells were dissolved in 150?l DMSO. The optical density was measured at 570nm against the mention wavelength of 650 nanometers. The per centum of viability was calculated based on the expression: Viability ( % ) = ( optical density of treated cells/ optical density of control cells ) x 100 % . The IC50 ( concentration that reduces cell viability to 50 % ) was derived from the dose-response curves. The selectivity index ( SI ) was calculated by divided the IC50 value of the normal MRC-5 cell line with the IC50 value of the cervical malignant neoplastic disease Ca Ski cell line. Sample which shows SI & A ; gt ; 3 will be considered to hold high selectivity ( B & A ; eacute ; zivin et al. , 2003 ; Aljewari et al. , 2010 ) .
In order to find whether the cytotoxicity is cytostatic or cytocidal, a recovery check was conducted whereby after the 72h incubation ( exposure period ) , the medium incorporating the compound was removed, washed with medium and replaced with medium entirely for a recovery period of 72h followed by add-on of MTT and measuring as described earlier. A sample is demoing cytostatic consequence when the IC50 in the recovery check was higher than that of the exposure check. Whereas for cytocidal consequence, the IC50 obtained in the recovery check is about similar to that shown by the exposure check ( Lee and Houghton, 2005 ; Ashidi et al. , 2010 ) .
Ca Ski cells were cultured at a denseness of 1-106cells/ml in 60mm civilization dishes. After 24h of fond regard, the cells were treated with 0.5 % DMSO ( control ) or 60?M of MAA for different clip periods. After 6-72h, the cells were harvested, washed with medium and the cell pellets were resuspended in medium. After incubation in 0.4 % trypan blue for 5min, feasible cells were counted utilizing a hemocytometer. At the indicated clip point straight before the cells were harvested, the cells were visualized under an upside-down phase-contrast microscope ( Motic ) to detect the cell morphological alterations upon intervention and exposure were taken.
The cell rhythm distribution and programmed cell death were assessed utilizing propidium iodide ( PI ) staining ( Nicoletti et al. , 1991 ) . Ca Ski cells were seeded in 60mm civilization dishes ( 1x106cells ) and left 24h for fond regard. The cells were so treated with 0.5 % DMSO ( control ) , 60, 80 and 100µM of MAA for 24h. The cells were besides treated with 60µM MAA for 6-72h. After the designated intervention period, both disciple and drifting cells were harvested and washed twice with PBS. Cell pellets were resuspended in 100ul of PBS and fixed with absolute ethyl alcohol and stored at -20 & A ; deg ; C for 24h. Fixed cells were washed twice with PBS and the cell pellets were incubated in a buffer incorporating 50µg/ml PI, 0.1 % Na citrate, 0.1 % Triton-X-100 and 100µg/ml RNase A for 45min in the dark at room temperature. The per centum of cells in the Sub-G1, G1, S, and G2/M-phases of the cell rhythm was so analyzed utilizing a FACSCalibur flow cytometer ( Beckton Dickinson ) . Data were acquired and analyzed utilizing CellQuest package ( Becton Dickinson ) .
A sum of 1×106 Ca Ski cells were seeded into 60mm civilization dishes and incubated for 24h for fond regard. Cells were so treated with DMSO ( 0.5 % ) or 20-100µM of MAA for 24h. For time-course survey, cells were treated with 60µM MAA for 0, 3, 6, 12 and 24h. After specific intervention period, cells were harvested and entire RNA was isolated utilizing the RNAqueous-4PCR kit ( Applied Biosystem ) harmonizing to the maker ‘s waies. The RNA concentration was determined utilizing spectrophotometry. The cistron look of PCNA was assessed by one-step SYBR Green comparative real-time PCR ( RotorGene System, Qiagen ) and normalized to GAPDH mention control elaborations. The primer sequences for PCNA and GAPDH were frontward 5’-GCCTGCTGGGATATTAGCTC-3 ‘ ; change by reversal 5’-CATACTGGTGAGGTTCACGC-3 ‘ and frontward 5’-CCAGGGCTGCTTTTAACTCTG-3 ‘ ; change by reversal 5’-CGTTCTCAGCCTTGACGGTG-3 ‘ severally. The PCR amplii¬?cation reactions was carried out in a entire volume of 25?l for 30 rhythms of 45 seconds at 95 & A ; deg ; C, 45 seconds at 56 & A ; deg ; C and 120 seconds at 72 & A ; deg ; C. The average i¬‚uorescence threshold value ( CT ) of each sample was obtained harmonizing to the maker ‘s guidelines, and used to find ?CT values whereby ?CT = CT mark cistron ( PCNA ) – Connecticut mention cistron ( GAPDH ) . The comparative crease alteration in PCNA look in the treated sample over the untreated control was calculated with the comparative a?†a?†CT method where a?†a?†CT = a?†CT sample – a?†CT control and calculated utilizing formula 2-??CT ( Livak and Schmittgen, 2001 ) .
All the consequences were presented as average ± criterion mistake ( S.E. ) of three experiments. Significant difference was analyzed by Student t-test. A P value & A ; lt ; 0.05 was regarded as a important difference from the matching control group.
Based on our old survey, the ethyl acetate fraction of L. indica demonstrated the strongest cytotoxic consequence on Ca Ski cells ( Wong and Kadir, 2011 ) . Hence, it was subjected to MTT assay-guided isolation. The consequences were summarized in Fig. 1. MTT trial on the first 9 fractions showed that Ca Ski cells were most susceptible to F8. Further separation of F8 yielded another 6 fractions ( F81 to F86 ) . Among the fractions, F83 was found to be the most effectual. Subsequent fractional process of F83 yielded another 6 fractions ( F831 to F836 ) . The active F835 was subjected to prep-TLC and this led to the isolation of two compounds. They were identified by spectroscopic methods ( 1H NMR, 13C NMR, DEPT-135 ) and by comparing with the published informations in literature ( Pegel and Rogers, 1985 ; Rogers and Thevan, 1986 ; Rogers, 1989 ) , to be ( 1 ) mollic acerb arabinoside ( MAA ) and ( 2 ) mollic acerb xyloside ( MAX ) . The chemical constructions were shown in Fig. 2.
Compound 1 was identified as 1?,3?-dihydroxy-cycloart-24-ene-28-oic acid 3-O- [ ?-L-arabinopyranoside ] , C35H56O8. Positive ESI-MS m/z: 627.3851 [ M+Na ] + . 1H NMR ( 125MHz, C5D5N ) : ? 0.47 ( 1H, vitamin D, J=4.0Hz, H-19A ) , 0.75 ( 1H, vitamin D, J=4.0Hz, H-19B ) , 0.85-1.66 ( 6 x CH3 ) , 3.39-4.42 ( arabinose protons ; H-1of aglycone ) , 5.01 ( 1H, vitamin D, J=6.6Hz, H-1 ‘ of arabinose ) , 5.21-5.5 ( 2H, m, H-3? , H-24 ) . Compound 2 was identified as 1?,3?-dihydroxy-cycloart-24-ene-28-oic acid 3-O- [ ?-D-xylopyranoside ] , C35H56O8. Positive ESI-MS m/z: 627.3851 [ M+Na ] + . 1H NMR ( 125MHz, C5D5N ) : ? 0.44 ( 1H, vitamin D, J=4.0Hz, H-19A ) , 0.72 ( 1H, vitamin D, J=4.0Hz, H-19B ) , 0.91-1.68 ( 6 x CH3 ) , 3.39-4.42 ( xylose protons ; H-1of aglycone ) , 5.08 ( 1H, vitamin D, J=6.6Hz, H-1 ‘ of wood sugar ) , 5.20-5.48 ( 2H, m, H-3? , H-24 ) . The 13C NMR informations of compounds 1 and 2 were shown in Table 1. The place of the sugar medieties were confirmed by HMBC correlativity of the anomeric protons to C3 of the aglycones and frailty versa. Their LC-MS spectra showed the same molecular ion extremum at m/z 627.3851, which corresponds to a molecular expression of C35H56O8.
Fig. 1. Flow-chart of bioassay-guided isolation of mollic acid arabinoside ( MAA ) and mollic acid xyloside ( MAX ) from the ethyl acetate fraction of L. indica. Each of the fractions was evaluated its cytotoxic consequence on Ca Ski cells utilizing MTT check. The IC50 values were agencies ± S.E. calculated from three experiments performed in triplicate.
Table 1. 13C-NMR spectra informations ( ? C in p.p.m, ; 75 MHz ) of compounds 1 and 2 ( C5D5N ) .
Fig. 2. The chemical constructions of the compounds isolated from L. indica via bioassay-guided attack.
MAA and MAX were evaluated for their cytotoxic consequence on Ca Ski cervical malignant neoplastic disease cells and MRC 5 normal cells utilizing MTT check. A 72h exposure to Ca Ski cells with MAA or MAX led to a important dose-dependent decrease in cell viability. Harmonizing to Fig. 3, the lessening in cell viability ranged from 20-95 % and 6-90 % when the cells were treated with 3.125-100µg/ml of MAA and MAX severally. The IC50 values for MAA, MAX and camptothecin ( CPT ) were summarized in Table 2. MAA and MAX were much less cytotoxic to the normal cells, as revealed by the comparatively higher IC50 values on MRC 5, whereas CPT displayed comparable IC50 value on Ca Ski and MRC 5 cells. The little different in IC50 value led to our tax write-off that CPT can non distinguish between normal and malignant neoplastic disease cells and killed both cells at about the same efficiency. This deficiency of tumour cell-specificity was besides reported by other bookman ( Iwasaki, 2006 ) .
The primary end of malignant neoplastic disease chemotherapy is to aim specific to malignant neoplastic disease cells and innocuous to normal cells. However, many anti-cancer drugs fail to run into this standard, as they can non know apart between malignant neoplastic disease and normal cells, which make them cytotoxic non merely to malignant neoplastic disease cells, but besides to normal cells. Therefore, development of fresh malignant neoplastic disease chemotherapeutic agent with a higher authority and specii¬?city against malignant neoplastic disease cells are desperately needed. It is interesting to observe that MAA and MAX exhibited about 20-fold and 17-fold higher IC50 values against MRC-5 when compared to CPT ( Table 2 ) . Furthermore, we besides determined the grade of their selectivity against Ca Ski cells over MRC-5 cells, by comparing the SI values of the compounds. As shown in Table 2, MAA exhibited the greatest selectivity ( highest index ) , bespeaking that the cytotoxicity to Ca Ski cells was approximately 8 times higher in selectivity over the normal cells. Good SI ( & A ; gt ; 3 ) was besides demonstrated by MAX, where Ca Ski cells was approximately 4 times more sensitive than normal cells to the cytotoxic consequence of MAX.
Fig. 3. Cytotoxic consequence of mollic acid arabinoside ( MAA ) and mollic acid xyloside ( MAX ) on Ca Ski cells. Cells were treated with 0.5 % DMSO ( control ) or increasing doses ( 3.125-100µg/ml ) of MAA or MAX for 72h. The cytotoxicity was measured by MTT assay as described in method. Camptothecin ( CPT ) was used as positive control. The informations were average values ± S.E. of three different experiments. The stars represented significantly different value from control ( * P & A ; lt ; 0.05 ) .
Table 2. IC50 [ in µg/ml ( µM ) ] and SI values of MAA, MAX and camptothecin ( CPT ) determined by MTT assay as described in methods. Data were agencies ± S.E. calculated from three experiments.
Since MAA exhibited higher SI and lower IC50 compared with MAX, it was selected for farther probes. In order to research its anti-cancer activity, the cytotoxicity, anti-proliferation and programmed cell death were analyzed. Previously, we have shown that intervention of MAA resulted in a conspicuous dose-dependent decrease of formazan formation in Ca Ski cells. This indicated that the cytotoxic action was mediated via break of mitochondrial dehydrogenase system inside the cells. The IC50 value obtained in the MTT recovery check is about the same to that shown by the exposure check ( informations non shown ) . Hence, it can be assumed that cytotoxic consequence appeared to be chiefly cytocidal.
The time-dependent consequence of MAA on cell growing was assessed by TBE check. As shown in Fig. 4, the control cells proliferated much faster compared to the MAA-treated cells, as demonstrated by the rapid exponential growing of the cells in the presence of 0.5 % DMSO. In contrast, when exposed to MAA, the cell growing was hindered. Treatment for 6h modestly inhibited the cell growing. Prominent cell growing deceleration was observed at 12h and 24h. After 24h, the cell figure started to diminish from the initial cell seeding denseness, bespeaking a more marked break of cell-membrane unity. Several cellular morphological alterations were observed during MAA intervention ( Fig. 5 ) . The cells remained elongated in form and attached at 6 and 12h, while at 24-72h, the cells shrunk to smaller rounded form and started to detach. This time-course TBE analysis and cell morphology observations suggested that MAA exerted both cytostatic and cytocidal effects on Ca Ski cells. Cytostatic consequence was apparent during early hours ( 6-24h ) of intervention with MAA. After 24h onwards, the cells died as a consequence of cytocidal consequence. This was in understanding with the MTT recovery check which showed cytocidal consequence at 72h.
Fig. 4. Anti-proliferative consequence of MAA on Ca Ski cells. Cells were seeded into civilization dishes and exposed to 0.5 % DMSO or 60µM MAA for 6-72h. At each clip point, the feasible cell Numberss were counted utilizing a hemocytometer as described in TBE assay in the methods. Each point represents means ± S Tocopherol from three experiments.
Fig. 5. Consequence of MAA on the morphological alterations of Ca Ski cells. Cells were seeded into civilization dishes and after 24h fond regard, the cells were incubated with 60µM MAA for 6-72h. The control represents cells without intervention at 0h. At each clip point, the cells were visualized under an upside-down phase-contrast microscope and photographed. Magnification: 100x.
Following, we investigated the consequence of MAA on cell rhythm distribution by flow cytometry to look into whether the anti-proliferative consequence was associated with any cell rhythm phase-specific apprehension. As depicted in Fig. 6, incubation with MAA at increasing exposure periods induced a sustained accretion of cells in the S-phase, which was significantly higher than the control. This accretion of cells in S-phase reached its upper limit ( 2.5-fold from the control ) at 12 and 24h, in which MAA was cytostatic and did non do decrease of cell viability ( Fig. 4 ) . Meanwhile, the S-phase population was about 2-fold significantly higher compared to command when treated with 60-100µM MAA for 24h. These consequences indicated that the ascertained cytostatic ( anti-proliferative ) consequence may be due to the S-phase apprehension which caused the disturbance of cell rhythm patterned advance.
Furthermore, DNA cell rhythm analysis was besides performed to look into whether the cell growth-inhibitory consequence of MAA could be related to the initiation of programmed cell death. Our consequences indicated that MAA caused a important addition of hypo-diploid cells in a clip and dose-dependent mode with a attendant lessening of the cells in the G1 stage ( Fig. 6 ) . These hypo-diploid cells, which revealed by the visual aspect of bomber G1 extremum, are an index of apoptotic cells ( Darzynkiewicz et al. , 2001 ) . Notably, the presence of apoptotic cells get downing from 12h and increased to 20-fold at 72h compared to the control. Furthermore, a about 30-fold addition was achieved during 24h incubation with 100µM MAA. Taken together, the initiation of programmed cell death may be responsible for the ascertained cytocidal consequence showed by MAA.
Fig. 6. Ei¬ˆect of MAA on cell rhythm stage distribution and programmed cell death in Ca Ski cells. ( A ) Cells were treated with 60µM MAA for 6-72h. Control corresponds to untreated cells for 6h and similar consequences were obtained at other incubation times. ( B ) Cells were treated with 60, 80 and 100µM or without ( control ) MAA for 24h. After intervention, cells were harvested, fixed, stained with PI and analyzed by i¬‚ow cytometry as described in methods. Cell rhythm distribution was shown in histograms from one representative experiment. ( C ) Percentage of cells in bomber G1, G1, S, and G2/M stages of the cell rhythm are showed in saloon charts. Consequences are average values ± S E of three experiments. Statistical signii¬?cance in comparing with the matching control is indicated by * P & A ; lt ; 0.05.
Previous studies have shown that PCNA is greatly expressed in most of the proliferating malignant neoplastic disease cells including cervical malignant neoplastic disease ( Chan et al. , 1983 ; Benbrook et al. , 1995 ) . PCNA is a cell proliferation biomarker which plays a polar function in the determination of the life or decease of the mammalian cells ( Hall et al. , 1990 ; Kelma, 1997 ; Paunesku et al. , 2001 ) . Hence, the consequence of MAA on the look degrees of PCNA was investigated. The cells were treated with 60µM MAA at different exposure clip periods and the comparative look of PCNA was measured. Consequences showed that MAA significantly suppressed the look of PCNA in a time-dependent mode ( Fig. 7 ) . Time class Q-PCR analysis showed PCNA suppression reached its extremum at 24h intervention of MAA. Cells were besides treated with increasing doses of MAA ( 20-100µM ) for 24h and consequences showed important suppression of PCNA in a dose-dependent mode. These informations suggested that the S-phase apprehension induced by MAA could be attributed to the down ordinance of PCNA look.
Fig. 7. Consequence of MAA on the PCNA look in Ca Ski cells. ( A ) Cells were treated with 60µM MAA for 0 ( control ) , 3,6,12 and 24h. ( B ) Cells were incubated in the absence ( control, 0.5 % DMSO ) or presence of 20-100µM MAA for 24h. After indicated clip, cells were collected, entire RNA was extracted and real-time PCR was performed as described in methods. The informations were from three independent experiments, which were expressed as average ± S.E. fold lessening in PCNA look compared to command, normalized with GAPDH. Statistical signii¬?cance in comparing with the matching control is indicated by * P & A ; lt ; 0.05.
To the best of our cognition, mollic acid glycosides have merely been isolated from Combretum species ( Pegel and Rogers, 1976 ; Pegel and Rogers, 1985 ; Rogers and Thevan, 1986 ; Rogers, 1989 ; Rogers and Coombes, 2001 ; Ahmed et al. , 2004 ; Facundo et al. , 2008 ) . This is the first clip that mollic acid glycosides were isolated from L. indica. It is notable that diverse anticancer surveies such as anti-proliferative, cytotoxic, anti-tumor, DNA-damaging and anti-mitotic activities have been reported in workss belonging to the household Combretaceae ( Pettit et al. , 1989 ; Dorr et al. , 1996 ; McGaw et al. , 2001 ; Fyhrquist et al. , 2006 ) . In one of the surveies, the writers suggested the possibility that the stray mollic acid glycoside was responsible for the ascertained strong cytotoxic consequence of Crombetum pepper tree on HeLa, T 24 and MCF 7 malignant neoplastic disease cells ( Fyhrquist et al. , 2006 ) . However, no farther survey was conducted to verify the compound ( s ) responsible for the cytotoxic action. Furthermore, the in vitro cytotoxic effects of mollic acid glycosides have non been studied. Here, we are the first to demo the cytotoxic consequence of mollic acid glycosides on malignant neoplastic disease cells. Previously, these types of compounds are besides known to exercise anti-molluscicide, hypo-glycaemic, analgetic, anti-inflammatory, anti-hypertensive and cardiovascular effects ( Pegel and Rogers, 1985 ; Ojewole, 2008a, B ; Ojewole and Adewole, 2009 ) .
MAA and MAX are belonging to the group of cycloartane triterpenoid glycoside. Recently, cycloartane-type triterpenes have received considerable attending for their cytotoxic potency ( Wang and Ma, 2009 ; Nuanyai et al. , 2009 ; Nian et al. , , 2010 ; Yokosuka et al. , 2010 ; Fang et al. , 2011 ) . Therefore, our findings here warrant the demand for farther probe on the anti-cancer potency of MAA, particularly for cervical malignant neoplastic disease. Elaborate surveies to place the mechanisms of action are in advancement.
Mollic acid glycoside ( MAA ) , a cycloartane triterpenoid glycoside was isolated for the first clip from L. indica via bioassay-guided attack. It was found to possess anti-cancer consequence for the first clip, which demonstrated strong and selective cytotoxic consequence on Ca Ski cervical malignant neoplastic disease cells while presenting lower toxicity to MRC-5 normal cells. The cytostatic ( anti-proliferative ) and cytocidal ( apoptotic ) effects of MAA with the coincident apprehension of the S-phase and suppression of PCNA look may explicate that MAA blocks the cell rhythm patterned advance to forestall farther cell proliferation accompanied by trigger of apoptotic response. These consequences showed that it is a strong campaigners for farther development as possible malignant neoplastic disease chemotherapeutic drugs.
The writers would wish to thank Miss Tan Hooi Poay from Forest Research Institute of Malaysia ( FRIM ) for her proficient aid in instrument operation and NMR spectral analyses. The writers would besides wish to show their gratitude to Mr. Yap Fon Kwei for providing the works stuff. This research was financially supported by a research fund from the University of Malaya ( PS282/2009C ) .