The reproduction of DNA is influenced by the topological province of the familial material.1 Topoisomerase is such sort of enzyme which can alleviate any torsional emphasis that develops in cellular Deoxyribonucleic acid molecules during reproduction. So a figure of nucleic acerb metamorphosis, such as reproduction, written text, chromatin remodeling, recombination and fix are be modified by the enzymes. This sort of enzyme are distinguished by their catalytic mechanisms. The Topo I Acts of the Apostless by bring forthing a transeunt single-stranded interruption in the dual spiral, while the Topo II Acts of the Apostless by bring forthing a transeunt double-stranded interruption. Furthermore, the Topo II is ATP dependant. 2 -5
In 1960s, Monroe Wall and colleagues found the rough infusions of Camptotheca acuminate were rather active in the L1210 mouse leukaemia life protraction assay among 100s of works infusions been tested.6 Camptothecin ( CPT ) was identified as most active compound, and the Na salt of which was tested clinically in the mid 1970s, but was discontinued because of its serious side effects. Until the Topo I was discovered as its cellular marks, the water-soluble derived functions of CPT, topotecan and irinotecan were successfully developed and used in clinic to handle ovarian malignant neoplastic disease and colon malignant neoplastic disease respectively.7 The mechanism of CPT was demonstrated as Topo I poison, which stabilize the covalent Topo I-DNA cleavage complex forming in the transesterification reactions to interrupt and rejoin DNA strand, Hence, decrease of cleavage composites leads to cut down cell decease. Two mechanisms were reported for a decrease of the Topo I cleavage composites: ( 1 ) Topo I mutations that property to the enzyme drug resistant, and ( 2 ) lessening topo I activity.8 Due to the clinic bound of CPT and the realisation of its alone mechanism, many non-CPT derived functions has been synthesized, like indolocarzazoles and indenoisoquinolines, some of which are now in clinical evaluation.9 Furthermore, several Topo II toxicant like amsacrine ( m-AMSA ) and etoposide,10, 11 which stabilize the covalent Topo II-DNA cleavage composite on both DNA strands, quinolone antibiotics moving as DNA gyrase inhibitors12 and a little category of compounds suppressing both Topo I and Topo II, including aclarubicin, intopolincine and some indenoisoquinolinones were developed and many of which have been used in clinic.13 – 15
Compared with the topoisomrease toxicants, the compounds interfering any of the other stairss in the catalytic rhythm are named catalytic inhibitors, which act at a measure upstream of DNA cleavage and covalent binding to DNA.16, 17 The topoisomerase catalytic inhibitors do non bring forth DNA strand interruptions and should be more active in cells with low topoisomerase degrees. The catalytic Topo II inhibitors contain a assortment of compounds that might interfere with the binding between DNA and topoisomerase, stabilise noncovalent DNA topoisomerase composite or inhibit Topo II-ATP binding.17
In an attempt to place quinolone alkaloids as topoisomerase inhibitors from the Evodia rutaecarpa and to understand their mechanism of action, eight alkaloids were isolated, two of which were quinazolinocarboline alkaloids, evodiamine and retaecarpine, and the other six compounds were quinolone alkaloids, and their repressive activities against topoisomerase were evaluated. We found three of them exhibited powerful repressive activities against human topoisomerase. Among them, evodiamine, which is the most active in primary screen, showed cytotoxicity against human leukemia K562 and THP-1 cell lines with IC50 values of 34.35 ?M and 90.87 ?M severally. Importantly, this is the first clip that the mechanism of evodiamine was demonstrated as catalytic inhibitor of both Topo I and Topo II, since this compound did non demo any grounds for the formation of the enzyme-DNA cleavage composite. However, the consequence of evodiamine on the single measure of the catalytic rhythm is still non to the full understood.
Consequences
Isolation and designation of quinolone alkaloids from Evodia rutaecarpa
The Evodia petroleum infusion were separated harmonizing to the literature18 with gradient system of methyl alcohol, acetonitrile and H2O. Eight fractions were collected utilizing reverse-phase HPLC with Waters semi-preparative column ( 7.8 millimeter x 300 millimeter, 7 ?m ) detected at i?¬ 254 nanometer, and the constructions of them were elucidated by 1-D, 2-D NMR and Mass spectroscopic methods. Although these compounds have been known for some old ages and the similar chromatograph obtained utilizing HPLC method one old ages before25, we got the different position with the construction of 6, which was identified as evocarpine by all the spectroscopic method in our survey, since two olifinic protons at ?H 5.35 correlating to two Cs at ?C 129.94 were assigned to alkene concatenation ( informations non shown ) .
Table 1. Structures of alkaloids isolated from Evodia rutaecarpa utilizing semi-preparative reverse-phase HPLC
1
2
Roentgen
3
4
5
6
7
8
Antiproliferation survey against human leukemia cell lines
The MTT-based check was used to measure the antiproliferative activities against THP-1 cell line for compounds ( 1 – 8 ) and m-AMSA. The viabilities of THP-1 cells for 5 vitamin D continuously treated were shown in Figure 1, from which, 1 and the positive control m-AMSA exhibited antiproliferative activities against THP-1 cell line among all the compounds.
Figure 1. Consequence of compounds ( 1 – 8 and m-AMSA ) with concentrations of 5 ng/?L on the antiproliferation of THP-1 cell line
Consequently, the IC50 values of 1 on the antiproliferation activity against human leukemia THP-1 and K562 cell lines were compared with two anti-tumor drugs, m-AMSA and etoposide for 1 H intervention. As shown in Figure 2, evodiamine ( 1 ) was more effectual against K562 cell line with an IC50 value of 34.35 ?M, which was better than the topoisomerase II toxicant etoposide with an IC50 value of 53.26 ?M. Evodiamine ( 1 ) , a characteristic quinazolinocarbolin alkaloid from Evodia, has been reported to suppress the invasion and metastasis of tumours, and induces cell decease in several types of malignant neoplastic disease cells, such as human ague leukaemia CCRF-CEM cells ( IC50 0.57 µM ) 26, human androgen independent prostate malignant neoplastic disease PC-3 cells ( IC50 1.53 µM ) 27, human chest malignant neoplastic disease MCF-7 cells ( IC50 6.02 µM ) 28, human melanoma A375-S2 cells ( IC50 15 µM ) 29 and murine fibrosarcoma L929 cells ( IC50 20.3 µM ) 30. It possessed lowest cytotoxicity against THP-1 cells among all the reported cell lines with an IC50 value of 90.87 ?M.
Figure 2. IC50
Effectss of quinolone alkaloids on topoisomerase activity
As the mechanisms of evodiamine have been demonstrated to heighten the polymerized tubulin levels26 and stabilise the Topo I -DNA cleavage composite as a Topo I poison28, to look into the mechanisms by which the quinolone alkaloids inhibited either topo I or topo II and thereby caused cytotoxicity, the consequence of all compounds on topoisomerase activity were examined by mensurating the relaxation of supercoiled Deoxyribonucleic acid of plasmid pBR 322 for Topo I in the absence or presence ethidium bromide ( EtBr ) and decatanation of kinetoplase DNA for Topo II. The gels without EtBr allow the sensing of compounds suppressing DNA relaxation by Topo I, while EtBr incorporating gels indicate whether or non the inhibiting activities of those compounds might be due to the effects on the Topo I-DNA cleavage composites. Compounds 1, 3 and 7 exhibited Topo I repressive activities ( Figure 3A ) , of which, 1 was the most effectual, since the relaxation of supercoiled DNA was wholly inhibited incubating with 100 ?M of 1. Compared with 1, CPT did non wind off DNA to any noticeable extent up to 100 ?M, while it increased the strength of nicked set ( Figure 3B, lane 3 ) , which indicated the formation of the enzyme-DNA cleavage composite with one strand of DNA broken. Furthermore, 1 did non demo any grounds increasing the strength of nicked set in the EtBr incorporating gel ( Figure 3B, lane 4 ) and therefore 1 might be non moving as Topo I poison.
Compounds 1 and 7 possessed Topo II inhibitory activities ( Figure 3C ) , of which, 1 ( evodiamine ) showed more effectual inhibitory activity compared with the Topo II toxicant m-AMSA as 90 % of catanated kDNA remained in the well ( Figure 3C, lane 4 ) when it incubated with 100 ?M of 1 ( evodiamine ) , while merely 17.8 % of catanated kDNA was in the well after incubating with 100 ?M of m-AMSA ( Figure 3C, lane 3 ) which was similar inhibitory activity with 7. However, 7 did non demo any cytotoxocity against two tried leukaemia cell lines ( informations non shown ) .
A
Bacillus
C
Figure 3. Consequence of compounds ( 1 – 7 ) with concentration of 100 µM on the relaxation of plasmid pBR 322 DNA ( 0.5 µg ) by 1 U of human Topo I. DNA samples were separated by cataphoresis on the 1 % agarose gel ( A ) with or ( B ) without ethidium bromide. The gels were photographed under UV visible radiation. Nck, nicked ; Rel, relaxed ; Sc, supercoiled. ( C ) Effect of 100 µM of compounds ( 1 – 7 ) on the decatanation of kDNA ( 0.2µg ) by 1 U of human Topo II.
The repressive activities of human Topo I and Topo II by evodiamine ( 1 ) were farther evaluated by Topo I relaxation assay and Topo II decatanation assay severally in concentration-dependant mode. The IC50 values of evodiamine ( 1 ) against human Topo I and Topo II were 60.74 ?M and 78.81 µM severally. Therefore, evodiamine ( 1 ) was identified as a double inhibitor against both Topo I and Topo II. More late, a figure of drugs have been identified that mark both Topo I and Topo II, and some of these drugs have been advanced to clinical test. They can move as poison-poison ( Intoplicine ) , poison-catalytic inhibitor ( Aclarubicine ) or catalytic-catalytic inhibitors ( F11782 ) .31 – 33 The mechanistic surveies were carried out to look into which type of inhibitors the 1 playing as.
A
C
Bacillus
Calciferol
Figure 4. IC50 rating of evodiamine ( 1 ) inhibitory activities against human Topo I and Topo II
Consequence of 1 on the Topo II-mediated DNA cleavage check
Topoisomerase II toxicants such as AMSA are known to stabilise the cleavage composite that leads to DNA strand interruptions, while catalytic inhibitors such as aclarubicin can protect cells against Topo II poison-induced DNA harm. As shown in the old consequences, 1 is a Topo I catalytic inhibitor. We besides tested whether 1 could execute as a toxicant and excite the DNA cleavage composites of Topo II utilizing the Topo II cleavage check. In contrast to m-AMSA, which stimulated the cleavage complex indicating as additive set in the gel, 1 had no consequence on Topo II cleavage activity even the concentration up to 100 µM and Topo II activity was partly inhibited incubating with therefore high concentration of enzyme ( 10 U ) .
Figure 5. 1 did non bring on the accretion of the Topo II-DNA cleavage composite. Supercoiled pBR 322 DNA ( 0.3 µg ) ( lane 1 ) was incubated with 10 U of human topo II in absence ( lane 2 ) or presence of AMSA or 1. Oc, Open circlar. Lin, Linear DNA.
Interaction between 1 and DNA
The drug-free DNA sample is negatively supercoiled and migrates through down the gel as a individual set. But its profile alterations significantly in the presence of intercalating agents. At low concentration, the DNA embolism of the agent between DNA base brace induces the relaxation of DNA, which shows easy migrates on the top of the gel. As the concentration further increased, the Deoxyribonucleic acid molecules wind in the oppposite manner so as to bring forth positive supercoils. When the Deoxyribonucleic acid is to the full positively supercoiled, it migrate as a individual set with an mobility near to that of the negatively supercoiled DNA. Therefore, the typical intercalating agent should demo a up-and-down profile with increasing concentration.
As indicated in Figure 6A, the DNA intercalator EtBr showed clearly the up-and-down profile with the increasing concentration when incubated with 10 U of human Topo I, while CPT and 1 did non consequence the check. DNase I footprinting ( Figure 6B ) indicated 1 did non adhere to DNA at the concentration up to 100 µM. The two Topo II toxicant, etoposide and AMSA did non adhere to DNA at the concentration of 100 µM, since etoposide was identified as non-DNA binder and AMSA is DNA intercalator.
A
Bacillus
Figure 6. ( A ) Supercoiled pBR 322 DNA ( 0.3 µg ) ( lane 1 ) was incubated with 10 units of human topoisomerase I in absence ( lane 2 ) or presence of consecutive dilution of EtBr ( lanes 3 to 7 ) , CPT ( lanes 8 to 11 ) or Evodiamine ( lanes 12 to 16 ) . ( B ) Deoxyribonucleic acid footprinting showed evodiamine did non adhere to DNA at the concentration up to 100 µM. Furthermore, the Topo II toxicant etoposide and m-AMSA did non adhere to DNA, since etoposide is known as DNA non-binder and m-AMSA is DNA intercalater.
Consequence of 1 on cell rhythm distribution in K562 cell line
To look into the consequence of 1 on cell rhythm patterned advance, K562 cells were exposed to 1 for 1 H, and so grew in drug free medium until trial. Cell rhythm distribution was determined by flow cytometric analysis. As shown in Figure 7, 1 clearly induced G2/M apprehension in exponentially cells treated by 20 ?M of drug. G2/M apprehension was demonstrated as consequence of topoisomerase II inhibitors, such as etoposide.25
Figure 7. Cell rhythm distribution analysis of K562 cells exposed to 20, 40 or 80 ?M of 1. Cell were treated for 1 H, following grew in drug free medium for 8, 24, 32, 48 and 72 H before trial.
We next evaluated the possibility that the cytotoxicity of 1 in K562 cells was non attributable to a higher degree of induced DNA harm. The potency of 1 to bring on DNA strand interruptions in K562 cells was assessed via Comet assay utilizing the Topo I poison CPT as a positive control. There is no any strand interruptions induced by 1 up to 100 µM, while CPT treated K562 cells exhibited extremely damaged in the check ( Figure 8 ) . The Topo I poison CPT achieve their cytotoxicity effects by stabilising the enzyme-DNA cleavage composite which leads to DNA strand interruptions in chromosomal DNA and consequences in cell decease. Consistent with the observation in Topo II-mediated DNA cleavage check, 1 did non do any DNA harm. So the mechanism of 1 inhibiting of topoisomerase was considered as both Topo I and Topo II catalytic suppression, but non topoisomerase toxicant action.
CPT
Control
10 ?M
1
Control
100 ?M
Figure 8. The comet check was applied to observe the DNA strand interruptions bring oning by evodiamine. The Topo I poison CPT showed extremely DNA damaged cells at the concentration of 10 ?M, while the DNA strand interruptions were non detected for the evodiamine treated cells even at the concentration up to 100 ?M.
Possible effects of 1 on the CPT resistant cell line
Topo I toxicants, such as CPT, are cytotoxic to the cells by pin downing cleavage composite instead than suppressing enzyme catalytic activity, which indicated that they turn Topo I into a cellular toxicant that induce DNA harm. Therefore, the decrease of Topo I activity is one of the first alteration in cells selected for CPT opposition, and conversely, sensitiveness of Topo I toxicants increase with the enzyme overexpression.34, 35 On the other manus, Figure 5 indicated 1 is more efficient inhibitory activity incubating with lower concentration of enzyme and the sensitiveness reduced with increasing of enzyme. Therefore, 1 might be effectual on the CPT resistant cell lines.
Figure 9. 100 ?M of evodiamine ( lane 3 to 7 ) or camptothecin ( lane 8 to 12 ) were incubated with consecutive dilution of human topoisomerase I and pBR 322 DNA ( 0.3 µg ) . Deoxyribonucleic acid samples were separated by cataphoresis on the 1 % agarose gel.
Discussion
Decision
Experimental Section
General Experimental Procedure
Chemistry
The methyl alcohol and acetonitrile used as nomadic stage and mass spectroscopy dissolvers were HPLC class and cowboies from Fisher Scientific ( UK ) . Distilled H2O used in extraction and HPLC were filtered by Water Purify System ( Millipore SimplicityTM, USA ) . All the other chemicals and dissolver were laboratory class and used without farther purification.
Extraction and Isolation of Quinolone Alkaloids
The dry fruit of Evodia pulverization ( ca. 50 g ) was soaked in ethyl ethanoate ( 800 milliliter ) for 30 min, and so extracted by ultrasonification for 45 min ( the extraction process was repeated twice for each sample and four samples were extracted ) . The ethyl ethanoate beds were filtered and combined, following that, the dissolver was evaporated by a rotary evaporator under decreased force per unit area to four give four brown petroleum infusions ( 1, 2, 3 and 4 ) . After fade outing in methyl alcohol and kept at 4 & A ; deg ; C for 24 H, xanthous crystals generated, which was identified as rutaecarpine by NMR spectroscopic method. The rutaecarpine crystals were separated out from the petroleum infusion ( 1, 2 and 4 ) , recrystallized by methylene chloride and weighted for output. Then the infusions without rutaecarpine crystals were separated by reverse-phase HPLC from Jasco ( Japan, dwelling of PU-980 intelligent HPLC pumps, UV-975 intelligent UV/VIS sensors and LC-980-02 treble gradient ) with the separate method harmonizing to the literature [ 18 ] on a Waters semi-preparative column ( 7.8 millimeter x 300 millimeter, 7 ?m ) . The separations were carried out utilizing a gradient system of methyl alcohol, acetonitrile and H2O ( 0 min: 2 % :38 % :60 % , 22 min: 5 % :38 % :57 % , 35 min: 30 % :38 % :32 % and 65min: 45 % :38 % :17 % ) with flow rate 4.5 mL/min. The sensor wavelength was set at 254 nanometers.
HPTLC quantification of rutaecarpine in petroleum infusion
Standard
The evodiamine and rutaecarpine criterions were supplied by SUTCM ( Shanghai, China ) and a solution of a 1 mg/mL of each criterion was prepared by fade outing the criterion in trichloromethane. The crystal ( XP-E-1 ) obtained from the evodiamine infusion was dissolved in trichloromethane to give a 1 mg/mL solution.
Developing dissolver
Hexane ( 14 milliliter ) was assorted with trichloromethane ( 4 milliliter ) and propanone ( 2 milliliter ) to give a entire volume of 20 milliliters developing dissolver.
Derivatizing reagent
Sulphuric acid ( 20 milliliter ) was gently added to frost cold methyl alcohol ( 180 milliliter ) , assorted carefully and allowed to chill down to room temperature.
HPTLC designation was carried out at room temperature utilizing a CAMAG HPTLC system ( CAMAG, Switzerland ) including an automatic TLC sampling station ( ATS 4 ) , automatic developing chamber ( ADC 2 ) , TLC visualiser ( 150904 ) and TLC package ( winCATS ) . The glass plates, HPTLC silica gel 60 F254, 20 ten 10 centimeter and electro-pneumatic TLC-sprayer were purchased from Merck ( Merck, Germany ) .
Test solutions and standard solutions ( 2 µL ) were applied with a set length of 8 millimeters each, 8 millimeter from the lower border and 22 millimeter from left and right borders of the home bases. The developing dissolver ( 20 milliliter ) were poured into an ADC 2 developing chamber and saturated for 15 min before development. The home base was dried before and after developing for 5 min. Migration distance was set at 80 millimeter. After developing, the un-derivatized home base was documented utilizing UV 254 and 366 lights. Then the home base was sprayed with the derivatizing reagent, dried for 10 min and placed onto a preheated hot home base ( 105 oC ) for 5 min. After chilling to room temperature, the home base was documented with white, 254 and 266 nm lights.
Word picture of Quinolone Alkaloids
NMR spectra were obtained utilizing Bruker Avance 500 NMR Spectrometer ( Bruker corp. UK ) . TMS ( tetramethylsilane ) is used as a mention for all NMR measurings. The pulse plans were Zg30 for normal 1H-NMR, Zgpg30 for normal 13C-NMR, and DEPT30 and DEPT90 for 13C DEPT spectra. The MS spectra were measured utilizing a Waters Q-Tof microTM ( Waters corp. USA ) mass spectrometer.
Evodiamine ( 1 ) : 1H NMR ( 500.13 MHz, CD3OD ) ? : 2.52 ( 3H, s, N-CH3 ) , 3.00 ( 2H, m, H-6 ) , 3.32, 4.90 ( 1H, each, m, H-5 ) , 5.95 ( 1H, s, H-3 ) , 7.17 – 8.16 ( 8H, m, Ar-H ) , 8.22 ( 1H, Br, N-H ) . 13C NMR ( 125.76 MHz, CD3OD ) ? : 20.44 ( C-6 ) , 42.58 ( C-5 ) , 40.83 ( N-CH3 ) , 113.22 ( C-12 ) , 119.64 ( C-7 ) , 120.8596 ( C-9 ) , 120.23 ( C-11 ) , 122.07 ( C-20 ) , 126.56 ( C-10 ) , 126.88 ( C-8 ) , 127.45 ( C-18 ) , 127.47 ( C-19 ) , 127.99 ( C-16 ) , 128.32 ( C-2 ) , 135.95 ( C-17 ) , 140.55 ( C-13 ) , 146.80 ( C-3 ) , 149.31 ( C-15 ) , 163.46 ( C-21 ) . [ M+H ] + m/z 304.1076. Intensify EF-2 was identified as. [ 65 ]
Rutaecarpine ( 2 ) : 1H NMR ( 500.13 MHz, CD3OD ) ? : 3.26 ( 2H, T, H-6 ) , 4.55 ( 2H, T, H-5 ) , 7.12 ( 1H, T, H-10 ) , 7.29 ( 1H, T, H-11 ) , 7.45 ( 1H, m, H-18 ) , 7.47 ( 1H, m, H-12 ) , 7.64 ( 1H, vitamin D, H-9 ) , 7.72 ( 1H, vitamin D, H-16 ) , 7.79 ( 1H, dt, H-17 ) , 8.23 ( 1H, Doctor of Divinity, H-19 ) , 8.54 ( 1H, Br, N-H ) . 13C NMR ( 125.76 MHz, CD3OD ) ? : 20.44 ( C-6 ) , 42.58 ( C-5 ) , 113.32 ( C-12 ) , 119.63 ( C-7 ) , 120.96 ( C-9 ) , 121.23 ( C-11 ) , 121.95 ( C-20 ) , 126.33 ( C-10 ) , 126.71 ( C-8 ) , 127.28 ( C-18 ) , 127.82 ( C-19 ) , 127.99 ( C-16 ) , 128.27 ( C-2 ) , 135.69 ( C-17 ) , 140.48 ( C-13 ) , 146.80 ( C-3 ) , 149.31 ( C-15 ) , 163.46 ( C-21 ) , [ M+H ] + m/z 288.3462. Intensify EF-3 was identified as rutaecarpien. [ 65 ]
1-methyl-2-nonyl-4 ( 1H ) -quinolone ( 3 ) : 1H NMR ( 500.13 MHz, CDCl3 ) ? : 8.45 ( 1H, Doctor of Divinity, H-5 ) , 7.67 ( 1H, m, H-7 ) , 7.51 ( 1H, vitamin D, H-8 ) , 7.38 ( 1H, m, H-6 ) , 6.25 ( 1H, s, H-3 ) , 3.75 ( 3H, s, N-CH3 ) , 2.72 ( 2H, T, H-1 ‘ ) , 1.69 ( 2H, m, H-2 ‘ ) , 1.43 ( 2H, m, H-3 ‘ ) , 1.37 – 1.26 ( 10H, m, H-4 ‘ , H-5 ‘ , H-6 ‘ , H-7 ‘ , H-8 ‘ ) , 0.88 ( 3H, T, H-9 ‘ ) . 13C NMR ( 125.76 MHz, CDCl3 ) ? : 177.86 ( C-4 ) , 154.86 ( C-2 ) , 141.95 ( C-8a ) , 132.02 ( C-7 ) , 126.70 ( C-5 ) , 126.57 ( C-4a ) , 123.29 ( C-6 ) , 115.29 ( C-8 ) , 111.23 ( C-3 ) , 34.81 ( C-1 ‘ ) , 34.13 ( N-CH3 ) , 28.58 ( C-2 ‘ ) , 31.89 – 22.69 ( Alkyl concatenation ) , 14.14 ( C-9 ‘ ) , [ M+H ] + m/z 286.3147, EF-4 was identified as 1-methyl-2-nonyl-4 ( 1H ) -quinolone. [ 31 ]
1-methyl-2- [ ( Z ) -5-undecenyl ] -4 ( 1H ) -quinolone ( 4 ) : 1H NMR ( 500.13 MHz, CDCl3 ) ? : 8.45 ( 1H, Doctor of Divinity, H-5 ) , 7.67 ( 1H, m, H-7 ) , 7.51 ( 1H, vitamin D, H-8 ) , 7.38 ( 1H, m, H-6 ) , 6.25 ( 1H, s, H-3 ) , 3.75 ( 3H, s, N-CH3 ) , 2.72 ( 2H, T, H-1 ‘ ) , 1.69 ( 2H, m, H-2 ‘ ) , 1.43 ( 2H, m, H-3 ‘ ) , 1.37 – 1.26 ( 10H, m, H-4 ‘ , H-5 ‘ , H-6 ‘ , H-7 ‘ , H-8 ‘ ) , 0.88 ( 3H, T, H-11 ‘ ) . 13C NMR ( 125.76 MHz, CDCl3 ) ? : 177.86 ( C-4 ) , 154.86 ( C-2 ) , 141.95 ( C-8a ) , 132.02 ( C-7 ) , 129.94 ( Olefinic ) 126.70 ( C-5 ) , 126.57 ( C-4a ) , 123.29 ( C-6 ) , 115.29 ( C-8 ) , 111.23 ( C-3 ) , 34.81 ( C-1 ‘ ) , 34.13 ( N-CH3 ) , 31.89 – 22.69 ( Alkyl concatenation ) , 14.14 ( C-11 ‘ ) , [ M+H ] + m/z 313.2115, EF-5 was identified as 1-methyl-2- [ ( Z ) -5-undecenyl ] -4 ( 1H ) -quinolone. [ 31 ]
1-methyl-2-dodecyl-4 ( 1H ) -quinolone ( 5 ) : 1H NMR ( 500.13 MHz, CDCl3 ) ? : 8.31 ( 1H, Doctor of Divinity, H-5 ) , 7.89 ( 1H, vitamin D, H-8 ) , 7.80 ( 1H, m, H-7 ) , 7.46 ( 1H, m, H-6 ) , 6.32 ( 1H, s, H-3 ) , 3.90 ( 3H, s, N-CH3 ) , 2.89 ( 2H, T, H-1 ‘ ) , 1.73 ( 2H, m, H-2 ‘ ) , 1.50 ( 2H, m, H-3 ‘ ) , 1.46 – 1.31 ( 12H, m, H-4 ‘ , H-5 ‘ , H-6 ‘ , H-7 ‘ , H-8 ‘ , H-9 ‘ ) , 0.89 ( 3H, T, H-11 ‘ ) . 13C NMR ( 125.76 MHz, CDCl3 ) ? : 179.61 ( C-4 ) , 159.00 ( C-2 ) , 143.52 ( C-8a ) , 133.95 ( C-7 ) , 127.00 ( C-5 ) , 126.69 ( C-4a ) , 125.18 ( C-6 ) , 117.89 ( C-8 ) , 111.15 ( C-3 ) , 35.70 ( C-1 ‘ ) , 35.49 ( N-CH3 ) , 31.89 – 22.69 ( Alkyl concatenation ) , 14.47 ( C-11 ‘ ) , [ M+H ] + m/z 315.1296, EF-6 was identified as 1-methyl-2-dodecyl-4 ( 1H ) -quinolone. [ 31 ]
Evocarpine ( 6 ) : 1H NMR ( 500.13 MHz, CDCl3 ) ? : 8.31 ( 1H, Doctor of Divinity, H-5 ) , 7.87 ( 1H, vitamin D, H-8 ) , 7.80 ( 1H, m, H-7 ) , 7.48 ( 1H, m, H-6 ) , 6.32 ( 1H, s, H-3 ) , 5.34 ( 2H, m, H-7 ‘ and H-8 ‘ ) , 3.90 ( 3H, s, N-CH3 ) , 2.89 ( 2H, T, H-1 ‘ ) , 1.73 ( 2H, m, H-2 ‘ ) , 1.50 ( 2H, m, H-3 ‘ ) , 1.46 – 1.31 ( 12H, m, H-4 ‘ , H-5 ‘ , H-6 ‘ , H-9 ‘ , H-10 ‘ , H-11 ‘ and H-12 ‘ ) , 0.89 ( 3H, T, H-13 ‘ ) . 13C NMR ( 125.76 MHz, CDCl3 ) ? : 179.61 ( C-4 ) , 159.00 ( C-2 ) , 143.52 ( C-8a ) , 133.95 ( C-7 ) , 130.9 and 130.7 ( Olefinic ) , 127.00 ( C-5 ) , 126.69 ( C-4a ) , 125.18 ( C-6 ) , 117.89 ( C-8 ) , 111.15 ( C-3 ) , 35.70 ( C-1 ‘ ) , 35.49 ( N-CH3 ) , 32.89 – 21.40 ( Alkyl concatenation ) ,14.47 ( C-13 ‘ ) , [ M+H ] + m/z 340.8739, EF-7 was identified as evocarpine. [ 31 ]
1-methyl-2- [ ( 6Z,9Z ) ] -6,9, -pentadecadienyl-4 ( 1H ) -quinolone ( 7 ) : 1H NMR ( 500.13 MHz, CDCl3 ) ? : 8.31 ( 1H, Doctor of Divinity, H-5 ) , 7.87 ( 1H, vitamin D, H-8 ) , 7.80 ( 1H, m, H-7 ) , 7.48 ( 1H, m, H-6 ) , 6.32 ( 1H, s, H-3 ) , 5.34 ( 2H, m, ) , 3.90 ( 3H, s, N-CH3 ) , 2.89 ( 2H, T, H-1 ‘ ) , 1.73 ( 2H, m, H-2 ‘ ) , 1.50 ( 2H, m, H-3 ‘ ) , 1.46 – 1.31 ( 12H, m, H-4 ‘ , H-5 ‘ , H-6 ‘ , H-7 ‘ , H-8 ‘ , H-9 ‘ ) , 0.89 ( 3H, T, H-15 ‘ ) . 13C NMR ( 125.76 MHz, CDCl3 ) ? : 179.61 ( C-4 ) , 159.00 ( C-2 ) , 143.52 ( C-8a ) , 133.95 ( C-7 ) , 130.2 ( Olefinic ) , 127.00 ( C-5 ) , 126.69 ( C-4a ) , 125.18 ( C-6 ) , 117.89 ( C-8 ) , 111.15 ( C-3 ) , 37.6 ( C-8 ‘ ) , 35.70 ( C-1 ‘ ) , 35.49 ( N-CH3 ) , 29.73 – 24.11 ( Alkyl concatenation ) , 14.47 ( C-15 ‘ ) [ M+H ] + m/z 366.9176, EF-8 was identified as 1-methyl-2- [ ( 6Z,9Z ) ] -6,9, -pentadecadienyl-4 ( 1H ) -quinolone. [ 65 ]
Dihydroevocarpine ( 8 ) : 1H NMR ( 500.13 0MHz, CD3OD ) ? : 8.31 ( 1H, Doctor of Divinity, H-5 ) , 7.87 ( 1H, vitamin D, H-8 ) , 7.80 ( 1H, m, H-7 ) , 7.48 ( 1H, m, H-6 ) , 6.32 ( 1H, s, H-3 ) , 5.34 ( 2H, m, ) , 3.90 ( 3H, s, N-CH3 ) , 2.89 ( 2H, T, H-1 ‘ ) , 1.73 ( 2H, m, H-2 ‘ ) , 1.50 ( 2H, m, H-3 ‘ ) , 1.46 – 1.31 ( 12H, m, H-4 ‘ , H-5 ‘ , H-6 ‘ , H-7 ‘ , H-8 ‘ , H-9 ‘ ) , 0.89 ( 3H, T, H-13 ‘ ) . 13C NMR ( 125.76 MHz, CD3OD ) ? : 179.61 ( C-4 ) , 159.00 ( C-2 ) , 143.52 ( C-8a ) , 133.95 ( C-7 ) , 127.00 ( C-5 ) , 126.69 ( C-4a ) , 125.18 ( C-6 ) , 117.89 ( C-8 ) , 111.15 ( C-3 ) , 35.70 ( C-1 ‘ ) , 35.49 ( N-CH3 ) , 31.89 – 22.69 ( Alkyl concatenation ) , 14.47 ( C-13 ‘ ) , [ M+H ] + m/z 342.4131, EF-9 was identified as dihydroevocarpine. [ 31 ]
Materials and Methods for Biological Assay
Cell civilization and cytotoxicity assay
Human K562 myelogenous leukaemia cells and THP-1 acute monocytic leukaemia cells were obtained from the American Type Culture Collection ( ATCC, UK ) . Cells were grown at 37 & A ; deg ; C in a humidified ambiance incorporating 5 % CO2 in RPMI-1640 medium with 10 % of foetal bovine serum and 2 millimeter of glutamine. The viability of cell line were determined utilizing the MTT [ 3- ( 4,5-dimethyl thiazol-2yl ) -2,5-diphemyltetrazolium bromide [ Sigma, UK ] assay. 19, 20 2 milliliter of cells with the concentration 5 – 105 cells/mL ( counted by hemocytometer ) in exponential growing were treated with changing concentrations ( 0.3 to 100 ?M ) of compounds under survey in extra for 1 H or 24 h. 1 % DMSO treated cells were taken as negative control. Following intervention, cells were pelleted ( 1500 revolutions per minute, 5 min ) , resuspend in 2 milliliter of RPMI-1640 medium, plated out at 200 ?L per good in U underside home bases and incubated for 96 H at 37 & A ; deg ; C. At the terminal of the incubation, 20 ?L of MTT ( 5 mg/mL in distilled H2O ) was added and incubated for 4 h. Formazan crystals formed were dissolved in 200 ?L of DMSO. The optical density was measured at 540 nanometer in the home base reader ( Varioskan Flash, Thermo Scientific, UK ) . The IC50 values were estimated by plotting the information on Grafit 5.0.4 version ( Erithacus Software, UK ) .
Topoisomerase I relaxation assay
Topoisomerase relaxation check and decatanation assay were carried out as antecedently described.21, 22 Supercoiled pBR 322 DNA ( 0.3 µg ) was incubated with 1 U of human Topo I ( Inspiralis, UK ) in 30 ?L of relaxation buffer ( 2 mM Tris-HCl pH 7.5, 20 millimeter NaCl, 25 µM EDTA and 0.5 % glycerin ) at 37 & A ; deg ; C for 30 min in the presence of changing concentrations ( 0.3 to 100 ?M ) of the compounds under survey. 100 µM of Topo I poison CPT was used as positive control.
5 U of the enzyme was used to analyze the intercalating ability of some compounds based on the Topo I relaxation assay.23
The ability of compounds to stabilise the covalent DNA enzyme reaction intermediate were evaluated by incubating supercoiled pBR 322 DNA ( 0.3 µg ) with 10 U of human Topo II for the relaxation check. After incubation with enzyme, the reaction was further incubated with 0.1 mg/mL proteinase K and 0.2 % SDS for 30 min at 37 & A ; deg ; C.
Topoisomerase II decatanation check
kDNA i??200 ngi?‰ was incubated with 1 unit of human Topo II ( Inspiralis, UK ) in check buffer ( 5 mM Tris-HCl pH 7.5, 12.5 millimeter NaCl, 1 millimeter MgCl2, 0.5 millimeter DTT and 10 ?g/mL albumen ) with 1 millimeters of ATP at 37 & A ; deg ; C for 30 min in the presence of different concentrations ( 3 to 100 ?M ) of the compounds under survey. The Topo II toxicants, amsacrine and etoposide, were served as positive control.
Gel cataphoresis
The topoisomerase catalytic reactions were stopped by add-on of 18 ?L of halt buffer ( 40 % saccharose, 100 millimeter Tris pH7.5, 10 millimeter disodium EDTA and 0.05 % bromophenol blue ) . Deoxyribonucleic acid samples were extracted by a mixture of Chloroform/iso amyl intoxicant ( 24:1 ) , followed by it running on a 1 % agarose gel at 10 V /cm in TAE buffer for 3 h. Gels were stained with ethidium bromide ( 0.5 ?g/mL in distilled H2O ) for 1 H and destained with distilled H2O for 30 min. Similar experiments were performed utilizing ethidium bromide ( 0.5 ?g/mL ) incorporating agarose gel to accomplish separation of nicked and relaxed species. The gel was visualized under UV visible radiation and photographed. For quantitative findings, the incorporate strengths of the ethidium bromide fluorescence of the sets ( supercoiled signifier for relaxation check and minicircled signifier for decatanation check ) were quantified and calculated utilizing ImageJ package ( Wayne Rasband, USA ) . The IC50 values were estimated by plotting the information on Grafit 5.0.4 version ( Erithacus Software, UK ) .
Comet check
The individual cell gel cataphoresis ( comet ) assay developed to let visual image of DNA strand break harm in single cells was performed as preciously described.24 2 milliliter of K562 cells with the concentration 2.5 – 104 cells/mL ( counted by hemocytometer ) in exponential growing were treated with assorted concentrations of compounds under survey for 1 h. 1 % DMSO treated cells were taken as negative control. 0.5 milliliter of cell suspension was assorted with 1 milliliters of pre-warmed low gelling temperature ( LGT ) agarose ( 1 % H2O ) . 1 milliliter of the mixture was quickly spread on the single-frosted glass microscope slid ( 25 – 75 millimeter, 1 millimeter midst ) pre-coated with 1 milliliters of type 1-A agarose ( 1 % H2O ) and covered with a coverslip ( 24 – 40 millimeter ) . Then 500 milliliters of ice-cold lysis buffer ( 0.1 M disodium EDTA, 2.5 M NaCl, 10 mM Tris-HCl pH 10.5 with NaOH and 1 % Triton X-100 newly added before usage ) was added to submerse all slides and incubated on ice for 1 Hs in dark. The slides were washed by ice-cold dual distilled H2O, fowllowed by it reassigning into the cataphoresis armored combat vehicle with 2 L of ice-cold base buffer ( 50 millimeter NaOH, 1mM disodium EDTA pH 12.5 ) and incubated for 45 min in dark to let unwinding of DNA. Electrophoresis ( 0.6 V/cm, 250 ma ) was so carried out at room temperature for 18 min in dark. After cataphoresis, slides were washed with neutralization buffer ( 0.5 M Tris-HCl, pH 7.5 ) , left for 10 min and rinsed twice with PBS. After drying nightlong at room temperature, slides were stained twice with 1 milliliters of propidium iodide solution ( 2.5 ?g/mL ) . All slides were dried in oven at 37 & A ; deg ; C and stored until image analysis. 50 indiscriminately selected single cells per slides were visualised and the olive tail minutes ( OTM ) were measured utilizing the comet imagination system ( ) with fluorescent microscope.
Cell rhythm analysis
Flow cytometry analysis of DNA content, 2 milliliter of K562 cells with the concentration 5 – 105 cells/mL ( counted by hemocytometer ) in exponential growing were treated with different concentrations ( 20 ?M, 40 ?M and 80 ?M ) of compounds under survey for 1 H and so resuspended in 2 milliliter of RPMI-1640 medium for clip class ( 0 H, 8 H, 24 H, 32 H, 48 H and 72 H ) . At the terminal of each clip class, cells were centrifuged ( 1500 revolutions per minute, 5 min ) and the pellets were suspended in 1 milliliter of PBS. 3 milliliter of absolute ethyl alcohol was added to the cells with whirl for arrested development. All the cells were stored at -20 & A ; deg ; C until analysis. On the twenty-four hours of analysis, cells were centrifuged ( 2000 revolutions per minute, 10 min ) , washed with PBS, suspended in 500 ?L of PI solution ( 50 ?g/mL propidium iodide, 100 ?g/ milliliter RNase A and 0.05 % Tritin X-100 ) , and incubated at 37 & A ; deg ; C for 30 min. After incubation, 3 milliliter of PBS was added, and the cells were pelleted ( 1500 revolutions per minute, 5 min ) , suspended in PBS at a concluding cell denseness of 5 – 105 cells/mL and transferred to a Falcon 2054 tubing ( BD Bioscience, UK ) for flow cytometry analysis. Analysis of 20,000 events was done on a CyAn ADP flow cytometer ( Beckman Coulter, UK ) utilizing Summit 4.3 version ( Dako Clolrado, Inc, USA ) and ungated information was gathered within 1 H.
Deoxyribonucleic acid footprinting
DNase I digestions were conducted in a entire volume of 8 ?L. The labelled DNA fragment ( 2 ?L, 200 counts s-1 ) was incubated for 30 min at room temperature in 4 ?L TN buffer ( 10 millimeter Tris Base and 10 millimeter NaCl, pH 7 ) incorporating the needed drug concentration. Cleavage by DNase I was initiated by add-on of 2 ?L of DNase I solution ( 20 millimeter NaCl, 2 millimeter MgCl2, 2 millimeter MnCl2 and DNase I 0.02 Unit of measurements, pH 8 ) and stopped after 3 min by snap stop deading the samples on dry ice. The samples were later lyophilized to dryness and resuspended in 5 ?L of formamide lading dye ( 95 % formamide, 20 millimeter EDTA, 0.05 % bromophenol blue and 0.05 % cyanol blue ) . Following heat denaturation for 5 min at 90 & A ; deg ; C, the samples were loaded on a denaturing polyacrylamide ( 10 % ) gel incorporating urea ( 7.5 millimeter ) . Electrophoresis was carried out for 2 H at 1650 V ( ~70 W, 50 & A ; deg ; C ) in 1- TBE buffer. The gel was so transferred onto Whatman 3MM and dried under vacuity at 80 & A ; deg ; C for 2 h. The gel was exposed overnight to Fuji medical X ray movie and developed on a Konica Medical Film Processor SRX-101A.
