APROVECHAMIENTO SOSTENIBLE DE LOS RECURSOS NATURALES

3.9.1 Instrumentation

NMR (Nuclear Magnetic Resonance) spectra were recorded on a JOEL 400 MHz FT NMR spectrometer at 400 MHz for 1H NMR (1H Nuclear Magnetic Resonance) and at 100.40 MHz for 13C NMR (13C Nuclear Magnetic Resonance). Internal standards used in 1H NMR spectra was TMS (: 0.00) for CDCl3; in 13C NMR

was CDCl3 (: 77.0).

GCMS (Gas Chromatography and Mass Spectra) analysis was performed using Agilent Technologies 6980N gas chromatograph equipped with a 5979 Mass Selective Detector (70 eV direct inlet); a HP-5ms (5 % phenyl methyl siloxane) capillary column (30.0 m x 250 µm x 0.25 µm) initially set at 60 C for 10 min, then programmed to 230

C at 3 C min-1 and held for 1 min at 230 C using helium as the carrier gas at a flow rate of 1 ml min-1.

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The total ion chromatogram obtained was auto integrated by ChemStation and the components were identified by comparison with an accompanying mass spectral database (NIST 05 MS Library, 2002). Only mass spectral fragmentation pattern that gave greater than 90 % match were accepted.

The GC (Gas Chromatography) analysis was performed on a Shidmadzu GC 14A equipped with a FID detector using fused-silica capillary HP-5mscolumn (30.0 m length x 250 µm; 0.25 m film thickness) with helium as carrier gas at a flow rate of 1 mlmin-1. Column temperature was programmed initially at 60 C for 10 min, then programmed to 230 C at 3 C min-1 and held for 1 min at 230 C.

3.9.2 Column chromatography

Column chromatography was performed using Merck Kieselgel 60 PF253 Art No

7734.1000 and 9385.1000 with particle size 0.063-0.200 mm and 0.040-0.0063 mm, respectively. The gel was made into slurry with solvent before it was packed onto the column and then allowed to equilibrate for at last an h before use. After the sample was introduced to the column, solvent with increasing polarity gradient was used to elute the chemical compounds from the column. Fractions collected were monitored by TLC and appropriate fractions were combined and where necessary subjected to further separation.

3.9.3 Thin Layer Chromatography (TLC)

Thin layer chromatography (TLC) was routinely used to detect and separate the various compounds. The extracts from chromatography was examined by TLC using

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precoated 20 x 20 cm glass plates, 0.25 mm thickness, silica gel F254 (Merck, Darmstadt, G.F.R). The TLC plates were spotted with a piece of fine glass capillary tube and then developed in saturated chromatography tanks with various solvent systems at room temperature. The spots were visualized by examination of the TLC plates under UV light (254 and/or 343 nm), followed by applying iodine vapour or spray reagent.

3.9.4 Preparative-Thin Layer Chromatography (Prep-TLC)

Preparative-Thin Layer Chromatography (prep-TLC) was used in the purification of compounds. The samples were introduced onto the plates as a continuous streak 2 cm above the base of the plates using capillary tubes. The plates were developed in a chromatographic tank saturated with the developing solvent at room temperature. The plates were then air-dried, and the desired band was scrapped out and extracted with a suitable solvent.

3.9.5 Identification of compounds in hexane extracts of P. bleo and P. grandifolia using GCMS

The hexane extracts of both Pereskia spp. showed strong antioxidant activities in the antioxidant screenings (Section 4.2). Thus, the hexane extracts of both Pereskia spp. were subjected to GCMS analysis to identify the chemical constituents in the extracts.

109 3.9.6 Extraction and isolation of chemical constituents from the bioactive ethyl acetate extract of P. bleo

Based on the results of bioactivity screenings, such as antioxidant activity (Section 4.2), antimicrobial activity (Section 4.3) and preliminary cytotoxicity screening (Section 4.4), the ethyl acetate extract of P. bleo was identified as the bioactive extract. The cell death elicited by the ethyl acetate extract also clearly demonstrated DNA fragmentation, indicating apoptotic cell death as the major mechanism involved (Section 4.5.2). In addition, LUX RT-qPCR analysis also showed that the apoptosis elicited by the ethyl acetate extract on KB cells was mediated largely via p53 although the role of caspase-3 and c-myc cannot be ruled out [Section 4.6.5 (i)]. Thus, further chemical investigation was directed to the ethyl acetate extract of P. bleo.

The extraction and fractionation procedures leading to the isolation of compounds is shown in Figure 3.4 and 3.5. Firstly, dried, ground leaves of P. bleo (1050.56 g) were extracted (3x) with methanol (1.5 L each time). The methanol- containing extract obtained was initially treated with charcoal, then filtered over Celite® and the filtrate was evaporated under reduced pressure to give a crude methanolic extract (99.44 g). Treatment with charcoal was necessary to remove the high content of chlorophyll present in the extract. The presence of chlorophyll interfered with efforts at chromatographic separation.

The crude methanol extract was then further partitioned with ethyl acetate and water using a separating funnel. The ethyl acetate-soluble layer was concentrated in vacuo giving an 18.34 g ethyl acetate fraction, which was subjected to flash silica gel column chromatography over Merck Kieselgel 60 (0.063-0.200 mm mesh size); elution starting with chloroform (10 L), and then with chloroform-methanol [9:1 (9 L)] and finally methanol (7.6 L). The chloroform (X), chloroform-methanol (Y) and methanol eluents (Z) were concentrated to give 4.89 g, 12.70 g and 3.04 g respectively, and then

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subjected to cytotoxicity screening. Further purification was directed to the fraction X as it was found to be active in the cytotoxicity screening.

The active chloroform fraction (X; 3.47 g) which was a dark brown residue obtained, was then purified over a column (3 cm diameter x 49 cm height) packed with 180 g of Merck Kieselgel 60 (0.063 - 0.200 mm mesh size) and the components were separated by successive elution, initially with hexane followed by hexane enriched with increasing percentages of dichloromethane, monitoring with TLC, gave several sub- fractions (A- L).

Compound 1 (21 mg) was isolated from the sub-fraction B [dichloromethane- hexane (1:9) fraction, 192.7 mg], after further purification using prep-TLC (preparative-TLC) with ethyl acetate: hexane (1:9) as developing solvent system. Repeated prep-TLC on the sub-fraction F [dichloromethane-hexane (1.6:8.4) fraction, 64.2 mg], using chloroformas the developing solvent yielded compound 2 (10.6 mg).

Subsequently, further elution of X fraction with dichloromethane-hexane (6:4) yielded sub-fraction G (475.5 mg), which was then subjected to silica gel column chromatography over Merck Kieselgel 60 (0.063-0.200 mm size mesh size), eluting initially with hexane, followed by hexane enriched with increasing percentages of dichloromethane. Compound 3 (41.2 mg) was isolated from the fraction obtained upon elution with dichloromethane-hexane (1.6: 8.4) on silica gel column.

Compound 4 (39.6 mg) and mixture A (20.5 mg) were obtained from sub- fraction I upon elution of X fraction with dichloromethane-hexane (8: 2). Lastly, elution with dichloromethane yielded sub-fraction J (206.7 mg) containing compound