Triterpene saponins of Maesa lanceolata leaves

Chemical investigation of Maesa lanceolata leaves aqueous MeOH extract has led to the isolation of eight new triterpene glycosides identified as 16-oxo-28-hydroxyolean-12-ene 3-O - β- glucopyranosyl-(1''→6')-β-glucopyranoside 1 , 16α, 28-dihydroxyolean-12-ene 3 - O -β-[(6''- O - galloylglucopyranosyl-(1''→2')][β-glucopyranosyl-(1'''→6')]-β-glucopyranoside 2, 16α, 22α, 28-trihydroxyolean-12-ene 3-O -[β-glucopyranosyl-(


Introduction
Maesa lanceolata Forsk (Myrsinaceae) is a well known plant used in the Kenyan indigenous system of medicine for the treatment of helminthes and bacterial infections. 1 In the previous communications, 2,3,4,5 we reported the isolation of hydroxylated-1, 4-benzoquinone derivatives from the plant various parts.Phytochemical studies undertaken by different group of workers elsewhere on the plant have resulted in the isolation and identification of various acylated triterpene saponins based on oleanane skeleton. 6,7,8Recently, flavonol glycosides have been reported from the plant leaves. 9The present paper discusses the isolation, structural elucidation and antibacterial activities of triterpene saponins 1-8 from the aqueous methanol extract of the plant leaves.

Results and Discussion
The aqueous MeOH extract of M. lanceolata subjected to column chromatography using sephadex LH-20 and silica gel, and further purification by preparative HPLC afforded triterpene saponins 1-8 together with known compounds 9-17.
Compound 1 analyzed for C42H68O13Na (HRMS 803.3456, [M+Na] + ) was positive to Liebermann-Burchard test and Molish reaction suggesting a triterpene moiety.It exhibited hydroxyl (3430-3150 cm -1 ), carbonyl (1705 cm -1 ) and a tri-substituted olefinic bond (1642 cm -1 ) absorption bands in the IR spectrum.The broad band decoupled 13 C and DEPT NMR spectra afforded 42 signals accounted for by 7 methyls, 12 methylenes, 15 methines and 8 quartenary carbon atoms.The 1 H and 13 C NMR data (Tables 1 and 3) of compound 1 were closely related to those of schimperinone 10,11 except for the presence of peaks originating from the sugar units, a fact confirmed by 1 H NMR two anomeric proton signals at δ 4.70 (d, J=7.5 Hz) and 4.40 (d, J = 7.6 Hz) with corresponding δc 103.6 and 102.8, respectively in the 13 C NMR spectrum.Acid hydrolysis afforded glucose as the sugar residue confirmed by TLC and PC co-chromatography with authentic sample.The large coupling constants of the anomeric protons (J = 7.6 Hz and 7.5 Hz) indicated that the sugars were present in the β-configurations.The presence of schimperinone as the aglycone was confirmed by comparing the 1 H, 13 C NMR and MS with the literature data. 11Unequivocal information on the ring system and the substitution mode in 1 was established from the 1 H, 13 C NMR and EI-MS data.In the EI-MS spectrum, characteristic peaks at m/z 208 [C14H24O] + (25 %), 207 [C14H23O] + (21%) and 248 [C16H24O2] + (11%) inferred retro-Diels-Alder cleavage of olean-12-ene derivative possessing a hydroxyl group or a sugar unit in rings A/B and a keto group together with a terminal oxymethylene in rings D/E part of the molecule. 11,12,13nalysis of the 1 H NMR spectrum revealed the presence of a disaccharide unit at C-3 which was assigned equatorial orientation due to the fact that the geminal proton centred at δ 3.50 appeared as doublet of doublet (J=11.5 and 5.0 Hz) and was in axial position.This interpretation was facilitated by the HMBC spectrum which exhibited a cross-peak between glucose-H-1' (δ 4.70) with C-3 (δ 81.4).The protons attached to each signal observed in the 13 C spectrum was deduced by analysis of DEPT spectrum and this data, in combination with the 1 H NMR spectral data established the oxygenated methylene carbon up field in the 13 C NMR at δ 70.6 signifying a terminal CH2OH group. 14Similarly, the position of hydroxymethylene group was assigned at C-17 on the basis of HMBC cross-peaks between H-28 (δ 3.76) and C-16 (δ 214.0) and between H-18 (δ 2.40) and C-16/C-28 (δ 70.6).
These results together with fragmentation pattern from EI-MS data confirmed the presence of schimperinone derivative containing a disaccharide at C-3.In the 13 C NMR spectrum, one glucose C-6 was downfield shifted at δ 67.3, suggesting glycosylation of the inner glucose by the terminal one on C-6 hydroxyl, a fact corroborated by HMBC correlation between the glc-C-6' (δ 67.3) with H-1'' (glc, δ 4.40).Therefore, based on the above spectroscopic consideration, compound 1 was characterized as schimperinone 3 Compound 2, obtained as an amorphous colorless powder showed the presence of hydroxyl (3460-3100 cm -1 ), carbonyl (1708 cm -1 ), double bond (1644 cm -1 ) and ether linkage (1050, 1020 cm -1 ) in the IR spectrum.The 13 C NMR spectrum revealed 55 carbon signals (Me-x 7, -CH2-x 9, CHx 6, -C-x 6, -CH2-O-x 4, CH-Ox 15, C=CHx 1, aromatic C-C-H x 2, aromatic C-C-OH x3, -CO-O-x 1 and aromatic C= x 1).The 1 H NMR data were similar to those of 3β, 16α, 28-trihydroxyolean-12-ene 15,16 17 Acid hydrolysis afforded glucose as the sugar residue identified on the basis of TLC and PC co-chromatography with authentic sample as well as GC analysis.Similarly, the aglycone was identified as 3β,16α,28-trihydroxyolean-12-ene (primulagenin A) after comparing its NMR and MS data with those already reported for the compound. 10,11Information on the ring system and the substitution pattern on the aglycone was provided by the EI-MS spectrum which displayed characteristic peaks at m/z 207 [C14H23O] + and 250 [C16H26O2] + signifying retro-Diels-Alder cleavage typical of olean-12-ene possessing a hydroxyl substituent or sugar moiety in rings A/B and two other hydroxyls in rings D/E part of the molecule. 10This was further supported by daughter ions at m/z 219 (22%) and 201 (41%) (due to successive loss of CH2OH and H2O from the m/z 250 peak) and 189 (100 %).In the 1 H NMR spectrum, an oxymethine proton at δ 3.50 (dd, J=11.6, 4.3 Hz, H-3) was coupled to two other protons and from decoupling experiments, it was shown to be part of -CH2-CH-(O-glc)-C(CH3)2-CHsystem analogous to the C-2 to C-5 region in oleanane skeleton. 18This allowed the oligosaccharide attached to the aglycone to be assigned to C-3 where it is in equatorial configuration, a fact further supported by the HMBC cross-peak between H-1' (δ 4.72) and C-3 (δ 80.1), and confirmed by NOESY cross-peaks between H-3 and Me-23 (δ 1.14 s) and also in turn with H-5.
Similarly, the existence of hydroxyl groups at C-16 and C-28 were deduced from the HMBC correlations between the peaks at δ 2.65 (dd, J=14.7, 4.5 Hz, H-18) and C-16 (δ 70.4), and between H-16 (δ 4.40) and C-28 (δ 66.5), respectively.The configuration at C-16 was established from NOESY experiments due to spatial proximity observed between H-16 and H-15β.The structure of the sugar chain at C-3 on the aglycone was achieved using 2D-NMR experiments and the results allowed the sequential assignments of all proton resonances within each sugar residue.Similarly, HSQC experiment was used to correlate the protons with corresponding carbons and this allowed the assignment of interglycosidic linkages.In the 13 C NMR, glycosylation shifts were observed for C-2' (δ 82.4) and C-6' (δ 68.1) of the inner glucose, thus suggesting that the terminal glucose moieties were linked to primary glucose through 1''→2' and 1'''→6'' bonds, respectively.The foregoing evidence was confirmed by HMBC correlations between the glucose-H-1''' ( 4.42) and the glucose-C-6' ( 68.1) and between the glucose-H-1'' ( 4.56) with glucose-C-2'.Similarly, the long range correlation between glucose-CH2-6'' (δ 4.14 and 3.87) with the galloyl-C-7 (δ 168.1) allowed the localization of the galloyl residue on the second glucose-C-6''.
Thus, on the basis of spectroscopic data, compound 2 was concluded to be 16α, 28dihydroxyolean-12-ene Compound 3, isolated as colorless amorphous powder showed a sodiated molecular ion peak at m/z 967.4562 [M+Na] + in the positive electron spray ionization-MS corresponding to C48H80O18 Na formula.The 1 H NMR spectrum revealed the presence of seven tertiary methyl groups (δ 0.86, 0.88, 0.92, 0.96, 1.00, 1.15, and 1.35), one trisubstituted olefinic proton (δ 5.  3) showed signals for a pair of olefinic carbons (δ 123.5 and 144.6) and three anomeric carbons (δ 102.1, 101.3 and 100.8).On acid hydrolysis, glucose and rhamnose were identified as the sugar residues by TLC and PC co-chromatography with authentic sample and GC analysis.Similarly, the aglycone, 16α, 22α, 28-trihydroxy -12-oleanene was suggested after comparison of its spectroscopic data ( 1 H, 13 C and MS) with those already reported. 19The relative stereochemistry of the substituents on the aglycone was determined by NOESY experiments.The configuration of H-3 was assigned as α on the basis of correlations between H-3/H-5 and Me-23, and the inter-proton coupling constant ( 3.56, dd, J=11.5, 4.2 Hz).Cross-peak observed between H-18 and H-22 indicated close spatial proximity between the two protons.The other significant NOESY correlation was observed between H-15β/H-16.These NOESY results are consistent with the structure in which the hydroxyls at C-16 and C-22 are both α-oriented and the C-3 substituent has β-configuration. 20The spin systems for the sugars were assigned on the basis of spectroscopic evidences obtained from 1 H-1 H COSY and HSQC while the interglycosidic linkages were evaluated using 13 C NMR and HMBC experiments.In the HMBC spectrum, long-range couplings ( 3 JHOH) were observed between proton signals at δ 4.60 (glc-H-1'') and 4.50 (rha-H-1''') with carbon resonances at δ 81.1 (C-2') and δ 67.9 (glc-C-6'), respectively, thus suggesting the presence of 2',6'-disubstituted glucose bearing another glucose and rhamnose as terminal sugars.The signal at δ 81.1 attributable to C-2' of primary glucose indicated glucopyranosyl (12)-glucopyranosyl arrangement (as in sophorosyl) while that at δ 67.9 attributed to C-6' of the same glucose signified rhamnopyranosyl-(16)-glucopyranosyl moiety (as in rutinosyl). 21he 13 C NMR data for the trisaccharide were in agreement with [α-rhamnopyranosyl-(1'''6')][β-glucopyranosyl-(1''2')]-glucopyranosyl moiety. 21,22Thus, based on the above spectroscopic evidences, compound 3 was deduced to be 16α,22α,28-trihydroxyolean-12-ene-3- Compound 4 was obtained as a colorless amorphous powder from aqueous MeOH.Its ESI-MS quasimolecular ion peak at m/z 1117.3425[M+H] + and the 13 C NMR data in combination with distortionless enhancement by polarization transfer (DEPT 45 0 , 90 0 and 135 0 ) suggested a molecular formula of C55H88O23.The IR spectrum showed significant absorption peaks for hydroxyl (3450 cm -1 ), ester carbonyl (1736 cm -1 ), aldehyde (1711 cm -1 ) and double bond (1650 cm -1 ) groups.The 13 C NMR spectrum (Table 3) exhibited 55 carbons of which 30 were assigned to the aglycone part, two to the acetyl group and 23 to the saccharide moiety.The seven sp 3 tertiary carbon signals at δ 30.9, 28.6, 26.6, 19.9, 17.2, 16.5, 15.4 and the three sp 2 hybridized carbons at δ 203.3, 144.3 and 124.8, together with the information from 1 H NMR (seven methyl proton singlets and a broad triplet vinyl proton at  5.36), suggested that the aglycone possessed an olean-12-ene skeleton with an aldehyde group. 19The combined interpretation of 1 H and 13 C NMR aided by HSQC allowed association of most protons with the corresponding carbon signals, and by the HMBC spectrum, which was vital in connecting the various spin systems, the aglycone was suggested to be 3β, 22α-dihydroxyolean -12-en-28-al , a fact corroborated by the EIMS peaks at m/z 514.2. 19The position of the oligosaccharide unit on the aglycone was established from HMBC experiments to be attached glycosidically at C-3 and from spin decoupling experiment it was in β-configuration. 18The presence of key HMBC correlations between H-16 (δ 4.34)/ H-18 (δ 2.56) and C-22 (δ 74.4); between H-16/H-18/ H-22 (δ 5.21) and C-28 (δ 203.3), and between H-22 and the acetyl group (δ 170.2) unambiguously confirmed the disposition of hydroxyl, acetyl and aldehyde groups at C-16, C-22 and C-28, respectively on the aglycone, a fact further confirmed by NOESY plot (Fig. 1).Acid hydrolysis yielded glucose, rhamnose and arabinose as the sugar residues identified by TLC and PC cochromatography after comparison with authentic sugar samples and also by GC-analysis.This was further corroborated by the 1   , were consistent with arabinose moiety containing two glucose and a rhamnose units.The formation of these fragment ions as outlined in Fig. 2 showed that arabinose moiety was the innermost sugar unit while one glucose and rhamnose were present as terminal ones.
Acid hydrolysis afforded glucose as the main sugar residue confirmed by TLC and PC co-chromatography with an authentic sample and GC analysis. 28The sequence of the sugar chain at C-3 was determined by analysis of 13 C NMR and HMBC spectra.In the 13 C NMR spectra the downfield peaks at δ 83.4 (glc I-C-4') and 82.3 (glc I-C-2') in comparison to kaempferol triglycoside 29 suggested that the inner glucose is glycosidated at positions C-2 and C-4 by two other glucose molecules.This was further confirmed by the HMBC experiments which showed correlations between the anomeric protons of glucose at δ 4.63 (glc-II-H-1'') with the glucose I-C-2 (δ 82.3), thus suggesting glucopyranosyl-(1''→2')-glucopyranosyl bioside previously observed in kaempferol 7-O-rhamnopyranosylsophoroside. 21The other HMBC cross-peak between δ 4.56 (glc-III-H-1''') and δ 83.4 (glc-I-C-4') signified the glucopyranosyl-(1'''→4')glucopyranosyl arrangement. 29Thus, the accrued spectroscopic evidence suggested that the trisaccharide is [glucopyranosyl-(1''→2')][glucopyranosyl-(1'''→4')]-glucopyranosyl attached at C-3, confirmed by HMBC correlation between δ 4.63 (glc-I-H-1') and δ 82.9 (C-3).Therefore on the basis of spectroscopic analysis, the structure of compound 6 was concluded to be 16α,22αdiacetyl-21β-angeloyloleanane-13β:28-olide Compound 7, analyzed for C52H84O19 (m/z 1035.3921[M+Na] + ) exhibited hydroxyl (3430-3250 cm -1 ), carboxylic group (1722 cm -1 ), an olefinic moiety (1644cm -1 ) and glycosidic bond (1041 cm -1 ) absorption bands in the IR spectrum.Its 1 H and 13 C NMR data (Tables 2 and 3) closely resembled those of aesculioside IIc previously isolated from Aesculus pavia 30 with a notable difference being the saccharide unit in the former compound.Detailed NMR spectroscopic data analysis indicated that the aglycone possessed an angeloyl group, evidenced by characteristic 1 H NMR peaks at  5.84 (q, J=7.3Hz), 1.82 (d, J=7.1Hz) and 1.76 (s), a fact further supported by 13 C NMR data ( 168.9, 136.5, 127.9, 20.6 and 16.4). 30,31,32,33 Inthe NMR spectrum, the observed downfield chemical shift at  5.20 (d, J=10.2 Hz) in comparison to aesculioside Ic 30 suggested that the angeloyl moiety was attached at this position and on the basis of the HMBC correlation it was assignable to H-21.The proton exhibited cross-peaks with C-21 ( 77.8) and C-16 ( 69.7).The stereochemistry of the aglycone was established from NOESY experiments and vicinal coupling of key protons (see Fig. 2 and Table 2). 28Acid hydrolysis yielded xylose, glucose and rhamnose as the sugar residues identified by TLC and PC co-chromatography with authentic samples and confirmed by the 1 H NMR single proton resonances at  4.86 (d, J=6.9 Hz), 4.60 (d, J=7.6 Hz) and 4.52 (d, J=1.2 Hz), respectively.The attachment of the oligosaccharide unit at C-3 of the aglycone was suggested by the downfield shift of the 13 C NMR C-3 peak at  84.6 and confirmed by HMBC correlation between H-3 and xylose-C-1 ( 105.4).The relative configuration at C-3 was evident from the NOESY crosspeaks between H-3 and Me-23 and also in turn with H-5 of the aglycone, thus indicating the βconfiguration of the C-3 substituent.The sequence of the carbohydrate chain was established from the following HMBC correlations: xylose-C-2 ( 81.9) with glucose-H-1'' ( 4.60) and rhamnose-H-1''' ( 4.52) with glucose-C-6 ( 66.7), thus suggesting glucopyranosyl-(1''2')rhamnopyranosyl-(1'''6'')-xylopyranoside moiety.Thus, the structure of 7 was established as16 α, 22α, 28-trihydroxy-21β-angeloylolean-12-ene 3β Compound 8, an amorphous colorless powder, had a molecular formula of C54H86O20 determined from its sodiated HRESIMS peak at m/z 1077.4532[M+Na] + , 13 C NMR and DEPT data.The HRESIMS is 42 units higher than that of compound 7, implying the presence of an acetyl group in the compound, a fact confirmed by the IR absorption peak at 1735 cm -1 and the 1 H NMR signal at  2.00 (with corresponding 13 C NMR data at  171.1 and 23.5).In fact, the 1 H and 13 C NMR and MS data of the compound resembled those of 7, except for the additional peaks from the acetyl functional group, thus indicating the replacement of one hydroxyl group by the acetyl moiety.The downfield chemical shift at  5.02 (d, J=9.6 Hz) was assigned to H-22 based on the HMBC cross-peaks between H-22 and C-28 and in turn with C-16, indicating that the acetyl group was at C-22.Acid hydrolysis afforded the aglycone, barringtogenol C, identified by NMR and MS data and comparison with reference data; 34, 35 the three monosaccharides rhamnose, arabinose and galactose, were also identified by the same method as depicted for compound 7.The localization of the oligosaccharide on the aglycone was provided by the 13 C NMR (Table 3), which is in complete agreement with those reported for saponariosde A 36 and 3-O-β-D-glucuronopyranosyloleanic acid. 37Support for this was provided by HMBC correlation between arabinose anomeric proton at  4.70 (d, J=4.4 Hz) with the aglycone C-3 at  83.7.
The sequence of the oligosaccharide chain at C-3 was established by a combination of 13 C NMR, HSQC and HMBC experiments.From the completely assigned 13 C-NMR, the branched nature of the sugar moiety was evident, and the noticeable 13 C shift difference between individual sugar residues and model compounds 38 suggested that arabinose was the branched centre, while rhamnose and galactose were in terminal positions.In the HMBC spectrum, the following inter-residue correlations were observed: H-1''' of rhamnose with C-3' of arabinose and H-1'' of galactose with C-2' of arabinose, thus confirming the [β-galactopyranosyl-(1''2')][αrhamnopyranosyl-(1'''4')]-α-arabinopyranoside moiety.
The sugar arrangement was further supported by the fragmentation pattern observed in the ESI-MS spectrum (see experimental section).Thus, compound 8 was concluded to be 16α

Antibacterial activity
Biologically active compounds are responsible for plants resistance against bacteria, fungi, viruses and other pests and this is demonstrated by the antibacterial activities reported in this study.The MeOH extract and pure isolates were assayed using eight clinically isolated bacteria comprising of four Gram +Ve (Staphylococcus aureus, Bacilus subtilis, Streptococcus pneumoniae and Enterococcus faecalis (syn:Streptococcus faecalis) and four Gram -Ve ( Salmonella typhii, Vibro cholerae, Eschericia coli and Pseudomonas aeruginosa).The MeOH extract of the plant showed varying degree of antibacterial activities against the tested bacteria species (Table 4) with promising result being recorded for V. cholerae (inhibition zone (28±0.1 mm) compared to other bacteria.This was followed closely by S. typhii which gave an inhibition zone of 260.2 mm.The pathogens S. aureus, B. subtilis and Enterococcus faecalis were found to be moderately sensitive with inhibition zones of 22±0.3, 20±0.4 and 180.0 mm, respectively.These results were found to be comparable to the standard antibiotics gentamycin and streptomycin which were used as the reference drugs.The microorganisms' P. aeruginosa and E. coli were observed to be less susceptible with inhibition zone of 140.2 and 100.2 mm, respectively.The behaviors of E. coli and P. aeroginosa could be as a result of enzyme destroying or inactivating the bioactive phytoconstituents.The activities appeared to be broad spectrum because it was independent on the gram reaction.The minimum inhibitory concentration (MIC) of the extract, pure compounds and the standard antibiotics are shown in Table 5.The MIC values for the extract against the organisms ranged between 100 and 1000 µg/ml with the highest activity of 100µg/ml recorded for V. cholerae, while the value for S. typhii was 125µg/ml.The gram positive bacteria S. aureus and B. subtilis gave MIC values 200 and 250 μg/ml, respectively while E. faecalis and S. pneumoniae both exhibited a MIC value of 500 µg/ml.The gram -Ve P. aeruginosa showed gave a MIC value of 500 µg/ml while that for E. coli was 1000 μg/ml.
Out of the 17 compounds isolated only four (5, 6, 7 and 8) showed activities against five strains of the microorganisms tested.Compound 6 was the most active and its MIC values ranged between 62.5-200 μg/ml with the highest activity reported for V. cholerae (MIC value 62.5 μg/ml).The compound was also found to be fairly potent to both S. aureus and S. typhii with MIC values 125 and 100 μg/ml, respectively.Compound 7 and 8 were the other metabolites with encouraging activities against some of the microorganisms studied.In this respect, compound 7 was more active than compound 8 with promising results being reported for S. typhii and V. cholerae exhibiting MIC values of 100 and 125 μg/ml, respectively.Compound 7 also exhibited a MIC value of 200 μg/ml for both S. pneumoniae and E. faecalis.The compound was, however, not active to S. aureus, B. subtilis, E. coli and P. aeruginosa even at greater than 200 μg/ml concentration.Compound 8, on other hand afforded MIC values of 200 μg/ml against S. typhii, V. cholerae and B. subtilis.The rest of the microorganisms were inactive to the secondary metabolite.Compound 5 was only active to S. aureus with MIC value of 200 μg/ml.The gram negative were more susceptible to the extract and pure metabolites, thus the demonstrated activity against the tested bacteria provides scientific basis for the local usage of the plant in the treatment of cholera 6 .

Experimental Section
General.Optical rotations were measured with JASCO DIP-370 digital Polarimeter.Melting points were determined using a Gallenkamp melting apparatus and are uncorrected.The UV and IR data were recorded on PYE UNICAM SP8-150 UV/Vis spectrophotometer and Perkins-Elmer FTIR 600 series.The ESI-MS data were taken in LCQ and JOEL JMS-700 M station mass spectrometer, respectively.EI-MS data were obtained on a MAT 8200 A Varian Bremen instrument.The 1 H NMR data were taken in DMSO-d6 and CDCl3-DMSO-d6 on a Brucker Ultra-shield-500 spectrometer operating at 500MHz and 125 MHz.Preparative high performance liquid chromatography (HPLC) was done on a JASCO model PU-2080 HPLC system equipped with a shodex R1-101 refractive index detector and YMC-pack RP-18 column (150 x20mm i.d).

Plant material
The leaves of Maesa lanceolata were collected near Kapsoit Trading Centre along the Kisumu-Kericho highway, Kenya in February 2006.Voucher specimens (leaves, fruits and twigs) were identified after comparison with authentic sample at the Botany Department, University of Nairobi.

Minimum inhibitory concentration (MIC).
Minimum inhibitory concentration (MIC) of crude extract and pure isolates was determined using broth micro-dilution technique 41,42,43 .Stock solution of extract was two fold diluted with RPMI 1000-1 µg/ml (final volume=100 µl) and a final DMSO concentration ≤ 1%.Pure compounds were dissolved in DMSO and different concentrations ranging between 200 and1 µg/ml prepared.Approximately 2ml of the concentrate from each dilution was added to 20 mL of molten agar (Oxoid Ltd) and uniformly mixed in a sterile Petri dish, then allowed to settle.A volume of 100µl of inoculum suspension was added to each well with exception of the sterility control where sterile water was added to the well instead.The plates were incubated at 37 0 C for up to 48 h.MIC was taken as the lowest concentration of extract or pure compound which resulted in total inhibition of the bacterial growth.The effects of the standard antibiotics (gentamycin and streptomycin) were taken as positive controls.
Acid hydrolysis.Compounds 1-8, 11, 12, 14, 15 and 16, each 10 mg), each in a mixture of 8% HCl (1 mL) and MeOH (5 mL) were separately refluxed for 2 h at 100 0 C, after which the reaction mixture cooled.After cooling, the mixture was extracted with EtOAc saturated with water.The EtOAc layer evaporated and the aglycone analyzed by NMR and MS and also data compared with the relevant literature.The water residues were reduced in vacuo to dryness, dissolved in H2O (1 mL) and neutralized with NaOH.The neutralized products were subjected to silica TLC analysis (eluent: EtOAc-MeOH-H2O-HOAc, 6:2:1:1) and PC (eluents: n-BuOH-HOAc-H2O, 4:5:1 and C6H6-n-BuOH-H2O-pyridine, 1:5:3:3).The chromatograms were sprayed with aniline hydrogen phthalate followed by heating at 100 0 C. The sugars were identified after comparison of their Rf values with authentic samples.Further confirmation of the sugar residues was performed according to the known method 44 whereby the reaction mixture was evaporated under a stream of nitrogen.Each residue was dissolved in 1-(trimethylsilyl) imidazole and pyridine (0.2 mL), and the solution was stirred for 5 minutes.After drying the solution, the residue was partitioned between H2O and CHCl3.The CHCl3 layer was subjected to GC using a L-CP-chirasil val column (0.32mm x 25 m).Temperatures of the injector and detector were 200 0 C for both.A temperature gradient system was used for the oven, starting at 100 0 C for 1 min and increasing up to 180 0 C at a rate of 5 0 C/min.Peaks of the hydrolysates were detected by comparison with retention times of authentic samples after treatment with 1-(trimethylsilyl)imidazole in pyridine.
C NMR signal at  83.1 indicating a glycosidation shift was in addition to the earlier mentioned HMBC evidence for compound 5, which is suggestive of the C-3 linkage to the sugar moiety.H-3 correlated with Me-23 and H-5 of the aglycone nucleus in the NOESY spectrum indicating a β-configuration of the C-3 oligosaccharide group.

Table 5 .
Minimum inhibitory concentration (MIC, µg/ml for extract and pure Compounds)