Isolation of Sarracenin from Root Barks of Strychnos Spinosa

Isolation of Sarracenin from origin Barks of Strychnos Spinosa On isolation of Sarracenin from Root utters of Strychnos spinosa and its Antimicrobial Properties.A known iridoid, Sarracenin, was isolated from the root bark of Strychnos spinosa. Its structure was elucidated by 1D and 2D-NMR experiments, and comparison with report data. This is the first time it has been isolated from this species. The compound showed significant antimicrobial activities against Staphylococcus aureus, Streptococcus pyogenes, Shigella dysenteriae, Klebsiella pneumonia, Candida albicans Candida tropicalis, Candida thrusei, and Candida stellatoidea, respectively.The genus Strychnos (Loganiaceae), consist of virtually 75 accepted species found through break through the tropics and subtropical Africa 1. Strychnos spinosa is commonly known as kaffir orange, Spiny monkey orange or Natal orange. It is utilized variously in African traditionalistic medicine for ailments such as dropsy, earache, snakebite, f ever, elephantiasis, fever epilepsy and rheumatism 2. The isolation and structural elucidation of the iridoid sarracenin from the root bark of this plant and its antimicrobial action against Staphylococcus aureus, Streptococcus pyogenes, Shigella dysenteriae, Klebsiella pneumonia, Candida albicans Candida tropicalis, Candida thrusei, and Candida stellatoidea, respectively, is hereby reported.Results and Discussion. The compound was obtained as fine, transparent, needle shaped crystals. It was find out as Sarracenin use 1D and 2D-NMR experiments, and comparison with reported data 3, 4, 5. Some revisions to reported chemical transformation assignments 3, 4 based on our DEPTq135, H, H-COSY, HMBC, HSQC and NOESY data are suggested.Table 1.1HNMR Data of Sarracenin in CDCl3 ( in ppm, J in Hz) based on fig. 1ADEPT spectra DEPTq 135 spectrum gave the proton chemical shifts 166.77 (quaternary or methylene), 150.08 (methine or methyl), 112.32 (quaternary or methylene), 91.68 (methine or methyl), 88.13 (methine or methyl), 68.99 (methine or methyl), 51.42 (methine or methyl), 35.06 (quaternary or methylene), 32.26 (methine or methyl), 22.06 (methine or methyl), 18.70 (methine or methyl). The conclusion by Miles et al, 3 that signals at 35.1 and 22.1 are collectible to methine (C-5) and methylene (C-6), respectively, does not agree with our results otherwise, we agree with their 13 C conclusions. Similarly, Wang et al, 4 report of chemical shifts at 91.7 as quaternary, 112.3 as methine, 18.7 as quaternary and 166.8 as methyl (Table 2.) is at variance with their acknowledgments on HMQC and HMBC data.In addition, Wang et al, 4 reports 1HNMR (400 MHz, CDCl3) 5.78 (d, J = 1.6 Hz), 1.34 (d, J = 6.5Hz, 3 H) as signals for protons at C-1 and C- 10, respectively no protons are located at those positions (Figure 1A). It would front a different numbering scheme was employ, however, two different numberings were considered (Figures 1A 1B) neither agreed completely with Wan g et al, 4. Those assignments would appear to have distorted conclusions on 1H, 1H-COSY, HMBC and HMQC data (Tables 1 2).1HNMR (400 MHz, CDCl3) 7.46 (s, 1H), 5.79 (t, J = 1.9 Hz, 1H), 4.99 (dd, J = 3.5, 0.8 Hz, 1H), 4.22 (q, J = 6.5 Hz, 1H), 2.98 (ddt, J = 10.7, 4.0, 1.9 Hz, 1H), 2.44 2.31 (m, 1H), 1.68 (dddd, J = 10.0, 5.3, 2.9, 1.0 Hz, 2H), 1.35 (d, J = 6.5 Hz, 3H).Table 2.13C-NMR Data of Sarracenin in CDCl3 ( in ppm) based on fig. 1AKey * = Major areas with ascertained variation.It was noted that the compound contained 11 signals using13C-NMR and DEPT spectra, including two Me, one CH2, six CH, and two quaternary carbons. Analysis of boilersuit NMR spectroscopic data revealed the signals at1 2EFigure 1A. Numbering of Sarracenin as by Miles et al, 3 and present work, B Numbering as on www.chemspider.com6 C Copernican HMBC correlations, D Important NOESY correlations, E Important 1H-1HCOSY correlations.1HNMR (400 MHz, CDCl3, TMS)7.46 (s, 1 H), 5.79 (t, J = 1.9, 1 H), 4.99 (dd, J = 3.5, 0.8, 1 H), 4.22 (q, J = 6.5, 1 H), 3.76 (s, 3 H), 2.98 (ddt, J = 10.7, 4.0, 1.9, 1 H), 2.37, 1.68 (m, dddd, J = 10.0, 5.3, 2.9, 1.0, 2 H), 1.35 (d, J = 6.5, 3 H) 13CNMR and DEPT (100 MHz, CDCl3, TMS) 166.77 (ester C=O), 150.08 (olefinicCH), 112.32 (olefinic quaternary carbon), 91.68 (CH), 88.13 (CH), 68.99 (CH), 51.42 (ester OCH3), 35.06 (methylene), 32.26 (CH), 22.06 (allylic CH), 18.70 (CH3)Antimicrobial activityThe antimicrobial activities of sarracenin were assayed against some pathogenic microbes obtained from the Department of Medical Microbiology A.B.U. Teaching Hospital, Zaria, Nigeria. The compound showed significant antibacterial and antimycotic agent activities against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Salmonella typhi, Shigella dysenteriae, Pseudomonas aeroginosa, Klebsiella pnuemoniae, Candida tropicalis and Candida stellatoidea (Table 3.). This points sarracenin out as a major medicinal principle of Strychnos spinosa and lends j ustification to its use in traditional medicine.ExperimentalCollection of appoint MaterialThe root bark of Strychnos spinosa was salt away from Katsina-Ala, Benue State, Nigeria, in August, 2013. The bark was air-dried and their size reduced with the aid of a wooden mortar and pestle. fall and IsolationThe ground material (750 g) was macerated for 72 hours using 500 mL each of hexane, chloroform, ethyl ethanoate and methyl alcohol. Preliminary antimicrobial screening revealed the ethyl acetate extract to be most active against adjudicate microbes. Thus the ethyl acetate extract (10 g) was separated by Vacuum liquid chromatography. A in truth gentle gradient elution was used from hexane through to ethyl acetate. Thirty fractions (25 ml each) were collected and allowed to evaporate to or so half their initial volumes. Fine needles were observed in fractions 20-25. These were monitored by TLC on ethyl acetate- methanol (11) solvent system and plates visualized using iodine vapour. The combined needles (221 mg) were further purified using SephadexLH20 with methanol-ethyl acetate proportionality (11) as solvent. 1HNMR, 13CNMR and 2DNMR experiments were carried out on the purified compound using 30 mg. Its melting point was 123 1240C determined using Electro thermal IA 9300 (Gallenkhamp capillary melting point apparatus with a thermometer).Antimicrobial AssayThe compound (0.01 mg) was weighed and turn in DMSO (10 mL) to obtain a concentration of 10 g/mL (This would subsequently be used to determine the antimicrobial activities of the plant). Mueller Hinton and Sabouraud dextrose agar were used as growth media for the microbes. All the media were prepared jibe to the manufacturers instructions, sterilized at 121 oC for 15 min and were poured into sterilized petri dishes, allowed to cool and solidify. Disc airing method was used to screen the initial crude extracts. Sterilized media were seeded with a standard inoculum (0.1 ml) of test microbe, Mueller Hin ton for the bacteria and SDA for the fungi. The inoculum was spread evenly over the surface of the media using a sterile swab. A well (6 mm) was cut at the center of the inoculated medium using a standard cork-borer (6 mm diameter). Solution of the extract (0.1 mL) was introduced into each well of the inoculated medium. The inoculated media were incubated at 37 oC for 24 hours for bacteria and at 30 oC for 7 days for the fungi, after which plates were observed for zones of inhibition of growth.Minimum Inhibitory Concentration of the compound was determined using the broth dilution method. Minimum bactericidal concentration and minimum fungicidal concentration (MBC and MFC) were also carried out to determine whether the test microbes were killed or only inhibited. Ciprofloxacin, Fulcin and Fluconazole were used as positive controls.Table 3. Antimicrobial action of sarraceninKey S = Sensitive, R = Resistant, = (No turbidity) No colony growth, = MIC or MBC or MFC, + = (Turbid) Scant y colony growth, ++ = defy colony growth, +++ = Heavy colony growthReferencesSitrit, Y., Loison, S., Ninio R, et al. (October 2003). Characterization of monkey orange (Strychnos spinosa Lam.), a potential unused crop for arid regions. J. 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