Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 13, Number 1, April 2024 | Pages: 135-139 | DOI: 10.14421/biomedich.2024.131.135-139 ISSN 2540-9328 (online) Isolation of Novel 6-methylideneoxecane-3,4,5,7,8,9-hexol from the Leaves of Rauwolfia vomitoria, Apocynaceae Azibanasamesa D.C Owaba1,*, Raji O. Rafiu1, Arueniobebh Frank2, Darlington D. Eboh3, Samuel J. Bunu1 1Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria. 2Department of Pharmacognosy and Herbal Medicine, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria. 3Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria. Corresponding author* azibanasamesa@gmail.com Abstract Rauwolfia vomitoria (Wennberg) belongs to the family Apocynaceae, the dried leaves were extracted successively using n-hexane, dichloromethane, 70% methanol, and concentrated in vacuo. Extracts were subjected to antibacterial assay and the butanol fraction was subjected to chromatographic purification to obtain NBF12 which was subjected to spectral analysis. The antibacterial of n-Hexane, dichloromethane, and 70% methanol extracts was inactive against the screened organisms assessed (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa). NBF12 is yellowish crystals (10.5 mg), Rf (0.69), UV, max Abs (248) The carbon 13C-NMR spectrum displayed ten carbon atoms; one methylene, two methylene oxide carbon, 6-carbinolic carbon (Sp3) and a quaternary carbon (SP2). The spectrum showed a tertiary carbon at δ131.09 ppm, due to C-6 and at δ 114.74 ppm due to exocyclic methylidene carbon (Sp2) (C=CH2) linked to the C-6 position. Based on the spectral data NBF12 is 6-methylideneoxecane-3,4,5,7,8,9-hexol with a molecular formula C10H18O7, molecular weight 250 g/mol. Keywords: Isolation; Rauwolfia vomitoria; antibacterial; 6-methylideneoxecane-3,4,5,7,8,9-hexol. INTRODUCTION Rauwolfia vomitoria (Wennberg) belongs to the family Apocynaceae and is a medicinal plant used for the management of unknown pyrexia by the Ijaws of Southern Nigeria. This is due to its versatile utility in traditional medicine. It has been reported as an aphrodisiac, antimicrobial, antipsychosis, antihypertensive, antianxiety, and antioxidant effect (Etim et al., 2018; Emencheta et al., 2020; Oyeniran et al., 2020; Balogun and Akintunde, 2022). This medicinal plant has a battery of chemical constituents isolated and reported in the literature which includes; ursolic acid, sistosterol, stigmasterol, reserpine, reserpinine, deserpidine, ajmalicine, and ajmaline, 2,6- dimethoxybenzoquinone (Ajayi et al., 2021). The research aimed to determine the antibacterial effect and to characterize the chemical constituents of R. vomitoria leaves. METHOD AND MATERIALS Chemicals and Reagents Methanol (Sigma U.K), Dichloromethane (Sigma U.K), n-hexane (Sigma U.K), Ethyl acetate (Sigma U.K), Dimethylsulphoxide (JHD), Sephadex LH-20 (Sigma), Silica gel 200-400 (Sigma U.K). Equipment UV spectrophotometry, NMR spectrophotometer 400 MHz (Agilent) Microorganism Staphylococus aureus NCTC6571, Bacillus subtilis NCTC8236, Escherichia coli ATCC25922, Psuedomonas aeruginosa ATCC 10145 Sample Collection The leaves of R vomitoria were collected from the wild at Otabi Community in Oloibiri District, Ogbia Local Government Area, Bayelsa State, Nigeria. Identification of Plant The plant was identified and authenticated by Prof. A.T Oladele of the Department of Pharmacognosy and Herbal Medicine, Faculty of Pharmacy, Niger Delta University, and Herbarium specimen was deposited in the Herbarium of Pharmacognosy and Herbal Medicine and herbarium number (NDUP/24/01) was assigned to it. Manuscript received: 03 March, 2024. Revision accepted: 20 May, 2024. Published: 22 May, 2024. https://doi.org/10.14421/biomedich.2024.131.135-139 136 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 135-139 Plant Preparation The fresh leaves of the plant were washed and cut into small portions before air drying at room temperature for 14 days. The dried leaves were pulverized using the electrical blender to coarse powder, weighed, and stored in an airtight glass bottle. Antibacterial Evaluation of the Extracts The antibacterial evaluation of the extract was carried out according to (Balogun and Akintunde, 2022). Staphylococus aureus NCTC6571, Bacillus subtilis NCTC8236, Escherichia coli ATCC25922, Psuedomonas aeruginosa ATCC 10145. A loop full of bacteria strains was inoculated in Nutrient agar media and was incubated for 48 hours. 80 mL of sterile molten Muller Hinton Agar was transferred from the Mckonkey bottle to the Petri dishes and allowed to solidify. The bacteria strain was diluted equivalent to 0.5 Mcfarland standard in 5 mL of 0.9% normal saline and 0.03 mL of the active test strain was transferred to the solid media, swirled allowed to dry. Wells were bored on the agar using a sterile cork borer (9 mm), and the 100 mg/mL (0.1, 0.2, 0.3, and 0.4 mL) stock concentration of each of the extracts were prepared in dimethylsulphoxide and water in a ratio (3:7). 10, 20, 30, and 40 mg of each extract was used against the bacteria strain using Ciprofloxacin 5µg/disc as the standard drug and was incubated at 37oC for 24 hours (Emencheta et al., 2020; Balogun and Akintunde, 2022; Karim et al., 2023; Dhital et al., 2024). Isolation 6-methylideneoxecane-3,4,5,7,8,9-hexol The methanol extract weighing 25 g was suspended in 200 mL of distilled water and it was partitioned sequentially using n-hexane, ethyl acetate, and n-butanol and the extracts were concentrated in vacuo using a rotary evaporator. The n-butanol fraction weighed 2 g was subjected to gel filtration using Sephadex LH-20 in a column (1.5 cm x 86 cm) and eluted with methanol (100%), 10 ml of the eluate collected to a total of 21 fractions coded (NBF 1-21). Based on the TLC profile NBF9-12 weighed 0.146 g was subjected to column chromatography using Silica gel as stationary phase (30 g, 200-400) with a dimension (1.5cm x 86 cm) and gradiently eluted with ethyl acetate, (100%); 95:5; 90:10; 80:20; 70:30; 65:35; 60:40; 50;50; 30:70; 10:90 (Ethyl acetate: Methanol) to 100% methanol and the progress of elution monitored using TLC in a solvent system ethyl acetate: methanol: water (100:16.5:13.5), fraction 12 gave a single spot on TLC, and concentrated in vacuo to give yellowish crystals weighed 10.5 mg which was coded NBF12, subjected to spectroscopic analysis. RESULT AND DISCUSSION Antibacterial Antibacterial evaluation of the n-hexane, dichloromethane, and 70% methanol extracts were screened at 10, 20, 30, and 40 mg and showed that the extracts were devoid of antibacterial activity because there was no zone of inhibition compared to the standard drug ciprofloxacin 5μg/disc which showed zone of inhibition; 24 mm, 21 mm, 22 mm and 21.5 mm against S aureus, B subtilis, E coli and P aeruginosa respectively, as illustrated in Table (1, 2 and 3). The results are in line with the report by Balogun and Akintunde, 2022 (Emencheta et al., 2020; Balogun and Akintunde, 2022). Table 1. Antibacterial activity of n-hexane extract. Zone of Inhibition (mm) S/N Agent/conc. S. aureus B. subtilis E. coli P. aeruginosa 1 10 mg - - - - 2 20 mg - - - - 3 30 mg - - - - 4 40 mg - - - - 5 Ciprofloxacin 5µg 24 21 22 21.5 Keys - = No zones of inhibition Table 2. Antibacterial activity of dichloromethane extract. Zone of Inhibition (mm) S/N Agent/conc. S. aureus B. subtilis E. coli P. aeruginosa 1 10 mg - - - - 2 20 mg - - - - 3 30 mg - - - - 4 40 mg - - - - 5 Ciprofloxacin 5µg 24 21 22 21.5 Owaba et al. – Isolation of novel 6-methylideneoxecane-3,4,5,7,8,9-hexol … 137 Table 3. Antibacterial activity of 70 % methanol extract. Zone of Inhibition (mm) S/N Agent/conc. S. aureus B. subtilis E. coli P. aeruginosa 1 10 mg - - - - 2 20 mg - - - - 3 30 mg - - - - 4 40 mg - - - - 5 Ciprofloxacin 5µg 24 21 22 21.5 Chemistry of 6-methylideneoxecane-3,4,5,7,8,9-hexol (NMR Analysis) Figure 1. 1H-NMR of Sample NBF12 138 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 135-139 Figure 2. 13C-NMR of Sample NBF12 Yellowish crystals (10.5 mg), Rf (0.69), UV, max Abs (248),1H-NMR (CD3OD, 400 MHZ, δ, ppm); 6.1, (s, 2H), 4.87 (s, 6H, OH proton), 4.48 (s, 4H), 3.69-3.27 (m, 6H). 13C-NMR (CD3OD, 400 MHZ, δ ppm); 130.04 (C-6); 114.74 (=CH2), 73.62 (C-4; C-8), 72.41 (C-2; C-10), 70.82 (C-5; C-7), 63.23 and 62.94 (C-3; C-9) respectively. OH OH CH2 OH OH OH OH 9 4 10 3 O 1 2 8 7 5 6 Figure 3. Chemical structure of 6-methylideneoxecane-3,4,5,7,8,9-hexol. The proton NMR spectrum of sample NBF12 exhibited four prominent peaks. A downfield signal resonates at δ 6.17 ppm integrated for 2H and assigned to methylidene protons linked to C-6 due to the exocyclic, unsaturated (C=CH2) group. An intense peak at δ 4.87 due to carbinol hydroxyl protons at (C-3; C-5and C-7; C- 9) integrated for 6H protons and intense multiplet peak at δ 3.69-3.27 ppm due to methine (CH) of carbinolic proton at C-3-5; 7-9) positions integrated for six protons (6H) as shown in Figure 1. The carbon 13C-NMR spectrum displayed ten carbon atoms; one methylene, two methylene oxide carbon, 6- carbinolic carbon (Sp3), and a quaternary carbon (SP2). The spectrum showed a tertiary carbon at δ131.09 ppm, due to C-6 and at δ 114.74 ppm due to exocyclic methylidene carbon (Sp2) (C=CH2) linked to C-6 position, the signal at δ 73.62 due to C-4 & C-8 position. The signal at δ 72.42 ppm, due to (C-2 and C-10) methylene-oxide group, and are chemically equivalent. The carbinolic signal at δ70.82 ppm is due to C-5; C-7 and the signal at δ 63.23 & 62.94 ppm is due to C-3 and C-9 respectively as illustrated in Figure 2. Based on the foregoing, sample NBF12 proposed as 6- methylideneoxecane-3,4,5,7,8,9-hexol with a molecular formula C10H18O7, molecular weight 250 g/mol (Kamentani et al., 1995; Bubb, 2003; Kalsi 2004; Sharma et al., 2005; Azogu, 2010). This is the first time of reporting this compound in R. vomitoria (Figure 3). CONCLUSION The extract was not active against the microorganism assessed and isolation from the n-butanol gave a novel 6- Owaba et al. – Isolation of novel 6-methylideneoxecane-3,4,5,7,8,9-hexol … 139 methylideneoxecane-3,4,5,7,8,9-hexaol and the first time of reporting this compound from Rauwolfia vomitoria. Acknowledgment: The authors are grateful to Prof. Kola’ K. Ajibesin and Prof. Augustine A. Ahmadu for their technical assistance in carrying out this study. Author’s Contribution: ADCO and ROR designed the work, ADCO and AF collected the Sample, ADCO, ROR, and AF carried out the isolation while DDE carried out the antibacterial assessment of the extracts. SJB performed a critical review and manuscript proofreading. The manuscript was written by ADCO and all authors read and approved the final copy. Competing Interest: The Authors have no competing interests. REFERENCES Ajayi OA. (2021). Phytochemical and GCMS analysis of bioactive components in ethanolic extract of Rauvolfia vomitoria leaves. Journal of Chemical Society of Nigeria, 46(4): 0656-0660. Azogu CP. (2010). Laboratory Organic Chemistry. 2nd Edition. Maybinson Book Publishers, New Jersey, USA. 269p. Balogun OD and Akintude SL. (2022). Antimicrobial activities of Rauwolfia vomitoria against selected organisms. International Journal of Innovative and Advanced Studies, 9(3): 73-77. Bubb WA (2003). NMR Spectroscopy in the study of carbohydrates: characterizing the structural Complexity. Wiley Periodicals, Inc Concepts Magn Reson Part A:19A (1): 1-19, DOI: 10.1002/cmr.a.10080. Dhital S, Amatya SP, Aryal S, Neupane P, Tamang NPM, Thanait P. (2024). Synthesis of Manganese Oxide nanoparticles using co-precipitation method and its antimicrobial activity. International Journal of New Chemistry, 11 (3), 243-253. Emencheta SC, Enweani BI, Oli AN, Ibezim EC, Imanyikwa IEO. (2020). Antimicrobial Evaluation of Plants Parts of Rauwolfia vomitoria. Journal of Complementary and Alternative Medical Research, 12(1); 11-20. Etim E I, Johnson EC, Bassey US, and Nwafor PA. (2018). Phytochemical and aphrodisiac studies of ethanol root extract of Rauwolfia vomitoria Afzel (Apocynaceae), Journal of Pharmacy and Bioresources, 15(2): 160 – 165. Fannang SV, Kuete V, Mbazoa CD, Momo JI, Van-Dufat HT, Tillequin, F, Seguin E, Chosson, E, Wandji J. (2011). A new acylated triterpene with antimicrobial activity from the leaves of Rauvolfia vomitoria, Chemistry of Natural Compounds, 47 (3): 20-25. Kalsi PS. (2004). Spectroscopy of Organic Compounds. 3rd Edition. New Age International Publishers, New Delhi, India, 183p. Kamentani S, Mizuno H, Shiga Y, Akunuma H. (1995). NMR of all-Carbon-13 sugars: An application in the development of an analytical method for a novel natural sugar, 1,5- Anhydrofructose. Journal of Biochemistry 119, 180-185. Karim M, Khan S, Ullah I, Gamaryani A, Hasnain M, Abbas SH, Nawaz H, Saeed Z, Zafar I, Ur R, Saeed SY and Malik, MUA. (2023). Etiology and antibiotic resistant pattern of urinary tract bacterial pathogen in district Mardan. Biomed Journal of Scientific and Technical Research, 52(5): 44207-44211. DOI: 10.26717/BJSTR.2023.52.008330. Oyeniran OH, Ademiluyi AO, Oboh G. (2020). Phenolic constituents and inhibitory effects of the leaf of Rauvolfia vomitoria, Afzel on free radicals, cholinergic and monoaminergic enzymes in rat's brain in vitro, J Basic Clin Physiol Pharmacol; 32(5):987-994, doi: 10.1515/jbcpp-2020- 0144. Sharma YR. (2015). Elementary Organic Spectroscopy: Principles and Chemical Application. S. Chand and Company PVT. Ltd., New York, 162p. THIS PAGE INTENTIONALLY LEFT BLANK