View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by International Institute for Science, Technology and Education (IISTE): E-Journals Journal of Natural Sciences Research ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.8, No.15, 2018 www.iiste.org Phenolic Glycoside and Steroid from the Roots of Comberetum Paniculatum Fikre Mammo1 Milkyas Endale*2 Dagne Addisu*1 Birhanu Bekele1 1.Department of Chemistry, College of Natural and Computational Sciences, Hawassa University, P.O.Box 05, Hawassa, Ethiopia 2.Department of Applied Chemistry, Adama Science and Technology University, P.O.Box 1888, Adama, Ethiopia Abstract Combretum paniculatum has been used widely in ethnomedicine where its root is traditionally used around Kaffa Zone and other central highlands of the country for the treatment of various illnesses, including conjunctivitis, eye ailments, leprosy, chronic diarrhea, dysentery, flatulence, vomiting and liver. Phytochemical screening tests of the methanol root extract revealed the presence of steroids, alkaloids, terpenoids, phenols, flavonoids, saponins, tannins, glycosides and the absence of anthraquinones. Silica gel chromatographic separation of the methanol extract gave cholest-5-en-3-ol (1) and mutiniside (2) for the first time from the roots. Complete characterizations of the isolated compounds were done with the help of spectroscopic techniques (UV-Vis, IR, 1D NMR). Keywords: Combretum paniculatum, phytochemical screening,cholest-5-en-3-ol, mutiniside Introduction Medicinal plants represent a rich source of antimicrobial agents (Khaing, 2011).Majority of rural population in Ethiopia still uses traditional medicine for their healthcare need (Kassayeet al, 2006). In Ethiopia, Combretum paniculatumi grow in the warm, moist areas of Kaffa, Jimma, Wollega and Shewa Ethiopia, and flowers in January and February. The local name of this plant is “baggo” (KafiNoono), “baggii” (Afaan Oromo) and “baye” (Amharic)(Figure 1) (Getahun, 1976). The sap expressed from flowers is used to treat conjunctivitis and eye ailments. It is also externally applied to treat leprosy (Schmelzer, 2012). Fig 1.Combretum paniculatum (Baggo) [Photo taken by Birhanu Bekele, Nov, 2017] Materials and methods Instrumentation Column chromatographic separation was carried out on silica gel (230-400 mesh size, Merck). Thin layer chromatography was done on silica gel 60 F-254, 0.2 mm thick layer on aluminum sheets for detection of spots. The UV-Vis spectrum was recorded on UNICAM UV-300 double beam spectrophotometer using CHCl3 as internal standard. The IR absorption spectrum was determined by Shimadzu 440 instrument using KBr disk in the range of 500-4000cm-1. The 1H NMR, 13C NMR, DEPT-135, spectra were recorded using Bruker Avance 400MHz spectrometer using TMS as internal standard. Chemical shift values for all NMR data are reported in parts per million (ppm) relative to internal standard. All the chemicals used were analytical grade. Plant material collection and authentication The roots of Combretum paniculatum were collected in November, 2017 from natural forest of Bonga town around Barta River 730 km from Hawassa. The plant species was identified by botanist Mr. Seyoum Robo at Mizan-Tepi University. Preparation of plant extract Powdered roots (500g) were soaked with CH3OH for 72hr with occasional shaking. The extract was filtered and concentrated using rotary evaporator at 40oC to give brown crude (45.6g, 9.12% yield). 75 Journal of Natural Sciences Research ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.8, No.15, 2018 www.iiste.org Isolation and purification of compounds Crude extract (30g) was subjected to silica gel column chromatographic separation (150g silica gel) and eluted with increasing gradient of ethyl acetate in petroleum ether. A total of 31 fractions (each 50 mL) were collected. Out of the 31 fractions (petroleum ether/ethyl acetate) collected. Fractions 14-18 revealed single spot showing yellow spot under UV light having the Rf value of 0.42 in (90:10) petroleum ether/ethyl acetate solvent system. After concentrating, the solid material left was repeatedly washed with n-hexane to yield compound 1 (20mg). The remaining sample in column chromatography was further washed with increasing gradient of methanol in chloroform and a total of 29 fractions were collected. Fractions 12-16 revealed single spotwith Rf value of 0.35 in (90:10) chloroform/methanol solvent system. After concentrating, the solid was washed repeatedly with n-hexane to yield compound 2 (12mg). Preliminary phytochemical screening Tests for steroids (Liebermann-Burchard test) Each extract (100 mg) was shaken with chloroform in a test tube; few drops of acetic anhydride was added to the test tube and boiled in a water bath and rapidly cooled in iced water. Concentrated H2SO4 (2 mL) was added alongside of the test tube. Formation of a brown ring at the junction of two layers and turning the upper layer to green shows the presence of steroids (Joshi et al., 2013). Test for alkaloids (Dragendroff’s Test) Crude extract (300mg) was mixed with 2mL of concentrated hydrochloric acid. The mixture was then filtered and mixed with small amount of amyl alcohol at room temperature. Few drops of dragendroff's reagent (Solution of Potassium Bismuth Iodide) was added to the acid layer and a reddish brown precipitate was observed (Ganjewala et al., 2009). Test for tannins (Gelatin Test) Small quantity of the extract was mixed with water and heated on water bath. To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins (Saklani et al., 2012). Detection of phenols (Lead acetate test) Crude extract (5mg) was dissolved in 1 milliliter of distilled water and 3 mL of 5% lead acetate solution was added. A bulky white precipitates indicated the presence of phenols (Harborne et al 1998). Test for flavonoids (Alkaline Reagent Test) Few drops of sodium hydroxide solution was added to the extract and formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids (Saklani et al., 2012). Test for saponins (Froth Test) Crude extract (0.1g) was dissolved in 20mL of water shaken in a graduated cylinder for 15 minutes. Formation of 1cm layer of foam indicates the presence of saponins (Roopashree et al., 2008). Detection of glycosides Glacial acetic acid (2mL) and 3 drops of 5% ferric chloride were added to 0.5 mL of an aqueous solutionof plan extract. Then, 1 mL of concentrated sulphuric acid was added. Formation of brown ring at the interface indicated presence of glycosides. Test of terpenes Methanol extract (0.25g) was mixed with 2mL of CHCl3 and 30mL of concentrated H2SO4 was added carefully to form a layer. Reddish-brown coloration of the interface was inspected (Debjyoti, 1995). Test for anthraquinones Methanol extract (0.5g) was boiled with concentrated hydrochloric acid for few minutes in water bath and filtered. The filtrate was allowed to cool and equal volume of CHCl3 was added to it. Few drops of ammonia were added to the mixture and heated in water bath. Formation of rose-pink color was inspected (Sofowora, 1982). Results and Discussion Phytochemical screening test results The results from the phytochemical screening of the methanol extract revealed the presence of steroids, phytosterols, alkaloids, terpenoids, phenols, flavonoids, saponins, tannins, cardiac glycosides and the absence of anthraquinones. Structural elucidation of isolated compounds Compound 1 was obtained as a white crystalline solid (20mg) from the methanol extract with Rf value of 0.42 in petroleum ether/ethyl acetate (90:10) solvent system. The UV spectrum (Appendix 1) indicated maximum absorbance (λmax) (chloroform) at 298 and 260 nm which indicated the presence σ-π and π-π* transitions, respectively. IR spectrum revealed (Appendix 2) broad absorption at 3425 cm-1 attributed to hydroxyl moiety. The bands at 1632 cm-1 and 1050 cm-1 can be attributed to olefinic C=C stretch and carbon-oxygen (C-O) stretch, respectively. The bands at 1464 cm-1 and 1387cm-1 also represent in-plane bending or scissoring of methyelenes 76 Journal of Natural Sciences Research ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.8, No.15, 2018 www.iiste.org stretching vibrations and in-plane O-H bend, respectively. Moreover, intense peaks at 2850 cm-1and 2930 cm1 indicate methyelenes (sp2) and methyls (sp3) C-H stretching vibrations, respectively. The 1H-NMR spectrum (CDCl3, 400MHz) (Appendix 3) revealed peaks at δ5.35 (t, 1H,H-6) and δ3.53 (m, 1H, H-3) attributed to olefinic proton adjacent to methylene and oxygenated methine (C-3), respectively. Moreover, five methyl groups at δ 0.68 (s, 3H, H-18), δ0.93 (s, 3H, H-19), δ0.92 (dd, 3H, H-21), δ0.83 (d, 3H, H-26) and δ 0.84 (dd, 3H, H-27) were observed. Peak observed at δ2.27 is attributed to methyelene proton (H-4) adjacent to olefinic carbon. The 13C-NMR spectrum (CDCl3, 150 MHz) (Appendix 4) showed twenty seven carbon signals assigned to five methyl, eleven methylene, eight methine groups and three quaternary carbon signals. The peak at δ71.8 (C-3) indicates sp3 oxygenated methine whereas peaks at δ140.7 (C-5) and δ121.7(C-6) are attributed to olefinic carbons (C-5,6). The DEPT-135 spectrum (Appendix 5) supported the presence of five methyl groups at δ19.0(C-18), δ11.9(C-19), δ18.8 (C-21), δ19.8 (C-26) and δ11.9 (C-27). Eleven methylene groups at δ37.2 (C-1), δ31.5 (C-2), δ45.8 (C-4), δ29.7 (C-7), δ21.1 (C-11), δ39.8(C-12), δ24.3(C-15), δ26.0 (C-16), δ33.9 (C-22), δ28.3 (C-23) and δ19.4 (C-25). Eight methine groups at δ71.8 (C-3), δ121.7 (C-6), δ31.9 (C-8), δ50.1 (C-9), δ56.8 (C-14), δ56.0 (C-17), δ36.2 (C-20) and δ23.1 (C-24). The absence of peak at δ140.7(C-5), δ36.5 (C-10) and δ42.3 (C-13) in the DEPT-135 spectrum which were observed in the 13C-NMR spectrum also confirmed the presence of three quaternary carbon atoms in compound 1. Thus, based on the above spectral data and comparison with literature (74-76), the structure of compound 1 is similar to cholest-5-en-3-ol (Tesemma et al., 2013; Mekoya, 2007) (Figure 2, Table 1). Table 1.1H-NMR (CDCl3, 400 MHz), 13C-NMR and DEPT-135 (150MHz) spectral data compound 1 13 C-NMR DEPT-135 Literature Position 1H-NMR (Tesemma et al., 2013; Mekoya, 2007) 1 13 H-NMR C-NMR 1 37.2 37.2 37.2 2 31.5 31.5 31.6 3 3.53 (broad m,1H) 71.8 71.8 3.52 71.8 4 2.27 45.8 45.8 45.9 5 140.7 140.7 6 5.35 (t, 1H) 121.7 121.7 5.34 121.7 7 29.7 29.7 29.2 8 31.9 31.9 31.9 9 50.1 50.1 50.2 10 36.5 36.5 11 21.1 21.1 21.1 12 39.8 39.8 39.8 13 42.3 42.3 14 56.8 56.8 56.8 15 24.3 24.3 24.3 16 26.0 26.0 26.2 17 56.0 56.0 56.1 18 0.68 (s, 3H) 19.0 0.65 19.0 19 0.93 (s, 3H) 11.9 0.98 11.9 20 36.2 36.2 36.1 21 0.92 (dd, 3H) 18.8 0.91 18.7 22 33.9 33.9 33.9 23 28.3 28.3 28.2 24 23.1 21.1 23.1 25 1.83 19.4 19.4 19.4 26 0.83 (d, 3H) 19.8 19.8 0.84 19.8 27 0.84 (dd, 3H) 11.9 0.87 11.9 77 Journal of Natural Sciences Research ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.8, No.15, 2018 www.iiste.org 22 21 27 20 24 18 23 12 11 13 9 14 19 2 3 HO 17 15 8 5 26 16 1 10 25 7 4 6 Fig 2. Structure of compound 1 (cholest-5-en-3-ol) Compound 2 was obtained as a yellow amorphous powder (12mg) from the methanol extract with Rf value of 0.35 under chloroform/methanol (90:10) solvent system. The UV spectrum (Appendix 6) showed in absorbance peak (λmax) (methanol) at 345 nm attributed to π-π* conjugation. The IR spectrum (Appendix 7) showed sharp and broad absorption bands at 1740 cm-1 and 3406cm-1 attributed to ester carbonyl and hydroxyl functional groups, respectively. The bands at 2845 and 2924cm-1 also revealed the presence of methyelenes (sp2) and methyls (sp3) C-H stretching vibrations. The medium bands at 1679cm-1 and 1057cm-1 indicate the presence of aromatic C=C and C-O stretching vibrations, respectively. Moreover, the bands at 1443cm-1, 1364 cm-1 and 760 cm-1attributed to CH2 bend, symmetrical CH3and CH2out-of-plane bending, respectively. The 1H-NMR spectrum (DMSO-d6, 400HZ) (Appendix 8) showed signals for the hexyl chain including methyl protons at δ0.84 (t, 3H, H-6'), six methylene protons at δ1.79-1.97 (m, 2H, H-2'), 1.30-1.45 (m, 2H, H-3'), 1.17-1.22 (m, 2H, H-4'),1.13-1.15 (m, 2H, H-5'), while the signal for oxygenated methylene protons appeared at δ4.07 (t, 2H, H-1'). The signal for the methoxyl group was observed as singlet at δ3.99. The meta-coupled aromatic protons appeared at δ 7.54 (d, J=2.1 Hz, 1H, H-6) and 7.75 (d, J= 2.1 Hz, 1H, H-2). The signals of a glucopyranose moiety appeared at δ5.28 (d, J =7.4 HZ, 1H, H-1''), 3.48-3.50 (m, 1H, H-2''), 3.39-3.42 (m, 1H, H3''), 3.34-3.37 (m, 1H, H-4''), 3.43-3.46 (m, 1H, H-5'') and oxygenated methylene protons at δ3.60 (dd, J=11.5, 3.0 HZ, 1H, H-6'') and 3.70 (dd, J=11.5, 4.9 Hz, 1H, H-6''). The anomeric proton of the glucopyranose moiety appeared at at δ4.1. The 13C-NMR (DMSO-d6, 150HZ) (Appendix 9) and DEPT-135 (Appendix 10) spectra (Table 3) showed 20 carbon signals with two methyl at δ14.4 (C-6') and δ56.4 (C-8), six methylene at δ66.2 (C-1'), 31.2 (C-2'), 29.5 (C-3'), 29.2 (C-4'), 22.6 (C-5') and 62.1 (C-6a''), seven methine at δ112.3 (C-2), δ105.1 (C-6), δ102.2 (C-1''), δ73.7 (C-2''), δ79.5 (C-3''), δ70.0 (C-4'') and δ76.9 (C-5'') and five quaternary carbons. The most downfield signals appearing at δ160.2 attributed to the ester carbonyl, whereas the quaternary carbons appearing at δ154.1, 153.8 and 139.8 were assigned to the oxygenated aromatic carbons. The signal for the anomeric carbon of glucopyranose moiety appeared at δ102.5. Thus, based on the above spectral data the structure of compound 2 was found to be phenolic glucoside given trivial name mutiniside in literature (Ali et al 2009) (Figure 3). 78 Journal of Natural Sciences Research ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.8, No.15, 2018 Table 3.1H (DMSO-d6, 400MHz) and literature 1 H-NMR Position 1 2 3 4 5 6 7 8 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 13 www.iiste.org C-NMR (DMSO-d6, 150MHz) data of compound 2 and reported 13 7.75(d, J= 2.1 Hz, 1H) C-NMR DEPT-135 112.3 154..1 139.8 153.8 105.1 160.2 56.4 66.2 31.2 29.5 29.2 22.6 14.4 102.2 73.7 79.5 70.0 76.9 62.1 7.54(d, J= 2.1 Hz, 1H) 3.99(s) 4.07(t) 1.79-1.97 (m, 2H) 1.30-1.45 (m, 2H) 1.17-1.22 (m, 2H) 1.13-1.15 (m, 2H) 0.85(t, 3H) 5.28(d) 3.70 (dd, 1H) Literature (Ali et al 2009) 1 13 H-NMR C-NMR 128.5 7.32(d) 111.6 151.6 132.3 145.1 7.00(d) 105.1 166.2 3.82(s) 50.5 4.12(t) 68.1 1.78-1.81(m) 30.1 1.32-1.36(m) 29.2 1.18-1.20(m) 26.7 1.15-1.17(m) 22.5 0.9(t, 3H) 14.0 5.02(d) 101.5 3.48-3.50(m) 74.3 3.39-3.42(m) 77.0 3.34-3.37(m) 70.0 3.43-3.46(m) 76.5 3.6(dd) 62.0 105.1 56.4 66.24 29.5 102.2 73.5 70.0 76.9 62.1 O 3 8 H3C 1 4 7 O 2' 5' 4' CH3 6' 6 O HO 3' 1' 2 HO 5 6'' O 5'' 4'' O 1'' OH 3'' OH 2'' OH Fig 3. Structure of compound 2 Conclusion and Recommendation This study is one of the few attempts to isolate phytochemical constituents from the roots of Combretum paniculatum of Ethiopian flora. The qualitative preliminary phytochemical screening of the methanol roots extract revealed the presence of steroids, alkaloids, terpenoids, phenols, flavonoids, saponins, tannins, glycosides and absence of anthraquinones. Silica gel column chromatographic separation of the methanol extract gave cholest-5-en-3-ol (1) and mutiniside (2). In agreement with the previous study, the wide traditional use of the plant may be attributed to its rich steroids and phenolic constituents. The finding of these pharmacologically important secondary metabolites from root extracts brings the attention of experts to look more on the medicinal importance of the plant. To the best of our knowledge, this is the first report on the presence of such kinds of compounds in the root of Combretum paniculatum. Acknowledgement Birhanu Bekele acknowledges Hawassa University and NORAD Project for the grant support to successfully carry out the project. We thank Department of Chemistry, Addis Ababa University for access to NMR, IR and UV-Vis instruments. We are also grateful to botanist Mr. Seyoum Robo for identification of the plant material. 79 Journal of Natural Sciences Research ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.8, No.15, 2018 www.iiste.org Reference Ali, S., Yasmeen, S., Afza, N., Malik, A., Iqbal, L., Lateef, M., Riaz, N., Ashraf, M. (2009). Mutiniside, new antioxidant phenolic glucoside from Abutilonm uticum. J. Asian. Nat. Prod. Res., 11:457-464. Debjyoti, D. (1995).Biochemistry 8th Ed., Academic publishers, Kolkata, p 39. Ganjewala, D., and Dipita, B..(2009). Effect of leaf positions on total phenolics, flavonoids and proanthocyanidins content and Antioxidant Activities in Lantana camara. Journal of Scientific Research. 2:363-369. Getahun, A. 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