Anthocleista vogelii Planch is a tree- and shrub-like plant of the genus Anthocleista in the Gentian family (Gentianaceae) and native mainly to tropical Africa, Madagascar and the Mascarene Islands. Anthocleista vogelii is commonly called “cabbage tree” and is a medicinal plant widely used in West Africa. The plant is traditionally used to treat various diseases such as diabetes mellitus, constipation, hernia, malaria, hypertension, hemorrhoidsO1 , syphilis, stomach aches and diabetes (Okorie, 1976; Olubomehin, Abo, & Ajaiyeoba, 2013).
Anthocleista vogelii is a medicinal plant with pharmacological activities such as laxative, analgesic, antiulcerogenic, antiplasmodial, antimicrobial, hypoglycaemic, antiobesity, anti-inflammatory, antitrypanosomal and spasmogenic activities (Anyanwu, Ur-Rehman, Onyeneke, & Rauf, 2015). A number of research on medicinal plants stops at the pharmacological activities of the extracts or fractions. This study sought to isolate and characterize compounds from Anthocleista vogelii root bark and O2 to investigate their pharmacological activities as it relates to their ethno- botanical O3 use.
RESULTS AND DISCUSSION
The n-butanol and chloroform fractions were concentrate were subjected to column chromatographic separation and purification on a PTLC to give Compounds 1 (7.2g) and 2 (3.9 g) respectively.
Figure 1: Structure of compound 1and 2 isolated from the root bark of A. vogelii Planch
Compound 1: The FT-IR data ?max : 2927.7, 2958.6, 2860.2 are C-H stretching vibrations, 1728.1 C=O of ester, 1276.8 and 1072.3 C-O of ester, 1461.9 of C=C of aromatic, 1579.6 C=N and 1124.4 C-N of aromatic amine, monosubstituted aromatic ring at 742.5,702.0, 651.9 cm?1 (Figure S1, Supporting Information).The 1H NMR spectrum of compound 1 (Table 1) exhibited two methyl groups at ?H 0.87 and 0.89 ppm, five methine protons at ?H 1.67, 7.52, 7.52, 7.68, and 7.69 ppm, eight methylene groups at ?H 1.18, 1.38, 1.29, 1.40, 1.41, 1.59, 1.91, 4.21 ppm (Figure S2, Supporting Information).
Consistent with the observations on 13C NMR spectrum with 17 carbon signals (Figure S3, Supporting Information), and DEPT-90 and -135 sub-spectra with two methyl, eight methylene, five methane, and four pyridine carbons (Figure S4 and S5, Supporting Information).
Further analysis of the HSQC data reveals a direct correlation of H-2, H-3´ and H-6´ (?H 7.52 and 7.68) to C2, C-3´ and C-6´ (?C 68.1, 124.1, and141.7) (Figure S6, Supporting Information). COSY data showed that there is a correlation between H-2 at ?H 4.66 and H-9 at ?H 1.67 indicating that the oxy-quaternary carbon is two bonds away from the methylene carbon at C-4 (?C 26.2) and the absence of further correlation is due that the underlying fact that the chemical environment of the carbon is bordered by oxo group and quaternary carbons (Figure S7, Supporting Information). In the key cross peak in the HMBC spectrum (Figure 2) (Figure S8, Supporting Information), the acetate unit is located at C-1´ of the isonicotinic acid methyl ester unit was verified by correlations from H-4, H-3´ and H-7´ (?H 4.21, 7.52 and 7.52) to C-1´ (?C 166.9).
Figure 2: Key HMBC (red curved arrows) and 1H?1H COSY (blue curved arrow) correlations of compounds 1 and 2.
In the NOESY spectrum (Figure 3) (Figure S9, Supporting Information), spatial correlations between H-2 at ?H 4.66 to H-3 at ?H 1.35, H-4 at ?H 1.35 and H-15 at ?H 1.35 indicated that H-11 and -15 are ?-oriented. The pyridine moiety of compound 1 (Isonicotinic acid 8-methyl-decyl ester) was confirmed by after acid hydrolysis. The structure of compound 1 is shown in Figure 1.
Figure 3: Key NOESY correlations of compounds 1
Sweroside, (4aS,5R,6S)-1-Oxo-5-vinyl-4,4a,5,6-tetrahydro-1H,3H-pyrano3,4-cpyran-6-yl ?-D-glucopyranoside (2): The FT-IR data ?max; 3417.6 O-H of alcohol and 1026.1 and 1001.0 C–O of the ?-D-glucopyranoside, C–H of alkane at 2914.2, 2061.8,C = O of ester absorption moved to lower wavenumbers due to conjugation at 1693.4, C = C of alkene at 1616.2, cm?1 (Figure S10, Supporting Information). 1H-NMR (DMSO-d) spectrum of compound 2 (Table 1) exhibited ten methane protons at ?H 2.69, 3.27, 3.27, 3.31, 3.44, 3.65, 3.76, 4.05, 4.67, 5.26, 5.31, 5.53, 5.54, and 7.58 ppm and four methylene protons at ?H 1.72 , 3.68, 4.35, 5.24 ppm (Figure S11, Supporting Information).
The 13C-NMR spectrum of compound 2 showed 16 signals; 10 carbon signals attributed to the aglycone part and 6 signals to the sugar moiety (Figure S12, Supporting Information). The DEPT-90 and -135 sub-spectra confirm the proton signals with four methylene and fourteen methine carbons (Figure S13 and S14, Supporting Information). The 1H-NMR spectrum showed a downfield doublet signal at ? 7.58 d, J = 7.4 Hz, indicating an oxyolefinic proton of the secoiridoids (Duke, 1985; Lim, 2014). Proton signal at ? 4.35,d, J = 6.5 was assigned to the methylene of C-12 by comparison with the data of Aberham, Pieri, Croom, Ellmerer, & Stuppner (2011) and Dwarika Prasad & Sati (2012). Also the two doublets at ? 5.50, d, J = 8.7, 9.4 Hz and ? 5.24, d, J = 9.4, 1.9 Hz that were assigned to two protons of a methylene group. The HSQC data indicated a direct correlation between 7.46 ,d, J = 2.4, 5.50 ,d, J = 8.7 and 9.4 to C-7 (151.4 ppm), C-11 (132.3 ppm) respectively(Figure S15, Supporting Information), while thepostion of the olefinic bond is confirmed by COSY data showing a correlation at H-7 (?H 7.46) and H-1 (?H 3.13) while the position of olefinic methylene hydrogen is verified by correction at H-12 (?H 4.35 ppm) and H-1 (?H 3.13 ppm) (Figure S16, Supporting Information). The HMBC data shows that the ?-D-glucopyranoside anomeric carbon is located at C-6 by the correlation between C-6(104.8 ppm) and H-2′ (?H 4.94 ppm) (Figure S17, Supporting Information) In addition, the sugar moiety includes resonances of an anomeric proton signal at ? 4.94, d, J = 8.0 Hz, together with five proton signals for the remaining ?-D-glucopyranoside protons. The NOESY spectral data are strongly in agreement with those reported for sweroside (Figure S18, Supporting Information) (De Oliveira et al., 2013; Devi Prasad et al., 2000).
Table 1. 1H and 13C (600 MHz) NMR Spectroscopic Data for Compounds 1 (Chloroform-d) and 2 (DMSO-d) (? in ppm, J in Hz)
?H (J in Hz)
?H (J in Hz)
1.31,dd,( 6.6, 4.4)
3.68 , dd,(8.8)
The results of screening C1 and C2 against lipase, ?-amylase and ?-glucosidase is shown in Table 2. Compound 1 was not active against PLE and ?-amylase, O4 but was moderately active against ?-glucosidase (IC50 = 40.28 ± 0.063 ?g/mL). Compound 2 had inhibitory activities against PLE and ?-glucosidase with IC50 values of 24.43 ± 0.096 and 10.28 ± 0.015 ?g/mL respectively, O5 but showed no activity against ?-amylase.
Table 2: Calculated IC50 values of lipase, ?-amylase and ?-glucosidase inhibitory activities
40.28 ± 0.063
24.43 ± 0.096
10.28 ± 0.015
0.068 ± 0.001
41.10 ± 0.031
2.59 ± 0.001
Values are mean ± SD, n = 3. NA- not applicable; (-) compounds did not inhibit 50 % of enzymes
In figure 4, the compound 1 significantly (p < 0.05) increased the feces O7 output compared to the normal and sodium picosulfate controls, but C2O8 had no significant changes when compared to the normal control, although, it was significantly (p < 0.05) decreased compared to the sodium picosulfate control. Dietary fat areO9 not directly absorbed by the intestine unless by the action of pancreatic lipase, and as such inhibitors of pancreatic lipases are proposed to function as antiobesity agents . Figure 4: Fecal output of rats after 8 h of administration of compounds Values are expressed as means ± SEM, * significantly different (p < 0.05) from control, # significantly different (p < 0.05) from sodium picosulfate. The compound 2O10 from A. vogelii should be explored as antiobesity agents for its activity against PLE. Also, the possibility of the use of C2O11 or its derivatives as an antidiabetic agent could be further researched as previous studies have shown that extracts of A. vogelii possessed ?-glucosidase inhibitory activity and by extension antidiabetic activity (Olubomehin et al., 2013). On the other hand, C1O12 acted as a potent laxative facilitating the excretion of feces from the animals and observation showed the feces were brown, mushy and non-uniform in texture. Thus, C1O13 might be the active ingredient in A. vogelii that made it popular among traditional healers in managing constipation. EXPERIMENTAL SECTION Plant material: The dried root bark of A. vogelii was harvested freshly from Umuekwune, Ngor-okpala, Imo State, Nigeria. The plant was authenticated by Dr.O14 Ihuma, J. O., Department of Biological Sciences, Bingham University, Karu, Nigeria. The plant was allocated voucher number (GA134-7421) and specimens were deposited.O15 Extraction and isolation: Powdered root bark of A. vogelii (10kg) was macerated in hexane (30L x 3) at room temperature. It was sieved using a muslin cloth and the marc was macerated in 20L methanol and water (80:20) repeated three times in 3 days. The resulting filtrates were concentrated to slurry using a rotary evaporator under reduced pressure at 40 oC. The slurry was equally divided into two portions (P1 and P2). P1 was extracted with water and partitioned with n-butanol (500 ml x 3) using separating funnel. The aqueous partition was laid aside and the n-butanol concentrate was subjected to column chromatography over silica gel eluting with CHCl3: EtOAc (100:0, 0:100), CHCl3: EtOH (100:0 to 10: 90) and EtOAc: EtOH (100: 0) to give many fractions 1-5, 3 spots; 4-5, 2 spots; 6-11, 0 spot; 12-41, 1 spot; 41-43, 0 spot. Compound 1 was found in 12-41 which was concentrated to give 7.2 g. P2 was acidified by adding 2 M H2SO4 and extracted with chloroform (500 ml x 3) as described by Harborne (1998) using a separating funnel. The chloroform partition was concentrated under reduced pressure included for column chromatography over silica gel eluting with n-hexane: EtOAc (100: 0, 90:10, 80:20, 70: 30) to give many fractions 1-2, 0 spots; 3-6, 3 spots; 7-14, 1 spot); 15-22, 3spots. Compound 2 O16 was from 7-14 which was concentrated to give 3.9 g. Compound 1, was isolated as a pale yellow viscus oil; UV (MeOH) ?max (log ?) 229 (1.395) nm; FTIR ?max 2927.7, 2958.6, 2860.2, 1728.1, 1461.9, 1380.9, 1276.81124.4, 1072.3, 742.5 cm?1 (Figure S1, Supporting Information); 1H and 13C NMR data, see Table 1; EIMS m/z 279.0 M+, establishing the molecular formula as C17H27NO2 (calcd for 277.402). Sweroside, (4aS,5R,6S)-1-Oxo-5-vinyl-4,4a,5,6-tetrahydro-1H,3H-pyrano3,4-cpyran-6-yl ?-D-glucopyranoside (2), is viscous light brown sweet smelling liquid; UV (MeOH) ?max (log ?) 230 (2.35) nm; FTIR ?max 3417.6, 2914.2, 2061.8, 1693.4, 1616.2, 1406.0, 1359.7, 1317.3, 1272.9, 1203.5, 1153.4, 1026.1, 1001.0, 900.7, 827.4 cm?1(Figure S10, Supporting Information); 1H and 13C NMR data, see Table 1; EIMS m/z 359.3 M+ (calcd for C16H22O9, 358.126). Pancreatic lipase inhibitory activity The inhibitory pancreatic lipase activity of the compounds were O17 measured using the method described by Kim, Jang, Kim, & Kim (2009) with some modifications. The enzyme buffer was made by adding 10 mM MOPS (morpholinepropanesulphonic acid) and 1 mM EDTA pH 6.8, while the assay buffer was Tris buffer (100 mM Tris-HC1 and 5 mM CaCl2, pH 7.0). To each well was added 164 ?l of assay buffer, 6 ?l pancreatic lipase solution (1mg/ml) from enzyme buffer, 20 ?l of either the compounds at different concentration (0, 2.35, 4.69, 9.38, 18.75, 37.5, 75, 150, and 300 ?g/ml) or orlistat and incubated for 10 min at 37°C. Then, 10 ?l substrate solution (10 mM p-NPB (p-nitrophenylbutyrateO18 ) in assay buffer) was added and incubated for 15 min at 37°C. The reaction was performed in triplicates and the absorbance was read at 405 nm using an ELISA reader. The Inhibition of lipase activity by the plant extracts wereO19 calculated from the formula below: Where A = activity without inhibitor; a = negative control without inhibitor, B = activity with inhibitor, and b = negative control with inhibitor. Alpha amylase O20 inhibitory activity The a-amylase inhibitory activity of compounds was carried out in a microtitre plate based on the starch-iodine test according to Xiao, Storms, & Tsang (2007). Exactly 25 µl of assay buffer, 0.02 M sodium phosphate buffer (pH 6.9 containing 6 mM sodium chloride), 20 µl of soluble starch (1%, w/v) and 20 µl of plant extracts/acarbose (0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 µg/ml) were incubated at 37°C for 5 min. Acarbose was used as a positive control at a concentration of 6 mg/ml. Then 15 µl of amylase solution (6 mg/ml) was placed into each reaction well and incubated for 15 min at 37°C. Thereafter, 20 ?l of 1 M HCl was added to stop the enzymatic reaction, and 100 ?l of iodine reagent (5 mM I2 and 5 mM KI) was added. The change in colour was noted and the absorbance was measured at 620 nm on a microplate reader. A dark-blue colour indicated the presence of starch (and a very active inhibitor); a yellow colour indicated the absence of starch (and inhibitor) while a brownish colour indicated partially degraded starch (and active/partially active inhibitor) in the reaction mixture. The calculation of percentage inhibition of alpha amylaseO21 : Alpha-glucosidase inhibitory activity Alpha-glucosidase inhibition was measured using adaptations of the procedures of Johnson, De Mejia, Fan, Lila, & Yousef (2013) and Kwon, Apostolidis, & Shetty (2008). To a 96-well plate in an orderly manner, 50uL of compound/acarbose, 50uL positive control, or 50uL reagent blank were added. Then, 100uL of a 1.0U/ml ?-glucosidase solution (in 0.1M sodium phosphate buffer, pH 6.9) was added. The plate was incubated at 25°C for 10 min. Then, 50uL of a 5 mM p-nitrophenyl-?-D-glucopyranoside solution (in 0.1M sodium phosphate buffer, pH 6.9) was injected into each well. The mixtures were incubated for 5 min at 25°C. The absorbance was recorded at 405nm before and after incubation. Percentage inhibition was calculated relative to the diabetes drug, acarbose, as the positive control, and to the negative control, which had 50uL of buffer solution in place of the compound. Laxative activity in vivo The compounds from A. vogelii were tested for laxative activity using the method reported by Capasso, Mascolo, Autore, & Romano, (1986). Thirty six O22 male Sprague-Dawley rats were placed into 6 groups (n=6). The rats were fasted O23 for 12 hours with water available ad libitum before the experiment. The rats in Group 1 (normal control) received only normal saline, Group 2 (standard drug) received 25mg/kg b.wt sodium picosulfate; Groups 3 and 4 received 25 and 50mg/kg b.w of compound 1 respectively; Groups 5 and 6 received 25 and 50mg/kg b.w of compound 2 respectively, and administration was orally using normal saline as the vehicle. The rats were placed in cages suitable for collection of fecesO24 after administration, and fecesO25 was O26 collected and weighed at the 8th hour. CONCLUSION ASSOCIATED CONTENT Supporting Information Supporting information including 1D and 2D NMR, EIMS, UV, and IR spectra of compounds 1 and 2 are available as supplementary material. AUTHOR INFORMATION Corresponding Authors. *Gabriel O. Anyanwu. Tel.- +2347030524887 Email- [email protected] or [email protected]; *Bamidele J. Okoli: Tel-+27767619418 Email- [email protected] ORCID Gabriel O. Anyanwu: 0000-0003-3110-2627 Bamidele J. Okoli: 0000-0001-7841-683X Author Contributions Kindly, include everyone's contribution. Acknowledgements This work was supported by The World Academy of Sciences (TWAS) and COMSATS Institute of Information Technology (CIIT), Pakistan under the 2014 CIIT-TWAS Sandwich Postgraduate Fellowship award to Mr.O27 Gabriel Anyanwu (FR number: 3240280470). Conflict of Interest The authors have declared that there is no conflict of interest. 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