Submitted : 26-08-2022 Revised : 18-10-2022 Accepted : 28-11-2022 Trad.
Med.
September-December 2022 Vol.
, p 210-217 ISSN-p : 1410-5918 ISSN-e : 2406-9086 Isolation and Identification of -sitosterol, 7-hydroxystigmast-22-en3,6-dione and 3, 24(S)-dihydroxycholesta-5, 25-diene-7-one from stem bark of Nauclea pobeguinii Adewusi John Adepoju1.
Sunday Olusegun Oladoye1*.
Ezekiel Temidayo Ayodele 1.
Adeola Victoria Falade1.
Gin Keat Lim2, and Chuan Wei OO2 1 Organic Chemistry Unit.
Department of Pure and Applied Chemistry.
Ladoke Akintola University of Technology.
Ogbomoso.
Oyo State.
Nigeria 2 School of Chemical Sciences.
Universiti Sains Malaysia, 11800 USM.
Pulau Pinang.
Malaysia
ABSTRACT
The stem bark of the Nauclea pobeguinii was collected, air-dried, and pulverized and was extracted with solvent of varying polarity .
-hexane, ethyl acetate, and ethano.
to obtain the crude extracts.
Silica gel column and thin layer chromatographic separation afforded three compounds whose structures were elucidated as -sitosterol .
, 7-hydroxystigmast-22-en-3.
6-dione .
, and 3, 24(S)-dihydroxycholesta-5, 25-dien-7-one .
by analysis of their chemical and spectral characteristic from 1D and 2D NMR.
FTIR and by comparing of data with those reported in the literature.
Keywords: lea pobeguinii.
Phytochemical.
Isolation.
-sitosterol.
INTRODUCTION
Over the years, bioactive compounds from plants have been investigated as potential alternative therapeutic options in the management of diseases and infections.
The attention on the medicinal plant is due to the presence of a large number of secondary metabolites with their novel mechanism of action and safety compared to synthetic drugs (Ashour et al.
, 2.
Nauclea species belong to the family Rubiaceae, which is the largest family of woody plants consisting of more than 13,000 species, the family is divided into sub-families including the Cinchonoideae which consist of the genus Nauclea that are often found in a tropical area, such as Africa and Asia (Haudecoeur et al.
, 2.
Nauclea pobeguinii (Hua ex Poby.
Merr.
locally called Opepe ira in western Nigeria is a deciduous shrub that is endemic to the swamp forest regions of the The tree grows to a height of about 30 m high and produces soft yellow sponge-like fruits.
has reportedly been useful in the treatment of jaundice, gonorrhea, fever, stomach ache, and epilepsy (Njoya et al.
, 2.
In Cameroon, the plant is used in the management of hyperglycemia, articular pain, stomach pains, and inflammation (Tsafack et al.
, 2.
Anti-helminthic application of stem bark water decoction of N.
diderrichi and N.
pobeguinii were also reported in Congo while its anti-malaria activity was recorded in Mali (Haudecoeur et al.
, 2.
Anti-abortifacient and the ability to treat sexual and reproductive dysfunction properties of N.
pobeguinii have also been reported in works of literature (Luzakibanza Methanolic bark and leaf extract of N.
pobeguinii was also reported to possess antiproliferative activity on cancer lines.
Phytochemical screening of the plant revealed the presence of alkaloids, saponins, tannins, phenols, flavonoids, and terpenoids (Adepoju et al.
, 2.
Numerous kinds of literature correlate the ethno-medicinal uses of N.
pobeguinii to their alkaloid content, including the alkaloid-Strictosamide responsible for its anti-proliferative and antimalarial activity.
Other phytochemicals previously isolated from N.
pobeguinii include magniflorine, augustine, strictosidine (Xu et al.
This study is aimed at further isolation and characterization of bioactive components from N.
pobeguinii stem bark.
METHODOLOGY
Materials Organic solvents used .
-hexane, ethyl acetate, chloroform, methanol, and ethano.
were purchased from Sigma-Aldrich Laborchemikalien GmbH.
Silica gel for Column Chromatography (Merck Kieselgel 60 PF253 Art No.
1000 and 1000 with the particle size 0.
063 - 0.
200 mm 040 - 0.
063 mm, respectivel.
, and silica gel .
Aluminium sheet were obtained from Merck.
Darmstadt.
Germany.
Other equipments used include a Rotary evaporator.
Ultraviolet lamp .
and 366 n.
model UVGL-58.
San Gabriel.
IR spectra were recorded on a Perkin AeElmer FTIR .
spectrum BX spectrometer, using Attenuated total reflectance (ATR) and absorption bands were measured in cm-1.
Proton Nuclear DOI: 10.
22146/mot.
77324 | Traditional Medicine Journal, 27.
, 2022 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.
0 International License.
Isolation and Identification of -sitosterol, 7-hydroxystigmast-22-en-3,6-dione Magnetic Resonance .
H-NMR), 13C Nuclear Magnetic Resonance .
C NMR), and 2D-NMR spectra were obtained with Bruker- FT-NMR Spectrometer .
MH.
Chemical shift () was recorded in ppm relative to tetramethylsilane signal (TMS) and Deuterated (CDCl.
solvents were The signals were described in terms of chemical shift with appropriate abbreviations for multiplicities such as s .
, d .
, t .
, q .
, and m .
Commercial silica gel 60 PF253 Art No 7734.
1000 and 1000 with particle size 0.
063 - 0.
200 mm and 040 Ae 0.
063 mm, respectively was applied for column chromatography (CC) and precoated silica gel plates .
F25 Merc.
were used for analytical thin layer chromatography (TLC).
Fractions were monitored by TLC and spots were detected under Ultra Violet (UV) light .
or 366 n.
Collection and preparation of plant material The stem bark of N.
pobegunii was collected from Forest Research Institute of Nigeria (FRIN).
Jericho.
Ibadan.
Oyo State.
South West.
Nigeria, it was identified and authenticated with the herbarium number FHI 108529.
The plant material was air-dried for one month, after which it was pulverized with an electrical blender and stored in a moisture-free container till further analysis.
Extraction of Phytochemicals Extraction was done by continuously soaking the dried ground stem bark .
with nhexane .
5 L), ethyl acetate .
0 L), and ethanol .
5 L).
Successive extraction was adopted by using n-hexane first to remove non-polar organic compounds, waxes, and fats.
The sample was drained out after one week.
This process was followed by extraction with ethyl acetate and ethanol to remove more polar compounds.
Each extract obtained was filtered and evaporated to dryness using a rotary evaporator at 40 AC to yield ethyl acetate .
and ethanolic .
extracts (Hesham et al.
, 2016.
Njoya et al.
Isolation of Phytochemicals from N.
crude extracts Ethyl acetate extract (EAS) .
was dissolved in chloroform, it was mixed with 20 g of silica gel, and homogenized in a beaker to form a loose powder.
This powder was loaded on a silica gel column and eluted with n-hexane, nhexane/chloroform, chloroform/methanol, and methanol to yield a total of 70 fractions of 100 mL Fraction 7 (EAS .
(CHCl3 100%) gave white powder on evaporation but was not pure when viewed under a UV lamp.
It was further purified Traditional Medicine Journal, 27.
, 2022 using column chromatography with the mobile phase of increasing polarity as follows.
n-hexane 100%, n-hexane/CHCl3 9:1, n-hexane/CHCl3 8:2, nhexane/CHCl37:3, n-hexane/CHCl3 6:4, nhexane/CHCl3 5:5, n-hexane /CHCl3 4:6, nhexane/CHCl3 3:7, n-hexane /CHCl3 2:8, nhexane/CHCl3 1:9.
CHCl3 100%.
A total of 82 fractions .
mL) each were obtained.
Fractions 51 Ae 55 (Hex.
CHCl3 3:.
formed white powder on The isolated compound showed a dark blue spot on the TLC plate when viewed under the UV lamp at 254 nm.
It was labeled compound 1 and the spectroscopic data was recorded.
Spectroscopic data suggested compound 1 to be sitosterol.
Fractions 3-6 (EAS 3-.
were combined and re-chromatographed on a silica gel column eluted with chloroform/methanol mixture to give 50 fractions of 30 mL each.
Fractions 11 and 12 gave similar TLC profiles and were therefore combined and concentrated.
The resulting yellow amorphous solid was labeled compound 2 and the spectroscopic data was recorded.
The data suggested compound 2 to be 7-hydroxystigmast22-en-3,6-dione.
Compound 3 was obtained from chromatographic fractionation of 6.
0 g of ethanolic extract (ETS) of N.
pobeguinii eluted with n-hexane, n-hexane/chloroform, chloroform/methanol, and methanol to give 114 fractions of 100 mL each.
Fractions 46 Ae 50 (CHCl3 100%) were pooled together based on their TLC profile, formed white powder, and coded as ETS 46 Ae 50.
Fraction ETS 46 Ae 50 was further chromatographed with a mini silica gel column using n-hexane and chloroform, 25 fractions of 30 mL each were collected.
Fractions 14-17 (ETS 14 Ae .
were combined based on their TLC profile.
This fraction was further purified with a silica gel column with nhexane/chloroform 2:8 as the mobile phase.
fractions of 30 mL each were collected and fraction 11 appeared as a single spot on the TLC plate.
This fraction was concentrated to obtain a white powder labeled as compound 3.
The spectroscopic data suggested compound 3 to be 3, 24(S)dihydroxycholesta-5,25-dien-7-one.
RESULTS AND DISCUSSION
Three compounds were successfully isolated and characterized from the ethyl acetate and ethanol stem bark extracts of N.
Structural elucidation of the compounds was done by spectroscopic techniques such as FTIR, 1H-NMR, 13C-NMR.
DEPT-135.
COSY.
HSQC, and a comparison of spectral data with those reported in The characterizations of the three compounds are therefore described as follows.
Adepoju Adewusi John Table I.
13C and 1H chemical shift values for -sitosterol recorded in CDCl3 .
MH.
Carbon no C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
Dept
CH2
CH2
CH(OH)
CH2
QC(=)
CH(=)
CH2
CH2
CH2
CH2
CH2
CH3
CH3
CH3
CH2
CH3
CH3
CH3
CH2
CH3
13C *
The chemical shift values (, pp.
were compared with what was obtained by Ododo et al.
, 2016
Assignments were made based on COSY and HSQC correlations.
Characterization of compound 1 (EAS51-.
Compound 1 .
hite powde.
with an Rf value of 0.
:7, n-hexane/CHCl.
which was positive to the Liebermann- Burchard test gave a violet-blue color that later turned green indicating its steroidal nature.
Further analysis by FTIR (Suppl.
revealed important absorption bands .
such as hydroxyl functional group (O - H) at 3430, alkyl chain at 2938 Ae 2848, olefinic band at 1655, cyclic methylene group (CH.
n at 1490, gemdimethyl (-CH(CH.
isopropyl at 1371 and C-OH of secondary alcohol at 1042.
The 1H AeNMR spectrum (Suppl.
2a and 2.
revealed the presence of fifty hydrogen atoms.
six methyls, eleven methylene, nine methines, and one hydroxyl proton as shown in Table I.
The two singlets at H 60 and H 0.
94 validate the presence of methyl protons H18 and H19 attached to the quaternary carbons C10 and C13, respectively.
Multiplet at H 48 was assigned to the H3 attached to the carbon .
that bears the OH group.
Also, the signal at H 5.
30 was assigned to the olefinic proton H6.
The 13C- NMR spectrum (Suppl.
exhibited 29 signals which were classified as six methyls, eleven methylene, nine methine, and three quaternary DEPT-135 spectrum (Suppl.
revealed 26 signals without signals for the three quaternary The main signals in the 13C-NMR spectrum are the signals at c 140.
77 and 121.
74 for the vinylic carbons C5 and C6 (C5 = C.
, respectively.
The signal at c 71.
83 was assigned to the carbon that is bonded to the hydroxyl group (C3 - OH).
The two angular methyl carbons C18 and C19 resonated at C 11.
99 and 21.
09, respectively.
COSY
spectrum (Suppl.
revealed the 1H Ae 1H correlation between H3/H1.
H3/H2.
H3/H4.
H6/H7, and H6/H4 while the HQSC spectrum (Suppl.
shows the correlation between carbon and protons attached to it.
There are correlation Traditional Medicine Journal, 27.
, 2022 Isolation and Identification of -sitosterol, 7-hydroxystigmast-22-en-3,6-dione Figure 1.
Structure of -sitosterol Table II.
13C -NMR and1H -NMR spectral values of 7-hydroxystigmast-22-en-3,6-dione compared with related stigmastane-3,6-dione in literature recorded in CDCl3 .
MH.
Carbon no C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
Dept
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
CH3
CH3
CH3
CH3
CH2
CH3
13C *
The chemical shift values (, pp.
were compared with what was obtained by Wei et al.
, 2004 previously.
Assignments were made based on COSY and HSQC correlations.
Traditional Medicine Journal, 27.
, 2022 Adepoju Adewusi John Figure 2.
Structure of 7-hydroxystigmast-22-en-3,6-dione Table i.
13C-NMR and 1H-NMR spectral values of 3, 24(S)-dihydroxylcholesta-5,25-dien-7-one compared with literature recorded in CDCl3 .
MH.
Carbon no C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
Dept
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
CH3
CH3
CH2
CH2
CH2
13C *
C-27
CH3
H, .
H, .
H, .
H,.
H) 27.
H) 93.
H) 83.
H) The chemical shift values (, pp.
were compared with what was obtained by Yang et al.
, 2011 previously.
Assignments were made based on COSY and HSQC correlations.
Traditional Medicine Journal, 27.
, 2022 Isolation and Identification of -sitosterol, 7-hydroxystigmast-22-en-3,6-dione between H18 Ae C18 at C 11.
H19 Ae C19 at C 09.
H6 Ae C6 at C 121.
74 and H3 Ae C3 at C 71.
When all these spectra data were put together and compared with the literature (Ododo et al.
, 2.
compound 1 was characterized as -sitosterol with molecular formula C29H50O (Figure .
Characterization of compound 2 (EAS11-.
Compound 2 was isolated as a yellow amorphous solid with an Rf value of 0.
CHCl.
which was also positive for the LiebermannBurchard test.
The IR absorption band .
(Suppl.
at 3489 is due to O Ae H stretching vibration, bands at 2922 Ae 2848 are due to stretching vibration of Ae CH3 and Ae CH2, 1714 is for the stretching vibration of carbonyl, band at 1655 showed the presence of carbon-carbon double bond (C = C), band at 1446 is due to cyclic methylene groups (CH.
n and band at 1371 are due to the presence of gem-dimethyl (- CH(CH.
The 1H- NMR spectrum (Suppl.
of compound 2 showed the presence of 46 protons, six high-intensity peaks which correspond to the methyl protons H18.
H26.
H27.
H29.
H21 and H19 at H 0.
6, 0.
75, 0.
76,0.
78, 0.
82 and 0.
The proton at H7 corresponds to the proton attached to the carbon that bears the hydroxyl group and resonated at H 4.
At H 5.
are the protons at H22 and H23 which revealed the presence of symmetric olefinic protons.
13C- NMR
of compound 2 showed a total of 29 signals classified as methyl, methylene, methine, and quaternary carbons.
DEPT-135 spectrum .
showed the presence of 24 signals instead of 25.
This is because of the two olefinic carbons C22 and C23 that overlapped at C 128.
The important signals in the 13C-NMR spectrum are at C 210.
15 which corresponded to C3 and C6.
The signal at C 128.
64 for the olefinic carbons C22 and C23.
The signal at C 64.
06 is due to C7 that bears the hydroxyl group.
Signal at C 54 and 13.
09 are for the two angular methyl carbons C18 and C19, respectively.
COSY spectrum (Suppl.
shows the correlation between H7/H8.
H22/H24.
H22/H20 through cross peak.
More so, the HSQC spectrum (Suppl.
reveals the correlation between carbons and protons.
There is correlation between H7 and C7 .
H22 and C22 C 128.
H18 and C18 C 11.
H19 and C19 C 13.
H5 and C5 C 56.
The HSQC spectrum also showed that C6 C 208.
17 and C3 C 210.
have no correlation with any proton which established the presence of two carbonyl carbons that are quaternary in the structure of compound Based on the spectra data of compound 2 and comparison with spectra data of stigmastane -3,6dione from literature (Wei et al.
, 2.
, compound Traditional Medicine Journal, 27.
, 2022 2 is proposed to be 7-hydroxystigmast-22-en-3,6dione.
The major differences in the spectra data of compound 2 and that of published data for stigmastane-3,6-dione are the chemical shifts for hydroxyl group at C7 and the double bond at C22 of the side chain.
Therefore, compound 2 was elucidated to be 7-hydroxystigmast-22-en-3,6dione with molecular formula C29H46O3.
Characterization of compound 3 (EAS.
Compound 3 with Rf value 0.
:8, nhexane/CHCl.
was isolated as a white powder which was positive for Liebermann- Burchard reagent confirming its steroidal or triterpenoid This was then validated by NMR spectra that the compound contains steroidal nucleus.
The IR spectrum (Suppl.
showed the important absorption bands .
Band at 3415 revealed O Ae H functional group, 1670 revealed the presence of conjugated carbonyl functional group (C=O), 1550 also revealed the presence of conjugated double bond, 2938 correspond to aliphatic or C-H stretching (CH.
, 1400 indicates the presence of CO in compound 3.
The proton NMR spectrum (Suppl.
showed the presence of 42 protons in the compound.
This spectrum also revealed four groups of methyl protons attached directly to the ring and the side chain .
ertiary methy.
of compound 3 at H 0.
68 (H.
H 0.
97 (H.
H 1.
(H.
and one doublet at H 1.
20 for H21.
multiplet at H 3.
46 is assigned to H3 that is directly attached to the carbon that bears the oxygen atom (C 70.
Also, a singlet at H 5.
65 corresponds to olefinic proton H6.
The Signal at H 4.
05 is also for the second oxygenated methine H24 while the doublet at H 5.
30 is assigned to the olefinic proton H26.
13C- NMR spectrum .
15a and 15.
showed the presence of 27 signals which include four methyl, eight methine, ten methylene, and five quaternary carbons.
13C spectrum revealed the signal of two carbons attached to oxygen .
at C 71.
02 (C.
and C 74.
02 (C.
two double bonds at C 170.
88, 122.
92 (C5.
and 95 (C.
, 120.
82 (C.
, one ketone C 198.
(C.
, two tertiary methyl C 10.
97 (C.
(C.
and two secondary methyl C 18.
41 (C.
66 (C.
DEPT-135 (Suppl.
revealed a total of 22 carbon atom signals.
Twelve signals on the positive side of the spectrum correspond to eight methine and four methyl carbons.
Ten signals were shown on the negative side corresponding to the number of methylene carbon.
COSY spectrum (Suppl.
revealed the correlation between H3/H2.
H3/H4.
H5/H4.
H24/H23.
H26/H27.
HSQC
spectrum (Suppl.
also revealed the correlation between carbons and protons in compound 3 as H18 and C18 at C 10.
H19 and C19 C Adepoju Adewusi John Figure 3.
Structure of 3, 24(S)-dihydroxycholesta-5,25-dien-7-one 84.
H3 and C3 C 71.
H6 and C6 C 122.
H26 and C26 C 126.
The spectrum also showed olefinic quaternary carbons without any correlation with protons C5.
C7, and C25 at C 88, 198.
92, and 139.
95, respectively.
The combined spectra data suggested that compound 3 is 3, 24-dihydroxycholesta-5,25-diene-7-one with molecular formula C27H42O3 which was further validated by comparison with the literature (Yang et al.
, 2.
to be 3, 24(S)-dihydroxycholesta5,25-dien-7-one.
CONCLUSION
Based on the chemical and spectral evidence and comparison of obtained results with literature data, the isolated compounds are identified as sitosterol .
, 7-hydroxystigmast-22-en-3,6-dione .
, and 3, 24(S)-dihydroxycholesta-5,25-dien-7one .
and to the best our knowledge compound 2 and 3 are being reported for the first time in N.
ACKNOWLEDGEMENTS
The authors acknowledge the financial support of the Tertiary Education Trust Funds (TETFun.
of Nigeria and facilities offered by the School of Chemical Sciences.
Universiti Sains Malaysia (USM).
Malaysia.
REFERENCES