Terpenoids of Boswellia neglecta oleo-gum resin

Article abstract of DOI:10.4314/bcse.v30i2.16

Oleo-gum resin exudate from Boswellia neglecta afforded a new ursane-type triterpene characterized as 3α-acetoxy-28-hydroxy-11-oxours-12-en-24-ioc acid (1) together with twelve known compounds. Their structural elucidation was accomplished using physical, chemical and spectroscopic methods.

Full text of DOI:10.4314/bcse.v30i2.16

Bull. Chem. Soc. Ethiop. 2016, 30(2), 317-323.
Printed in Ethiopia
DOI: http://dx.doi.org/10.4314/bcse.v30i2.16
ISSN 1011-3924
 2016 Chemical Society of Ethiopia
SHORT COMMUNICATION
TERPENOIDS OF BOSWELLIA NEGLECTA OLEO-GUM RESIN
Lawrence Onyango Arot Manguro1*, Samuel Otieno Wagai2 and Joab Otieno Onyango3
1Chemistry Department, Maseno University, P.O. Box 333-40105 Maseno, Kenya
2 Botany Department, Rongo University College, P.O. Box 103-4040 Rongo, Kenya
3Department of Chemical Sciences and Technology, Technical University of Kenya, P. O. Box
52428-00200 Nairobi, Kenya
(Received April 24, 2015; revised June 29, 2016)
ABSTRACT. Oleo-gum resin exudate from Boswellia neglecta afforded a new ursane-type triterpene
characterized as 3α-acetoxy-28-hydroxy-11-oxours-12-en-24-ioc acid (1) together with twelve known compounds.
Their structural elucidation was accomplished using physical, chemical and spectroscopic methods.
KEY WORDS: Boswellia neglecta, Burseraceae, Oleo-gum rein, Triterpenes
INTRODUCTION
Boswellia species (Burseraceae) are found in areas from the sea level up to 1000 m, usually in
rocky slopes and gullies with growth heights of 3-12 m [1]. In Kenya, the genus Boswellia is
represented by three species namely; Boswellia neglecta S. Moore (syn: B. hildebrandtii Engl.),
B. carterii and B. rivae Engl. (syn: B. boranensis) and are acclimatized in the arid and semi-arid
regions of the country [2]. They are known for frankincense which is an oleo-gum resin exudate
obtained from incision or damage to the plants [3]. The resin of B. neglecta is burnt to repel
insects, used in perfumery, food and beverage flavoring and as antiinflammatory [4, 5]. The
resin also finds usage as incense during ceremonies [6]. Previous phytochemical studies on the
plant resin resulted in the identification of monoterpenes with α-thujene, α-pinene and terpin-4-
ol as major constituents [7], diterpenes [8], triterpenes [9-11]. In this communication,
phytochemical analysis of the steam distilled residue of the plant oleo-gum resin resulted in the
isolation of one new ursane-type triterpenes characterized as 3α-acetoxy-28-hydroxy-11-oxours-
12-en-24-oic acid (1) together with known compounds 3α,11α-dihydroxyurs-12-en-24-oic acid
(2), 11-oxo-β-boswellic acid (3), α-Boswellic acid (4), β-boswellic (5), ursolic acid (6), lupeol
(7) [8], β-amyrin (8) and α-amyrin (9), α-amyrone (10) [9], quercetin (11) [12], olean-12-en-3-
O-β-glucoside (12) [13] and catechin-3-O-glucoside (13) [14]. Compounds 1, 2, 11, 12 and 13
are reported from B. neglecta gum resin for the first time.
__________
*Corresponding author. E-mail: kamanguro@yahoo.com

318
Lawrence Onyango Arot Manguro et al.
30
29
21
12
26
R3
22
R2
19
16
11
R4
28
1
9
27
3
5
7
R
R1
23
1 R = OAc, R1 = COOH, R2 = R3 = O, R4 = CH2OH
EXPERIMENTAL
Instrumentation, solvents and chemicals. Melting points were determined using Gallenkamp
melting point apparatus and are uncorrected. The IR spectra were run on Pye-Unicam SP8
spectrophotometer using KBR pellet. The NMR data were measured in CDCl3 and CDCl3-
DMSO-d6 on a Brucker NMR Ultrashield TM operating at 500 and 125 MHz, respectively, with
TMS used as internal standard. The mass spectral data were obtained using a Varian MAT 8200
A instrument. Silica gel 60G (0.02-0.7 mm Mesh) Merck was used for medium pressure
chromatography. All solvents used were of analytical grade. Glucose, galactose and rhamnose
used as reference standards were bought from Kobian Kenya Ltd. On the other hand, authentic
standards of quercetin and catechin were stocks at the Natural Products section, Department of
Environmental Science, Policy and Management, University of California at Berkeley,
California, USA.
Oleo-gum resin source. The resin samples were collected in Wajir County, Kenya and supplied
by Ms. Rose Chiteva of Non-Wood Forest products, Kenya Forestry Research Institute
(KEFRI). Authentification of the plant was done by Mr. Norman Gachathi (Taxonomist) of the
same institution and voucher specimen (No: KEFRI/BN-03/2010) was deposited in the
herbarium of Non-Wood Forest Products Programme.
Extraction and isolation of compounds. The residue from hydrodistillated resin (1 kg) was cold
extracted with CH2Cl2 (3 x 4 L) and MeOH (3 x 4 L), each lasting four days, separately
evaporated under reduced pressure yielding yellow-brown (35 g) and dark brown (70 g)
materials, respectively.
Fractionation of CH2Cl2 extract. A portion of the extract (30 g) was adsorbed onto silica gel and
then subjected to column chromatography (3.0 x 60 cm, SiO2 500 g, pressure ≈ 1 bar) using n-
hexane-CH2Cl2 gradient (increment 10%) up to 100% CH2Cl2 and elution concluded with ethyl
acetate, collecting 20 mL each. The process afforded various sub-fractions (I-VI) as determined
by TLC profiles [solvent systems: n-hexane-CH2Cl2 (1:4, 1:2, 1:1) and n-hexane-EtOAc, 3:2)].
The sub-fraction I (fractions 1-20) showed no spot and solvent recovered. Sub-fraction II
(fractions 30-60) produced colorless oil and was kept under freezing conditions a waiting GC-
MS analysis.
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The sub-fraction III (fractions 65-90, 7 g) was further subjected to medium pressure
chromatography using n-hexane-EtOAc (4:1), collecting 10 ml each to afford α-amyrone (10), a
mixture of α-and β-amyrins (9 and 8, 200 mg) and lupeol (7, 34 mg). Sub-fraction IV (fraction
93-130, 4 g) showed two spots Rf 0.46 and 0.42 (solvent system: n-hexane-EtOAc, 3:2) and was
further purified by medium pressure chromatography to give ursolic acid (6, 44 mg) and a
mixture of α- and β-boswellic acids (5 and 4, 50 mg). Sub-fraction V (fractions 140-170, 6.0 g)
afforded three spots on TLC analysis (solvent system: n-haxane-EtOAc, 1:1) with Rf values
0.31, 0.24 and 0.19, respectively. Further purification using 0.2% MeOH in CH2Cl2 afforded 11-
oxo-β-boswellic acid 3 (Rf 0.31, 25 mg), 2 (Rf 0.24, 30 mg) and 1 (Rf 0.19, 40 mg). Subfraction
VI (fractions 174-195, 5 g) was loaded onto silica gel column and further purified by medium
pressure chromatography (2.5 x 50 cm, SiO2 150 g, pressure ≈ 1 bar) to give further compounds
1 and 2 in 70 mg and 40 mg, respectively.
Fractionation of MeOH extract. Medium pressure chromatographic separation of MeOH extract
constituents (approx. 65 g, 3.5 x 90 cm, SiO2 500 g, pressure ≈ 1 bar) using a stepwise gradient
mixture of CH2Cl2-MeOH (97:3; 95:5; 9:1; 4:1; 2:1) and finally with MeOH afforded 250
fractions each 20 mL. The fractions were grouped into three pools (I-III) depending on TLC
profiles. Pool I (fractions 20-40, 4 g) was repeatedly fractionated over silica gel column using
CH2Cl2-MeOH (97:3) to give quercetin (11, 47 mg). Pool II (fractions 64-135, 3.5 g) showed
one major spot and was further purified by crystallization (CH2Cl2-MeOH, 98:2) to give olean-
12-en-3-O-β-glucoside (12, 76 mg). Fractions 142-242 [constituted pool III (4.5 g)] and on
further purification using CH2Cl2-MeOH (95:5) followed by same solvent system in the ratio 9:1
afforded further 12 (35 mg) and catechin 3-O-β-glucoside (13, 45 mg).
Acid hydrolysis of compounds 12 and 13. A solution of 12 and 13 (each 10 mg) in a mixture of
8% HCl (1 mL) and MeOH (20 mL) were separately refluxed for 2 h. The reaction mixtures
were reduced in vacuo to dryness, dissolved in H2O (2 mL) and neutralized with NaOH. The
neutralized products were then subjected to TLC analysis (eluent: EtOAc-MeOH-H2O-HOAc,
6:2:1:1). The chromatograms were sprayed with aniline hydrogen phthalate followed by heating
0
at 100 C for 2 min. The presence of glucose was confirmed after comparison with reference
samples (glucose, galactose and rhamnose). Similarly, the presence of aglycones quercetin and
catechin were confirmed by TLC co-chromatography with authentic samples.
0
Compound 1. An amorphous white powder with m.p. 204-206 C; [α]25D -20.60 (MeOH, c 0.6).
IR νmax (KBr) cm-1: 3415, 3000-2500, 1740, 1710, 1680, 1640, 1580, 1450, 1340, 1250, 1210,
1110, 1028, 960; 1H NMR (CDCl3+ drop DMSO-d6) ppm: 5.32 (1H, brs, H-12), 4.75 (1H, dd, J
= 8.4, 3.6 Hz, H-3), 3.32 (1H, d, J = 11.0 Hz, H-28), 3.0 (1H, d, J = 11.0 Hz, H-28), 2.25 (1H, s,
H-9), 2.10 (3H, s, OAc), 1.98 (1H, m, H-2), 1.75 (1H, m, H-19), 1.65 (1H, m, H-1), 1.45 (2H,
m, H-2 and H-16), 1.43 (d, J = 12.0 Hz, H-18), 1.42 (1H, dd, J = 12.0, 3.1 Hz, H-5), 1.33 (3H, s,
CH3-27), 1.20 (2H, m, H-21), 1.15 (1H, m, H-16), 1.14 (3H, s, CH3-26), 1.10 (1H, m, H-1), 1.06
(3H, s, CH3-23), 0.98 (3H, s, CH3-25), 0.88 (3H, d, J = 6.6 Hz, CH3-30), 0.80 (3H, d, J = 6.5 Hz,
CH3-29); 13C NMR (CDCl3+ drop DMSO-d6) ppm: see Table 1; ESIMS m/z (rel. int.) 528
[M]+(4), 487 (5), 368 (1), 448 (3), 289 (7), 280 (21), 257 (14), 248 (21), 221 (8), 220 (6), 218
(100), 217 (12), 208 (2), 203 (50), 135 (60). HRMSHRSMS m/z: 528.7357 (calculated for
C32H48O6, 528.7366).
Compound 2. White crystals from CH2Cl2-MeOH (99:1), m.p. 189-193 0C; [α]25D -360 (MeOH, c
0.5). IR νma1x (KBr) cm-1: 3425, 3100-2500, 1705, 1642, 1570, 1454, 1380, 1257, 1208, 1100,
1028, 898; H NMR (CDCl3+ drop DMSO-d6) ppm: 5.45 (1H, d, J = 3.6 Hz, H-12), 3.70 (1H,
dd, J = 8.3. 3.4 Hz, H-11), 3.40 (1H, dd, J = 8.3, 2.4 Hz, H-3), 2.18 ((1H, d, J = 9.0 Hz, H-9),
1.95 (1H, m, H-2), 1.70 (2H, m, H-1and H-6), 1.55 (1H, dd, J = 11.6, 3.4 Hz, H-5), 1.47 (1H,
Bull. Chem. Soc. Ethiop. 2016, 30(2)

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Lawrence Onyango Arot Manguro et al.
d, J = 12.3 Hz, H-18), 1.40 (2H, m, H-21), 1.30 (3H, s, CH3-27), 1.25 (1H, m, H-16), 1.20 (3H,
s, CH3-23), 1.18 (1H, m, H-1), 1.10 (3H, s, CH3-26), 1.02 (3H, s, CH3-25), 0.95 (3H, s, CH3-28),
0.93 (3H, d, J = 6.5 Hz, CH3-30), 0.86 (3H, d, J = 6.7 Hz, H-29); 13C NMR (CDCl3+ drop
DMSO-d6) ppm: see Tables 1 ESIMS m/z ; (rel. int.) 472 [M]+ (2), 456 (4), 454 (8), 436 (2),
238 (15), 234 (22), 220 (15), 218 (100), 216 (10), 203 (50), 189 (30), 160 (13), 55 (70).
HRSMS m/z: 472.7146 (calcd. for C32H48O4, 472.71457).
Compound 12. White amorphous powder with m.p. > 250 0C; IR νmax (KBr) cm-1: 3350, 2980,
1
2850, 1660, 1380, 1190, 1038, 980; H NMR (CDCl3 + drop DMSO-d6) ppm: 5.31 (1H, t, J =
3.5 Hz, H-12), 3.40 (1H, dd, J = 12.2, 4.5 Hz, H-3), 1.95 (1H, m, H-15), 1.84 (1H, m, H-18),
1.72 (1H, s, H-1), 1.65 (1H, m, H-19), 1.55 (1H, m, H-2), 1.75 (1H, m, H-9), 1.46 (1H, m, H-5),
1.20 (H, m, H-1), 1.43 (d, J = 12.0 Hz, H-18), 1.42 (1H, dd, J = 12.0, 3.1 Hz, H-5), 1.33 (3H, s,
CH3-27), 1.32 (1H, m, H-2), 1.24 (3H, s, CH3-27), 1.10 (3H, s, CH3-25), 0.99 (3H, s, CH3-23),
0.96 (3H, s, CH3-26), 0.90 (3H, s, CH3-30), 0.86 (3H, s, CH3-29), 0.82 (3H, s, CH3-28), 0.78
(3H, s, CH3-24); glc: 5.01 (1H. d, J = 7.2 Hz, H-1’), 3.77 (1H, m, 6’a), 3.64 (1H, m, 6’b), 3.56
(1H, m, H-5’), 3.47 (1H, m, H-2’), 3.36 (1H, m, H-4’), 3.28 (1H, m, H-3’); 13C NMR (CDCl3 +
drop DMSO-d6) ppm: see Table 1; ESIMS m/z (rel. int.) 426 [M]+ (10), 208 (15), 218 (100),
203 (56), 189 (45), 135 (32), 55 (86), 41 (75).
0
Compound 13. An amorphous white powder with m.p. > 250 C; IR νmax (KBr) cm-1: 3450,
2950, 1870, 1660, 1018, 860; 1H NMR (CDCl3 + drop DMSO-d6) ppm: 7.03 (1H, d, J = 1.2 Hz,
H-2’), 6.80 (1H, dd, J = 8.5, 1.2 Hz, H-6’), 6.67 (1H, d, J = 8.5Hz, H-5’), 5.95 (1H, d, J =
2.1Hz, H-6), 5.89 (1H, d, J = 2.1 Hz, H-8), 5.10 (1H, d, J = 7.4 Hz, H-1’’), 4.85 (1H, br s, H-2),
4.1 (1H, m, H-3), 3.98 (1H, m, H-6’’a), 3.62 (1H, m, H-6’’b), 3.46 (1H, m, H-4’’), 3.41 (1H, m,
H-3’’), 3.37 (1H, m, H-2’’), 3.20 (1H, m, H-5’’); 13C NMR (CDCl3+drop DMSO-d6) ppm: 79.4
(C-2), 66.7 (C-3), 30.0 (C-4), 157.8 (C-5), 95.8 (C-6), 156.3 (C-7), 96.0 (C-8), 156. 0 (C-8a),
132.6 (C-1’), 116.1 (C-2’), 144.9 (C-3’), 145.0 (C-4’), 116.4 (C-5’), 120.2 (C-6’’); Glc: 102.3
(C-1’’), 74.0 (C-2’’), 76.7 (C-3’’), 70. 8 (C-4’’), 76.4 (C-5’’), 61.6 (C-6’’); ESIMS m/z: (rel.
int.) 290 (57), 373 (24).
RESULTS AND DISCUSSION
Compound 1 was isolated as white amorphous powder with a molecular formula C32H48O6 as
evidenced by EI-MS which exhibited a [M]+ ion peak at m/z 528 (9 unsaturation equivalents). It
showed a positive Liebermann-Burchard test and Molish reaction suggesting a triterpene
skeleton [12]. The IR spectrum showed characteristic absorptions attributable to hydroxyl (3415
cm-1), ester carbonyl (1740 cm-1), carboxylic cid (1710 cm-1), conjugated keto (1680 cm-1) and a
1
double bond (1640 cm-1) functional groups. The H NMR spectrum of 1 displayed five tertiary
methyls (δ 2.10, 1.33, 1.14, 1.06 and 0.98, each singlet including methyl from acetoxy group),
two secondary methyls (δ 0.88, d, J = 6.6 Hz and 0.80, d, J = 6.5 Hz) and a trisubstituted
olefinic proton (δ 5.32, s), which are characteristic of acetylated ursane-type triterpenes related
to boswellic acids [15, 16]. The 13C NMR spectrum (Table 1) of compound 1 displayed 32
distinct peaks accounted for by 7 methyls, 7 methines, 9 methylenes and 9 quaternary carbons in
the 135 DEPT spectrum. Careful analysis of both 1H and 13C NMR data of the compound taking
into consideration the fragmentation pattern in the EI-MS (Figure 1) suggested that compound 1
is a 3α-acetoxy-11-keto-β-boswellic acid derivative possibly with acetoxy and carboxylic acid
groups in rings A/B [m/z 280 (C16H24O4)], while an oxo moiety together with terminal
hydroxymethylene are in rings D/E [m/z 248 (C16H24O2) [8, 17].
1
The H NMR spectrum confirmed the presence of acetoxy group at C-3 and was in axial
orientation as evidenced by the narrow peak half-height width w1/2 (3.6 Hz) of the equatorially-
positioned geminal proton which appeared relatively downfield at δ 4.75 [18, 19), an
interpretation further substantiated by HMBC correlation between H-5 (δH 1.42) and C-3 (δc
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321
75.6). Similarly, the H-3 showed an HMBC cross-peak with a peak at δc182.1 which further
supported the presence of carboxylic group in ring A at either C-23 or C-24 positions. The
1
characteristic H-1H proximity (NOESY) between CH3-23 and H-5 allowed the assignment of
the functional group at C-24 as previously observed with boswellic acids [8, 16].
+
.
+
HO
O
H
McLafferty
(I)
CH2OH
CH2OH
rearrangment
AcO
CO2H
CO2H
OAc
m/z = 528
+
O
HO
+
H
CH2OH
CH2OH
CH2OH
+
H
+
O
m/z =289
HO
OH
m/z =134
+
.
O
RDA
HO
cleavage
.
+
CH2O
H
CH2OH
AcO
(II)
CO2H
m/z = 280
m/z = 248
- CH2OH
AcO
CO2H
m/z =528
O
+
.
CO2H
m/z = 232
m/z = 217
Figure 1. Possible fragmentations of compound 1.
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Lawrence Onyango Arot Manguro et al.
Table 1. 13C NMR of compounds 1, 2 and 12.
H
1
2
12
1
2
3
4
5
6
7
8
9
36.2
24.8
75.6
44.0
50.4
18.3
34.0
41.9
58.0
35.0
205.3
132.7
165.5
44.3
30.7
26.6
34.4
56.5
40.0
43.0
26.9
36.9
24.1
182.1
16.0
17.2
20.3
68.8
16.9
28.8
169.8
20.5
37.1
27.7
70.1
43.0
48.0
20.0
33.5
39.4
47.9
36.9
72.1
122.5
141.4
42.1
29.5
25.5
33.8
55.7
38.0
41.4
28.1
37.9
24.2
179.3
16.5
16.8
21.7
23.5
15.9
25.8
37.5
26.0
76.2
41.2
54.6
20.8
33.9
40.5
48.0
36.3
24.4
123.7
143.2
43.4
28.5
26.2
34.0
58.1
39.2
40.7
30.8
41.0
28.7
22.0
16.1
16.6
23.6
27.0
18.0
21.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
MeCO
CH3CO
Glc
1’
104.0
74.3
77.6
71.5
75.9
62.6
2’
3’
4’
5’
6’
On the other hand, the singlet peak at δ 5.32 (H-12) correlated with C-11 (δc 205.3)/C-8 (δ
58.0) and in turn with C-13 (δc165.5)/C-14 (δc44.3) and on this basis, the position of the keto
group was assigned to C-11; a fact further supported by EI-MS peaks at m/z 289 [C19H29O2]
originating from McLafferty rearrangement and 248 [C16H24O2] due to retro-Diels-Alder
cleavage. Similarly, in the HMBC spectrum, the terminal hydroxymethylene protons showed
long range correlations with carbons at C-18 (δc 56.5)/C-16 (δc 26.6)/C-22 (δc 36.9) suggesting
the location of the group at C-17 [20]. This was further supported by the medium intensity
diagnostic EI-MS fragmentation peaks which appeared at m/z 289 and 248. The latter fragment
lost a terminal hydroxymethylene group to form an m/z 218 (100) leading to stable tertiary
carbenium, thus confirming the –CH2OH to be at C-17 (8).
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323
Thus, on the basis of spectroscopic data and also comparison with literature data, structure
of compound 1 was deduced as 3α-acetoxy-28-hydroxy-11-oxours-12-en-24-oic acid (3α-
acetoxy28-hydroxy-11-keto-β-boswellic acid).
CONCLUSION
Compounds 2-9 isolated from B. neglecta oleo-gum resin was consistent with other reports of
boswellic acids of ursane-type triterpene [8].
ACKNOWLEDGMENTS
This work was supported by Third World Academy of Sciences (TWAS). The authors are
thankful to Mr. Gachathi and Ms Rose Chiteva of Non-Wood Forest Products programme,
Kenya Forestry Research Institute for identification and collection the plant materials. The
Institute of Organic Chemistry, Technical University of Munich, Germany is acknowledged for
the spectroscopic data.
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