Natural Product Research

Formerly Natural Product Letters

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A new dammarane type triterpene glucoside

from the aerial parts of Gouania longipetala
(Rhamnaceae)

Soh Désiré, Nkwengoua Ernestine, Tchebemou Bakang Bruno, Sidjui Sidjui

Lazare, Dzo Defokou Ulrich, Mehreen Lateef, Bernd Schneider, Muhammad
Shaiq Ali & Nyassé Barthélemy

To cite this article: Soh Désiré, Nkwengoua Ernestine, Tchebemou Bakang Bruno, Sidjui Sidjui
Lazare, Dzo Defokou Ulrich, Mehreen Lateef, Bernd Schneider, Muhammad Shaiq Ali & Nyassé
Barthélemy (2019): A new dammarane type triterpene glucoside from the aerial parts of Gouania
longipetala (Rhamnaceae), Natural Product Research, DOI: 10.1080/14786419.2019.1690483

To link to this article: https://doi.org/10.1080/14786419.2019.1690483

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Published online: 29 Nov 2019.

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1690483

A new dammarane type triterpene glucoside from the

aerial parts of Gouania longipetala (Rhamnaceae)
Soh D
esir

ea,b,c, Nkwengoua Ernestineb, Tchebemou Bakang Brunob, Sidjui Sidjui
Lazare , Dzo Defokou Ulrichb, Mehreen Lateefe, Bernd Schneiderf,
c,d

Muhammad Shaiq Alic and Nyass
e Barth
elemyb

a
Department of Chemistry, Higher Teacher Training College, TWAS Research Unit (TRU), University of
Bamenda, Bamenda, Cameroon; bLaboratory of Medicinal Chemistry & Pharmacognosy, Faculty of
Science, Department of Organic Chemistry, University of Yaound
e I, Yaound
e, Cameroon; cH. E. J.
Research Institute of Chemistry, International Center for Chemical & Biological Sciences (ICCBS),
University of Karachi, Karachi, Pakistan; dFaculty of Sciences, Department of Organic Chemistry,
Institute of Medical Research and Medicinal Plant Studies, TWAS Research Unit (TRU), University of
Yaounde I, Yaound
e, Cameroon; eMultidisciplinary Research Lab, Bahria University Medical and
Dental College, Karachi, Pakistan; fMax Planck Institute for Chemical Ecology, Jena, Germany

ABSTRACT ARTICLE HISTORY

3-O-b-D-glucopyranosyl gouanogenin A (1), a new naturally occur- Received 22 April 2019
ring dammarane class of triterpene glucoside, has been isolated Accepted 3 November 2019
from the aerial parts of Gouania longipetala along with six known
secondary metabolites 2–7. Their structure was elucidated KEYWORDS
through spectroscopic data including 1 D- and 2 D-NMR. The com- Gouania longipetala; 3-O-
b-D-glucopyranosyl gouano-
pounds 1 and 6 showed significant antioxidant potential in DPPH genin A; antioxidant
radical scavenging assay. On the other hand, the compound 4 activity; urease inhibition
revealed potent inhibitory potential against the enzyme urease,
while 1 and 3 were significantly active.

1. Introduction
Gouania is a genus of flowering plants belonging to family, Rhamnaceae. It comprises
about 50–70 species native to tropical and subtropical regions of Africa, America and
Southern Asia, these are mostly shrubs or lianas (Sven et al. 2011). One of the species
of this genus is Gouania longipetala (Hemsl) which is a scandent shrub or liana mainly

CONTACT Soh D
esir
e desiresoh75@gmail.comdesiresoh@yahoo.fr

Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1690483.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 S. DÉSIRÉ ET AL.

found in closed forests, forest margins, and in jungle regrowths (Burkill 1985). The
stem and/or leaves of the plant are used in traditional medicine in different African
countries including Cameroon for the treatment of a variety of human ailments includ-
ing swelling, pain, edema venomous stings, gout, febrifuges, women fertility, heart dis-
eases, mellitus and malaria (Burkill 1985; Abbiw 1990; Focho et al. 2009; Njamen et al.
2013). Biological studies have demonstrated the antibacterial, antioxidant, anti-inflam-
matory, antidiabetic, antilipidemic and estrogenic effects of the extracts of this plant.
(Ekuadzi et al. 2012; 2014; Dzeufiet et al. 2015; Ezeja et al. 2015). The ethnopharmaco-
logical and chemotaxanomic significance of the genus Gouania prompted us to under-
take pharmacochemical studies on G. longipetala. Herein we reported the isolation of
3-O-b-D-glucopyranosyl gouanogenin A (1), a dammarane type triterpene glucoside
isolated for the first time as a natural product following earlier report of its formation
as an additional product of incomplete enzymatic hydrolysis by Edward J. Kennelly
and Walter H. Lewis 1993. In addition, six known secondary metabolites namely
Joazeiroside A (2), isolated for the first time from the genus Gouania, alphitolic acid 3
(Kuang et al. 1989; Aguirre et al. 2006), lupeol 4 (Burns et al. 2000; Jain and Bari 2010;
Abdullahi et al. 2013), betulinic acid 5 (Sholichin et al., 1980; Siddiqui et al. 1988;
Aguirre et al. 2006; Nyasse et al. 2009), gouanic acid B 6 (Giacomelli et al. 2007),
b-sitosterol-3-O-b-D-glucoside 7 (Tania and Kar 2017), have also been isolated and
determined. The structure of 3-O-b-D-glucopyranosyl gouanogenin A was character-
ized by extensive spectroscopic studies, while the known secondary metabolites were
identified through comparison of physical and spectral data with the literature. The
antioxidant and urease inhibitory potentials of compounds 1–4 and 6 have also been
ascertained.

2. Result and discussions

Compound 1 was isolated as white powder, m.p 186–188
C; ½a25 D ¼ þ27.40; UV
(MeOH) kmax (log E) 213 (0.503), 298 (2.027) nm, revealing the presence of extended
conjugation. The IR spectrum displayed absorption bands for -OH (3300 cm1) the car-
bonyl of: a c-lactone (1787 cm1), the conjugated ketone (1686 cm1) and conjugated
olefinic (1600 cm1) functionalities. The HR-ESI-MS showed quasi-molecular [M þ H]þ
peak at m/z 633.3956 consistent with molecular formula C36H56O9 (calcd for
C36H56O9 633.4003).
The 1H NMR (Table 1) exhibited the singlets of six tertiary methyls at dH 0.83, 0.86,
1.00, 1.05, 1.90, 1.92 and the doublet of a secondary methyl at 1.17 (d, 3H, J ¼ 6.8 Hz,
H-21). The 1H NMR spectrum further displayed signals of 5,5-dimethyl-2(E),4-dienone
moiety at dH 7.50 (dd, 1H, J ¼ 11.6 and 15.2 Hz, H-23), 6.14 (d, 1H, J ¼ 15.2 Hz, H-22),
and 6.04 (d, 1H, J ¼ 11.6 Hz, H-24). The presence of this moiety was further confirmed
by the base peak in the EI-MS at m/z 109 (Kennelly et al. 1993). A c-lactone was evi-
dent by the observance of geminally coupled oxymethylene protons as doublets at dH
4.41 and 4.23 (J ¼ 10.4 Hz) as well as doublets of geminal a methylene proton at dH
2.69 and 2.49 (J ¼ 19.2) (Yoshikawa et al. 1996). The signal of an anomeric proton was
observed as doublet at dH 4.31 (d, 1H, J ¼ 8.0 Hz, H-1’) suggesting its b and axial
configuration (Figure 1).
 NATURAL PRODUCT RESEARCH 3

Figure 1. Structures of compounds 1–7.

The 13C NMR (Table 1) (BB and DEPT) spectra showed the presence of 36 signals com-
prising seven methyl nine methylene thirteen methine and seven quaternary carbons. The
downfield signals at dC 206.3 and 179.9 could be attributed to the lactone and conjugated
carbonyl functionalities while the olefinic carbons resonated at dC 150.4, 140.9, 128.0 and
125.3, respectively. An oxymethine carbon resonated at dC 90.4 while the oxymethylene
carbon was observed at dC 72.0. All these data suggested dammarane type skeleton for
compound 1. The sugar moiety was located at C-3 on the basis of the correlation between
4 S. DÉSIRÉ ET AL.

Table 1: 1H and 13
C NMR (400 MHz, in CD3OD) data for compound 1
Position
dH (m; J in Hz) dc
1 0.96 (m) 1.68 (m) 39.4
2 1.48 (m) 1.52 (m) 27.1
3 3.17 (dd, J ¼ 8.0, 4.0) 90.4
4 – 40.2
5 0.81 (d, 12.0) 56.3
6 1.49 (m) 1.65 (m) 18.9
7 1.36 (m) 1.55 (m) 35.2
8 – 42.3
9 0.74 (d, J ¼ 9.2) 54.0
10 – 37.8
11 1.34 (m) 1.58 (m) 21.9
12 1.31 (m) 1.66 (m) 26.5
13 1.91 (dd, J ¼ 9.2, 4.3) 46.7
14 – 53.4
15 2.49 (d, J ¼ 19.2) 2.69 (d, J ¼ 19.2) 35.0
16 – 179.9
17 2.92 (dd, J ¼ 4.8, 4.2) 44.7
18 0.86 (s) 19.0
19 0.83 (s) 16.7
20 – 206.3
21 1.17 (d, J ¼ 6.8) 16.5
22 6.14 (d, J ¼ 15.2) 128.0
23 7.5(dd, J ¼ 11.6, 15.2) 140.9
24 6.04 (d, J ¼ 11.6), 125.3
25 – 150.4
26 1.92 (s) 26.8
27 1.90 (s) 19.8
28 1.05 (s) 28.3
29 1.00 (s) 18.4
30 4.23 (d, J ¼ 10.4) 4.41 (d, J ¼ 10.4) 72.0
Glucose –
1’ 4.31 (d, J ¼ 8) 106.6
2’ 3.17 (m) 75.1
3’ 3.41 (m) 78.2
4’ 3.30 (m) 71.6
5’ 3.24 (m) 77.6
6’ 3.65, 3.85 (2dd, J ¼ 11.8, 5.2) 62.7

the proton signal at dH 3.17 (H-3) and the anomeric carbon at dC 106.6. The structure was
finally assigned on the basis of 1H,1H- Cosy and HMBC experiments (Figure 2). In the HMBC
spectrum, the correlations were observed between the following protons and carbons: dH
1.90 (H-27) with dC 19.8, 125.3, 150.4, dH 1.92 (H-26), with dC 26.8, 125.3, 150.4, dH 6.04
(H-24) with dC 19.8, 26.8, 128.0; dH 7.50 (H-23) with dC 125.3, 128.0, 150.4, 206.32, dH 6.14
(H-22) with dC 125.3, 206.3, dH 2.90 (H-17) correlated with dC 16.5, 46.7, 53.4, 206.3, dH 1.17
(H-21) showed connectivity to dC 44.7, 46.7, 206.3, dH 1.90 (H-13) with dC 16.5, 53.4, dH
2.49, 2.69 (CH2-15) connected with dC 42.3, 46.7, 53.4, dH 4.23, 4.41 (CH2-30) displayed
correlations with dC 42.3, 53.4, 179.9.
The 1H-NMR spectrum of compound 1 showed the anomeric proton as doublet at
dH 4.31(d, 1H, J ¼ 8.0 Hz, H-1’) which correlated with signal of anomeric carbon at dC
106.6 in the HSQC spectrum, suggesting the presence of a sugar moiety with b glyco-
sidic linkage. Acid hydrolysis provided the aglycone which could be identified as
D-glucose through sign of its optical rotation as well as comparison of GC retention
times of its trimethylsilyl ethers with those of a standard sample. The stereochemical
 NATURAL PRODUCT RESEARCH 5

Figure 2. Key 1H–1H COSY ( ) and HMBC ( ) correlations of compound 1.

H H
H
H
H3C 19 18 CH3
13
17 24 CH3
CH2OH 22
29 CH 11 26
O
3
10 8 12 H CH3 23
9 14 15
2
O 4 1 6 27
HO 3 5 7 H H O H CH3
HO H 30 16
H3C 28 H O O
HO
H H
H H H
H

Figure 3. Key NOe effects in compound 1.

assignments shown for compound 1 were made with the help of NOESY and found
typical of glucoside of dammarane-type triterpene. In these class of triterpenes,
a lactone at C-14 originates through oxidation of Me-30 which is biogenetically
a-oriented. Further evidence to this effect was provided by close similarity of chemical
shifts of various carbons of dammarane skeleton with those of ebelin-lactone and
related saponins (Kennelly et al. 1993; Yoshikawa et al. 1995, 1996; Muhammad et al.
2016). In the NOESY spectrum of compound 1 NOe effects between the b-axial Me-29/
Me-19, Me-19/Me-18, Me-18/H-13 and between the a-axial H-3/H-5 and H-5/H-9,
the b-equatorial orientation of H-17 is deduced by the NOe effect observed with H-13
confirm the expected stereochemistry (Yoshikawa et al. 1995; Muhammad et al. 2016)
(Figure 3). The configuration of the side-chain is deduced from the observed NOe
effects between H-22 and H-24, H-24 and H-27, H-23 and H-26. Based on these eviden-
ces, the structure of compound 1 could be assigned as 3-O-b-D-glucopyranosyl
gauanogenin.

2.1. In vitro biological screening of the isolated compounds

The activity of compounds isolated from Gouania longipetela were evaluated for their
potential against various enzymes involved in Diabetes, Alzheimer’s disease, ulcer and
other cancer related process by various in vitro biological assays (Table 2).
6 S. DÉSIRÉ ET AL.

Table 2. IC50 values of pure compounds in antioxidant activity and enzyme inhibition.
IC50 ± SEM (lM)
Urease Butrylcholinesterase Chymotrypsin Lipoxygenase
Compounds Antioxidant inhibition Inhibition inhibition inhibition
1 56.4 ± 0.21 26.7 ± 0.26 >100 ± 0.28 >100
2 69.5 ± 0.87 63.4 ± 0.45 49.5 ± 0.47 19.6 ± 0.55
3 68.2 ± 0.34 29.2 ± 0.14 75.2 ± 0.26 Nil
4 63.2 ± 0.09 12.5 ± 0.21 >100 Nil
6 52.5 ± 0.12 61.2 ± 0.24 58.5 ± 0.28 17.5 ± 0.65
BHA 44.2 ± 0.06
Thiourea – 21.6 ± 0.12 – – –
Eserine – – 7.8 ± 0.27 – –
Chymostatin – – – 5.7 ± 0.14 –
Baicalein – – – – 22.6 ± 0.08

Among all compounds, compound 4 showed remarkable urease inhibition with IC50
value of 12.5 ± 0.21 mM as compared to standard thiourea which shown to have IC50
value of 21.6 ± 0.12 mM. It indicates that compound 2 is more potent than standard
and required in less concentration to inhibit 50% percent urease enzyme involved in
progression of ulcer. In addition to compound 4, compound 1 and 3 also showed
excellent antiurease activity with IC50 value of 26.7 ± 0.26 mM and 29.2 ± 0.14 mM
respectively when compared with standard IC50 value. Structure chemistry relationship
of compound 4, 3 and 1 revealed that the presence of number and position of func-
tional group (–OH and double bond), (-OH, double bond and –COOH), ketone double
bond and lactone favours the inhibition of specific enzyme. When the activities of
compounds were analysed against butyrylcholinesterase, the compounds 2 and 6
showed IC50 value of 49.5 ± 0.47 mM and 58.5 ± 0.28 mM as compared to standard
eserine with IC50 7.8 ± 0.27 mM. It indicates that these compounds are effective against
bacterial cholinesterase enzyme involved in Alzheimer’s Disease but required incom-
parably higher concentration than standard aspirin to inhibit the enzyme with less
potency. The active compounds 2 and 6 have –OH group, double bond and present
of cyclic ether and –COOH functional group at 15, 16, 18, 23 and 2, 14, 17, 20, 24 pos-
ition respectively. The overall conformation of these compounds may participate in
inhibition of butyrylcholinesterase enzyme. Butyl cholinesterase enzyme can be the
essential target in the treatment of Alzheimer’s disease because in Alzheimer’s disease
patients, Butryl cholinesterase accumulated in higher concentration in brain especially
in neurons, glial cells, neurotic plaques and tangles in addition to acetylcholinesterase
(Perry et al. 1978; Greig et al. 2001). Therefore, Butyrylcholinsterase inhibitors can be
effective to treat dementias and Alzheimer’s disease. It is suggested that effective
compounds 2 and 6, if produce no or less side effects as compared to other available
drugs, they can be used as future candidates for drug development therefore toxico-
logical studies can be recommended to be done for these lead compounds in future.
Evaluation of Chymotrypsin Inhibition Activity of compounds shows that compound
2 and 6 exhibit good protease inhibitory potential against chymotrypsin with IC50
value of 19.6 ± 0.55 mM and 17.5 ± 0.65 mM as compared to standard chymostatin (IC50
5.7 ± 0.14 mM). It shows that these compounds 2 and 6 can be potential serine inhibi-
tors. NS3 serine protease of HCV and HIV protease are key enzymes that catalyse
 NATURAL PRODUCT RESEARCH 7

replication of virus and are the target for anti-HCV and anti-HIV drugs (Patick and
Potts 1998) therefore these compounds can be effective as anti-viral compounds and
can be further used for anti-viral studies in future.
All the compounds 1–6, showed moderate antioxidant activity as compared to
standard butylated hydroxyanisole (BHA) while devoid of lipoxygenase inhibition activ-
ity. Lipoxygenase are the family of enzymes in the biosynthesis of leukotriens that are
postulated to play an important role in the pathophysiology of several inflammatory
diseases but results showed that these compounds are not involved in inhibition of
lipoxygenase metabolism.
It is concluded that compound 1 and 3 have potential as antiulcer agents while
compound 2 and 6 are found to active against enzyme involved in progression of
Alzeimers disease and its potential of being serine protease inhibitor made it potential
candidate for antiviral studies.
Compound 2 and 6 showed promising potential against the enzyme butrylcholines-
terase, so these compounds can be effective against Alzheimer disease. Therefore,
future studies would also be planned to explore their potential against acetylcholin-
esterase. On the other hand, compound 2 and 6 also showed chymotrypsin inhibition
which indicated that it has potential against proteolytic enzyme and can be used in
future to treat infectious diseases.

3. Experimental
3.1. General experimental procedures
Column chromatography was carried out on silica gel (70–230 and 230–400 mesh, E.
Merck, Darmstadt, Germany). Thin layer chromatography was performed on percolated
0.5 mm thick aluminium sheets (Merck Si gel 60, F254 and visualised by spraying with
ceric sulphate solution followed by heating at 120
C on a hot plate. Melting points
were recorded on a Barnstead Electrothermal apparatus (Digital Melting Point IA-90)
and are uncorrected. The mass spectra where recorded on a JEOL MS Route instru-
ment and nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX-
400 instrument, 1H and 13C NMR probes operating at 500 and 125 MHz, respectively,
with tetramethylsilane as an internal standard. Optical rotations were recorded on a
JASCO P-2000, polarimeter (Japan). The UV spectra were measured on a HP 8451 A
diode array spectrophotometer.

3.2. Plant material

The stem and leaves of Gouania longipetala were collected in April 2016 in Bali
Nyonga, Mezam division in the North West region of Cameroon by Dr. Tacham Walter
Ndam, a Botanist of the Faculty of Science, The University of Bamenda. The collected
plant material was identified at the National Herbarium of Cameroon in comparison
with a voucher specimen number 60787 HNC.
8 S. DÉSIRÉ ET AL.

3.3. Extraction and isolation

The stems and leaves of Gouania longipetala were air dried and ground into fine pow-
ders. The powders from stem (2.35 kg) and leaves (1.32 kg) were macerated with
methylene chloride/methanol (1:1, v/v) and methanol, respectively, for 72 h. The
extracts were filtered and concentrated on a thin film rotary evaporator to obtain
crude extract (75.10 g) from stem and (129.22 g) from the leaves. The crude extract of
stem (60 g) was subjected to column chromatography over silica gel (230–400 mesh)
and eluted with hexane, hexane/EtOAc (7.5:2.5), hexane/EtOAc (7:3), hexane/EtOAc
(1:1), EtOAc, EtOAc/MeOH (9:1) and MeOH to afford seven fractions which were com-
bined on the basis of their thin layer chromatography (TLC) profile into two pools F1
(16 g) and F2’ (28 g), respectively. The fraction F1 (16 g) was subjected to column chro-
matography over silica gel (230–400 mesh) and eluted with n-hexane/EtOAc (1:0–17:3),
to afford lupeol 4 (10.02 mg); gouanic acid B 6 (10.10 mg) from the top and the tail
fractions, respectively Further elution with n-hexane/EtOAc (1:0–8:2) afforded betulinic
acid 5 (12.6 mg). The fraction F2’(28g) was also subjected to column chromatography
eluting with mixtures of n-hexane/EtOAc in increasing order of polarity. The fraction
which eluted with n-hexane/EtOAc (12:8) afforded b-sitosterol-3-O-b-D-glucoside (7)
(43.03 mg). Further elution with increasing ethyl acetate concentration afforded 3-O-
b-D-glucopyranosyl gouanogenin A (1) (83.01 mg)) and a semi-pure powder which was
subjected to column chromatography using CH2Cl-MaOH (9:1) to furnish Joazeiroside
A 2 (90.05 mg) The 75.45 g of crude extract of leaves was suspended in water and
extracted successively with ethyl acetate and n-butanol to afford two major sub-frac-
tion GL1(35.32 g) and GL2 (30.52 g) respectively.
GL1(30.32 g) was subjected to column chromatography over silica gel (230–400
mesh) eluting with n-hexane and mixtures of n-hexane/EtOAc in increasing order
of polarity to obtain five fractions: F1 (n-hexane), F2 [n-hexane/EtOAc (19:1- 4:1)], F3
[n-hexane-EtOAc (3:1-1:1)], F4 [n-hexane/EtOAc (13:7-0:1)], F5 [EtOAc, EtOAc/MeOH
(19:1)]. The fraction F2 (8.01 g) was rechromatographed and eluted with n-hexane/
EtOAc (9:1) to provide the alphitolic acid 3 (77.23 mg).

3.4. Acid hydrolysis

A solution of 1 (3 mg) in methanol (4 ml) containing 1 N HCl (2 ml) was refluxed for
4 h. Then the mixture was concentrated, diluted with water, and extracted with ethyl
acetate. The organic phase was a mixture of aglycone products which could not
be worked up due to paucity of material. The aqueous phase was concentrated to
obtain the sugar residue which could be identified through sign of its optical rotation
(½a23
D ¼ 51.9) as well as comparing the GC retention time of its Me3Si ether (a anomer
tR 4.2, b anomer tR 7.9) with that of a standard sample.

3.5. Determination of DPPH radical scavenging activity

The free radical scavenging activity was measured by 1,1-diphenyl-2-picryl-hydrazil
(DPPH) using a method previously described (Gulcin et al. 2005). The solution of DPPH
of 0.3 mM was prepared in ethanol. Five microlitres of each sample of different
 NATURAL PRODUCT RESEARCH 9

concentration (62.5 lg–500 lg) was mixed with 95 ll of DPPH solution in ethanol. The
mixture was dispersed in a 96 well plate and incubated at 37
C for 30 min.
The absorbance at 515 nm was measured by microtitre plate reader (Spectramax
plus 384 Molecular Device, USA) and the percent radical scavenging activity was
determined in comparison with the methanol treated control (Basar et al. 2015). BHA
was used as standard.

3.6. Urease inhibition assay

Reaction mixtures comprising 25 lL of enzyme (Jack bean Urease) solution and 55 lL
of buffers containing 100 mM urea were incubated with 5 lL of test compounds (1 mM
concentration) at 30
C for 15 min in 96-well plates (Mehta et al. 2003). Urease activity
was determined by measuring ammonia production using the indophenol method as
described by Weatherburn. Briefly, 45 lL each of phenol reagent (1% w/v phenol and
0.005% w/v sodium nitroprusside) and 70 lL of alkali reagent (0.5% w/v NaOH and
0.1% active chloride NaOCl) were added to each well. The increasing absorbance at
630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA).
All reactions were performed in triplicate in a final volume of 200 lL. The results
(change in absorbance per min) were processed by using SoftMax Pro software
(Molecular Device, USA). Assays were performed at pH 8.2 (0.01 M K2HPO4.3H2O, 1 mM
EDTA and 0.01 M LiCl2). Percentage inhibition was calculated from the formula
100–(ODtestwell/ODcontrol)  100. Where OD stands for optical density. Thiourea was
used as the standard inhibitor of urease.

3.7. Lipoxygenase inhibition assay

Lipoxygenase inhibiting activity was measured by a modified spectrophotometric
method developed by Tappel, 1962. Lipoxygenase enzyme solution was prepared and
the enzyme concentration in the reaction mixture was adjusted to give rates of 0.05
absorbance/min. The reaction mixture contained 160 lL (100 mM) of sodium phos-
phate buffer (pH 8), 10 lL of test solution and 20 lL of LOX solution in buffer. The
contents were mixed and incubated for 10 min at 25
C. The reaction was then initi-
ated by the addition of 10 lL substrate solutions (linoleic acid, 0.5 mM, 0.12% w/v
tween 20 in a ratio of 1:2) and the change in absorbance at 234 nm was followed for
6 min. The concentration of test compounds that inhibited lipoxygenase activity by
50% (IC50) was determined by monitoring the effect of increasing concentrations of
compounds in the assays on the degree of inhibition. The IC50 values were calculated
by means of EZ-Fit, Enzyme kinetics Program (Perrella Scientific In., Amhherset, USA).

3.8. In vitro a-chymotrypsin assay

The a-chymotrypsin inhibitory activity was performed using a previously described
method (Cannell et al. 1988). Briefly, a-chymotrypsin (9 units/mL in 50 mM Tris–HCl
buffer, pH 7.6; Sigma Chemical Co. USA) was pre-incubated with the test compounds
at the concentration varying from 5 to 500 mM for 20 min at 25
C. 100 mL of substrate
10 S. DÉSIRÉ ET AL.

solution (N-succinyl-phenylalanine-p-nitroanilide, 0.01–0.06 mM in 50 mM Tris–HCl buf-

fer, pH 7.6) was added to start the enzyme reaction. The absorbance of released p-
nitroaniline was continuously monitored at 410 nm until a considerable color change
was achieved. The final DMSO concentration in the reaction mixture was 7%.
Best inhibition was found at concentration 3.02  105 g/100 mL.
The percentage (%) inhibition was calculated as follows: (E – S)/E x 100) where E is
the activity of the enzyme without test compound, and S is the activity of enzyme
in the presence of the test compound. The concentrations of test compounds that
inhibited the hydrolysis of substrate up to 50% (IC50) were determined by monitoring
the effect of various concentrations of these compounds in the assays. The IC50 values
were then calculated using the EZ-Fit Enzyme Kinetics program (Version Perrella
Scientific Inc., Amherst, USA).

3.9. Enzyme inhibition assay

Butrylcholinesterase inhibition activity was determined by method as described by
Ellman et al. Horse serum butryl cholinesterase enzyme, EC3.1.1.8 (Sigma, USA) was
prepared by dissolving the enzyme in phosphate buffer (100 mM, pH 8.0). The enzyme
concentration in reaction mixture was adjusted to 0.2 U per well. Sodium phosphate
buffer (180 lL, pH 8.0) and buffered Ellman’s Reagent (DTNB, 5, 5-dithiobis [2-nitroben-
zoic acid] 0.1 M NaHCO3, 17.85 mmol/L, 10 lL) was added in wells labeled as blank (B
substrate and B enzyme), control and test. Test compound solution (of various concen-
trations of 5–500 lM, 10 ll) was added in each well labeled as test. Then, 20 lL of
butrylcholinesterase solution was added in each well including B enzyme, control and
test. The contents were mixed and incubated for 15 min at 25
C. The reaction was
initiated by the addition of 10 lL substrate solution butrylcholinesterase iodide
(10 mM) in each well except B enzyme. The absorbance was measured at 412 nm.
The IC50 values were determined by monitoring the inhibition effects of various
concentrations of under investigation compounds and this was calculated by means
of EZ-Fit, Enzyme Kinetics Program (Perrella Scientific In., Amhherset, USA).

4. Conclusion
As a result of the phytochemical studies on the aerial parts of Gouania longipetala,
one naturally occurring dammarane class of triterpene glucoside, has been isolated
along with six known secondary metabolites and the biological activities has been
evaluated. The results from this investigation support the traditional usage of Gouania
longipetala for the management of infection diseases.

Acknowledgements
The author SD are grateful to the Third World Academy of Sciences (TWAS) and
The International Centre for Chemical and Biological Sciences for the award of ICCBS-TWAS
fellowship 2016 (Fr. 3240293185) and wish to thank Dr Tacham Walters for his assistance in the
collection and identification of plant samples.
 NATURAL PRODUCT RESEARCH 11

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Third World Academy of Sciences (TWAS) and The International Center for Chemical and
Biological Sciences for the award of ICCBS-TWAS fellowship 2016.

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