mostly in the heartwood and the bark (Pecha & Perez
2015). Previous studies on the compounds present in
P. juliflora revealed the presence of some alkaloids;
juliprosopine, juliflorine, prosoflorine and juliprosine
(Dos Santos et al. 2013; Henciya et al. 2017). The few
flavonoids in P. juliflora that have been reported so
far include mesquitol, catechin, quercetin, luteolin, 4’O-methylgallocatechin and kaempferol (Chepkwony
et al. 2020; Khandelwal, Sharma, & Agarwal 2016;
Sirmah et al. 2009).
2 EXPERIMENTAL
2.1 Reagents and chemicals
All solvents (water, formic acid, acetonitrile and
methanol) were HPLC grade apart from acetone
and dichloromethane which were of analytical grade
and were all purchased from Merck Company,
Germany.
2.2 Sample preparation and extraction
The P. juliflora stem samples were collected from
three different areas in Kenya; Baringo (0
◦ 28
0
N,
35
◦ 58
0
E), Turkana (03
◦ 09
N, 35
◦ 21
E) and Garissa
Counties (0
◦ 27
09
S, 39
◦ 38
45
E). The samples consisted of small trees aged less than 4 years and big
trees aged more than 8 years. The stems were further divided into five parts which included the bark,
sapwood, heartwood, knot wood and the pith. These
samples were air dried, then crushed and ground by
a Fritsch pulverisette 9 laboratory vibrating cup mill
at 1,200 rpm and finally sieved through a 115-mesh
sieve.
Serial extractions of the different samples were
done using the Accelerated Solvent Extractor Dionex
(ASE) using dichloromethane and acetone solvents in
order of increasing polarity. An ASE Dionex extraction
method was developed according to the method used
by Sirmah et al. (2009) with some modifications. The
extractions were performed on 8 g of sample powder at
100
◦ C using a 34 mL cell size. Each extraction entailed
three static cycles of 5 minutes each. The samples were
later concentrated to dryness by a rotary evaporator.
2.3 LC-ESI-MS/MS analysis
Liquid chromatography-mass spectrometry analyses
of samples were carried out using a Shimadzu (Noisiel, France) LC-20A ultra-HPLC (UHPLC) system
equipped with an auto sampler and interfaced to a
PDA UV detector SPD-20A, followed by an LC-MS
8030 triple-quadruple mass spectrometer. The separation was carried out at a flow rate of 0.4 mL/min on a
Luna C18 analytical column (inner diameter, 150 mm
by 3 mm; Phenomenex, Le Pecq, France) using a 10
minutes gradient as follows: starting from 2 % of acetonitrile which contained 0.1 % formic acid in water
also containing 0.1 % formic acid solution, acetonitrile proportion was increased linearly to 20 % in 3
min then to 80 % in 6.25 min. Initial conditions were
then reached in 0.25 min. The injection volume was
1 µL. UV-visible spectra were recorded between 190
and 800 nm.
Negative and positive electrospray mass spectrometric analyses were performed at a unit resolution
between 100 and 2,000 m/z at a scan speed of 15,000
U/s. The desolvation line and heat-block temperatures were 250
◦ C and 400
◦ C, respectively. Nitrogen
was used as a nebulizing (3 liters/min) and drying
(15 liters/min) gas. The ion spray voltage was +/4,500V. Approximately 10 mg of each of the acetonic
extracts were dissolved in 1000 µl of methanol before
aspiration into the LC-MS/MS.
3 RESULTS AND DISCUSSION
3.1 General
The identification of the phenolic compounds present
in P. juliflora by the LC-ESI-MS/MS was done in the
negative ion mode which described the corresponding
deprotonated pseudomolecular ions. This is because
it has been demonstrated that the negative ion mode
has higher sensitivity and selectivity compared to the
positive ion mode detection which generates a higher
background signal (Cuyckens & Claeys 2004; Gu et al.
2003; Sun, & Miller 2002). Another reason is because
of the free phenol groups present in the compounds
identified since acids deprotonate easily in the negative ion mode while they form adducts with cations
in the sample or mobile phase in the positive mode
(Swatsitang, Swatsitang, Robards, & Jardine 2000).
The analysis of the acetonic extracts revealed that
the major class of phenolic compounds found in P.
julifora was the flavonoids which either occurred as
aglycones and / or their glycosylated forms. According
to Sherwood and Bonello (2013), the sugar unit of phenolic glycoside serves to improve solubility for storage
in cell organs. Reference standards were used to
substantiate the identification of peaks whenever available otherwise, identification of the compounds was
obtained by comparing their molecular ions obtained
by the theoretical molecular weights from literature.
The LC chromatograms from the different regions
were studied and analyzed as shown in Figure 1.
The selected chromatograms are examples of some
of the many LC chromatograms obtained and show the
peaks corresponding to the different compounds that
were tentatively identified. Some peaks were found in
higher percentages in one geographic region compared
to the others while some peaks were found only in some
regions and were absent in some.
3.2 Phenolic glycosides
Peak 1, was tentatively identified as coniferin (abietin).
This is a glycosylated phenolic compound that usually
serves as an intermediate in cell wall lignification. It is
believed to aid in the transport of monolignols (Wang,
Chantreau, Chantreau, & Hawkins 2013). This peak
144
2015). Previous studies on the compounds present in
P. juliflora revealed the presence of some alkaloids;
juliprosopine, juliflorine, prosoflorine and juliprosine
(Dos Santos et al. 2013; Henciya et al. 2017). The few
flavonoids in P. juliflora that have been reported so
far include mesquitol, catechin, quercetin, luteolin, 4’O-methylgallocatechin and kaempferol (Chepkwony
et al. 2020; Khandelwal, Sharma, & Agarwal 2016;
Sirmah et al. 2009).
2 EXPERIMENTAL
2.1 Reagents and chemicals
All solvents (water, formic acid, acetonitrile and
methanol) were HPLC grade apart from acetone
and dichloromethane which were of analytical grade
and were all purchased from Merck Company,
Germany.
2.2 Sample preparation and extraction
The P. juliflora stem samples were collected from
three different areas in Kenya; Baringo (0
◦ 28
0
N,
35
◦ 58
0
E), Turkana (03
◦ 09
N, 35
◦ 21
E) and Garissa
Counties (0
◦ 27
09
S, 39
◦ 38
45
E). The samples consisted of small trees aged less than 4 years and big
trees aged more than 8 years. The stems were further divided into five parts which included the bark,
sapwood, heartwood, knot wood and the pith. These
samples were air dried, then crushed and ground by
a Fritsch pulverisette 9 laboratory vibrating cup mill
at 1,200 rpm and finally sieved through a 115-mesh
sieve.
Serial extractions of the different samples were
done using the Accelerated Solvent Extractor Dionex
(ASE) using dichloromethane and acetone solvents in
order of increasing polarity. An ASE Dionex extraction
method was developed according to the method used
by Sirmah et al. (2009) with some modifications. The
extractions were performed on 8 g of sample powder at
100
◦ C using a 34 mL cell size. Each extraction entailed
three static cycles of 5 minutes each. The samples were
later concentrated to dryness by a rotary evaporator.
2.3 LC-ESI-MS/MS analysis
Liquid chromatography-mass spectrometry analyses
of samples were carried out using a Shimadzu (Noisiel, France) LC-20A ultra-HPLC (UHPLC) system
equipped with an auto sampler and interfaced to a
PDA UV detector SPD-20A, followed by an LC-MS
8030 triple-quadruple mass spectrometer. The separation was carried out at a flow rate of 0.4 mL/min on a
Luna C18 analytical column (inner diameter, 150 mm
by 3 mm; Phenomenex, Le Pecq, France) using a 10
minutes gradient as follows: starting from 2 % of acetonitrile which contained 0.1 % formic acid in water
also containing 0.1 % formic acid solution, acetonitrile proportion was increased linearly to 20 % in 3
min then to 80 % in 6.25 min. Initial conditions were
then reached in 0.25 min. The injection volume was
1 µL. UV-visible spectra were recorded between 190
and 800 nm.
Negative and positive electrospray mass spectrometric analyses were performed at a unit resolution
between 100 and 2,000 m/z at a scan speed of 15,000
U/s. The desolvation line and heat-block temperatures were 250
◦ C and 400
◦ C, respectively. Nitrogen
was used as a nebulizing (3 liters/min) and drying
(15 liters/min) gas. The ion spray voltage was +/4,500V. Approximately 10 mg of each of the acetonic
extracts were dissolved in 1000 µl of methanol before
aspiration into the LC-MS/MS.
3 RESULTS AND DISCUSSION
3.1 General
The identification of the phenolic compounds present
in P. juliflora by the LC-ESI-MS/MS was done in the
negative ion mode which described the corresponding
deprotonated pseudomolecular ions. This is because
it has been demonstrated that the negative ion mode
has higher sensitivity and selectivity compared to the
positive ion mode detection which generates a higher
background signal (Cuyckens & Claeys 2004; Gu et al.
2003; Sun, & Miller 2002). Another reason is because
of the free phenol groups present in the compounds
identified since acids deprotonate easily in the negative ion mode while they form adducts with cations
in the sample or mobile phase in the positive mode
(Swatsitang, Swatsitang, Robards, & Jardine 2000).
The analysis of the acetonic extracts revealed that
the major class of phenolic compounds found in P.
julifora was the flavonoids which either occurred as
aglycones and / or their glycosylated forms. According
to Sherwood and Bonello (2013), the sugar unit of phenolic glycoside serves to improve solubility for storage
in cell organs. Reference standards were used to
substantiate the identification of peaks whenever available otherwise, identification of the compounds was
obtained by comparing their molecular ions obtained
by the theoretical molecular weights from literature.
The LC chromatograms from the different regions
were studied and analyzed as shown in Figure 1.
The selected chromatograms are examples of some
of the many LC chromatograms obtained and show the
peaks corresponding to the different compounds that
were tentatively identified. Some peaks were found in
higher percentages in one geographic region compared
to the others while some peaks were found only in some
regions and were absent in some.
3.2 Phenolic glycosides
Peak 1, was tentatively identified as coniferin (abietin).
This is a glycosylated phenolic compound that usually
serves as an intermediate in cell wall lignification. It is
believed to aid in the transport of monolignols (Wang,
Chantreau, Chantreau, & Hawkins 2013). This peak
144