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Morphological, phytochemical and anti-hyperglycemic evaluation of

Brachychiton populneus

Article in Revista Brasileira de Farmacognosia · July 2019

DOI: 10.1016/j.bjp.2019.05.001

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Original Article

Morphological, phytochemical and anti-hyperglycemic evaluation of

Brachychiton populneus
Alia Y. Ragheb a , Mona E.S. Kassem a , Moshera M. El-Sherei b
, Mona M. Marzouk a,∗
,
Salwa A. Mosharrafa a , Nabiel A.M. Saleh a
a
Department of Phytochemistry and Plant Systematics, National Research Centre, Giza, Egypt
b
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Giza, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Brachychiton populneus (Schott & Endl.) R.Br., Malvaceae, is one of five Brachychiton species cultivated in
Received 27 October 2018 Egypt. Little information was found concerning the morphological, phytochemical and biological investi-
Accepted 17 May 2019 gations of B. populneus. Morphological investigations of B. populneus were performed on fresh and dried
Available online xxx
leaves. Air-dried, ground leafy branches were extracted with 70% methanol/water yielding B. populneus
extract. Seventeen flavonoids were isolated and identified using different chromatographic and spectro-
Keywords: scopic techniques; eleven of them were reported for the first time from this plant. Potential activity of
Brachychiton
B. populneus extract against alloxan inducing oxidative stress and diabetes in male rats was preliminary
Macromorphology
Micromorphology
investigated (four groups of ten rats /group). B. populneus extract (500 mg/kg bw i.p.) exhibited significant
Flavonoids acute anti-hyperglycemic activity with blood glucose levels of 227.3 and 157.6 mg/dl after 4 and 24 h,
Anti-hyperglycemic activity respectively, compared to alloxan and standard Diamicron (5 mg/kg bw p.o.) groups, as well as to a nor-
Chemosystematics moglycemic control group at p < 0.05. The extract reverted the body weight values of the alloxan-induced
diabetic rats to that of control animals after 24 h. In addition, B. populneus extract counteracted the effect
of the oxidative stress induced by alloxan causing significantly increase in the glutathione content level
(2.35 mmol/l) and relative decrease in the malondialdehyde level (21.31 nmol/l) and nitric oxide content
(1.98 ␮mol/l) in serum after 24 h of treatment compared to alloxan-induced diabetic rats (1.01 mmol/l,
118.9 nmol/l, 4.69 ␮mol/l, respectively) and to normoglycemic control at p < 0.05. These effects appear
to be related to the flavonoid principles. The intergeneric relationship of the genus Brachychiton and
other related genera assessed well-supported differentiation between them. Furthermore, a significant
dissimilarity was observed at interspecific level.
© 2019 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Farmacognosia. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).

Introduction antioxidant and free radical scavenging effects (Dapkevicius et al.,

2002). On the other hand, flavonoids have wide structural diver-
Recently, in developing nations and industrialized countries the sity, a broad variety of biological effects and have been isolated
occurrence of diabetes is escalating. Imbalance between Reactive in a great scale from Brachychiton species (Jahan et al., 2014; El
Oxygen Species (ROS) and the antioxidant defense system medi- Sherei et al., 2016). They could also be used as taxonomic markers
ated oxidative stress is a key factor in the mechanism of several at interspecific and intergeneric levels (Crawford, 1978).
diseases including diabetes (Baynes and Thorpe, 1999). One of the The genus Brachychiton Schott and Endl., Malvaceae, contains
possible therapies for this condition is to enhance the antioxidant about 31 species growing wild in Australia (30 species) and New
resistance system by a suitable antioxidant remedy (a part from Guinea (one species) (Huxley et al., 1992). Based on some dis-
traditional treatment). Flavonoids and phenolic phytochemicals tinctive morphological features, that genus with five other genera
are considered to promote optimal health by protecting cellu- (Firmiana Marsili, Hildegardia Schott and Endl., Pterocymbium R.Br.,
lar components against free radical induced damage, due to their Pterygota Schott and Endl. and Scaphium Schott and Endl.) was
treated under the genus Sterculia (El Sherei et al., 2016). Some
biological activities have been reported for a number of species
belonging to the genus Brachychiton, viz; antidiabetic (Desoky and
∗ Corresponding author.
Youssef, 1997; Kassem et al., 2002; Abou Zeid et al., 2017), digestive
E-mail: mm.marzouk@nrc.sci.eg (M.M. Marzouk).

https://doi.org/10.1016/j.bjp.2019.05.001
0102-695X/© 2019 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Farmacognosia. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Ragheb, A.Y., et al. Morphological, phytochemical and anti-hyperglycemic evaluation of Brachychiton
populneus. Revista Brasileira de Farmacognosia (2019), https://doi.org/10.1016/j.bjp.2019.05.001
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system and urinary tract disorders (Keay, 1989), antischistosomal Botanical study
(Yousif et al., 2007), antimicrobial (Newbold et al., 1997; Yousif
et al., 2007; Abdel-Megeed et al., 2013), antioxidant (Abdel-Megeed The macromorphological investigations were described from
et al., 2013) and anti-inflammatory (Agyare et al., 2012). fresh materials or after keeping in 70% ethanol containing 5%
Five Brachychiton species are cultivated in Egypt; B. acerifolius glycerin by naked eye, with a hand lens or low magnification
(Cunn. ex G. Don) Macarthur, B. australis (Schott and Endl.) A. Ter- using a stereomicroscope. The leaves were air dried, reduced to
rac., B. discolor F. Muell., B. populneus (Schott and Endl.) R. Br. and fine powder and kept for microscopic examination. For detailed
B. rupestris (T. Mitch. ex Lindl.) K. Schum. B. populneus [Synonyms: micromorphological studies, mature leaves were fixed in FAA (40%
Sterculia diversifolia G. Don., B. populneus subsp. populneus (Schott formaldehyde solution, acetic acid, 70% ethanol (5:5:90). Trans-
and Endl.) R. Br. and Poecilodermis populnea Schott and Endl] is verse sections of lamina and petiole were investigated through
a small to medium sized ornamental tree, cultivated in gardens hand-microtome cross sections at 8–10␮. The sections were
and roadsides and commonly known as “Kurrajong or Bottelboom stained with safranin-light green or crystal violet-erythrosine com-
Tree”. The bark was used as fiber, and the soft spongy wood for bination according to the conventional methods (Abd-Elgawad and
making shields. The leaves are also used as emergency fodder Alotaibi, 2017), ruthenium red for testing mucilage and FeCl3 for
for drought-affected animal stock. The seeds are used as a coffee testing tannins (Pakravan et al., 2007). Epidermal peels of upper
supplement by roasting and crushing (Anderson, 2016). Petronici and lower surfaces were made. They were stained in 1% aque-
et al. (1970) identified eight fatty acids (palmitic, heptadecenoic, ous safranin solution 4–8 min, washed in water to remove excess
stearic, oleic, linoleic, eicosenoic, malvalic and sterculic acids) stain before mounting on clean slides in 10% glycerol (Metcalfe and
in B. populneus fruit, while Desoky and Youssef (1997) reported Chalk, 1979).
the isolation of three flavonol aglycones (kaempferol, quercetin
and isorhamnetin) and five flavonol glycosides (kaempferol Extraction, isolation and structure elucidation
3-O-␤-glucoside, kaempferol 3-O-rutinoside, kaempferol 3-O-(2 -
rhamnosylrutinoside), isorhamnetin 3-O-rutinoside and quercetin Air-dried, grounded leafy branches of B. populneus (1.7 kg)
3-O-arabinoside) from the stem bark. Recently, Batool et al. (2018) were extracted three times by repeated percolation (40–60 ◦ C)
specified rutin, catechin and myricetin and proposed the presence with 70% methanol/water till exhaustion then evaporated under
of antioxidant and hepatoprotective mediators in the methanol reduced pressure affording 80 g B. populneus extract (BPE). BPE
extract of leaves. Except for these publications, no previous phy- (70 g) was defatted with petroleum ether (40–60 ◦ C) then subjected
tochemical or biological investigations have been reported for B. to a polyamide column (150 × 5 cm). Stepwise gradient elution
populneus. Additionally, there are no micro-morphological and was carried out starting with water as eluent then decreasing
chemosystematic surveys concerning the studied species. the polarity by increasing the concentration of methanol (Mabry
Accordingly, it was deemed of interest to evaluate the morpho- et al., 1970). Fractions of 100 ml each were collected. Similar
logical features and detailed flavonoid investigations which could fractions were combined according to their paper chromatogra-
play a role in supporting the classification status, as well as, anti- phy (PC) properties using H2 O, 15% HOAc (H2 O–HOAc 85:15) and
hyperglycemic evaluation. BAW (n-BuOH–HOAc–H2 O 4:1:5, upper layer) as eluents to give
six main fractions (A-F). Fraction A (100% H2 O; 11 g) was re-
chromatographed over a Sephadex chromatographic column (CC)
(35 × 2.5 cm). Elution was carried out using a solvent system of
Material and methods methanol/water (1:1 v/v). It was subjected to preparative paper
chromatography (PPC) twice using 15% HOAc as eluent, then on a
General Sephadex LH-20 column (35 × 2.5 cm) using MeOH to yield com-
pounds 1 (42 mg) and 2 (49 mg). Fraction B (20% MeOH/H2 O; 8.5 g)
1 H NMR experiments were recorded on a Jeol EX-500 spec-
was re-chromatographed over a Sephadex CC (35 × 2.5 cm) using
trometer: 500 MHz (1 H NMR), 125 MHz (13 C NMR). UV:UV H2 O followed by PPC using BAW as eluents, then on a Sephadex LH-
spectrophotometer (Shimadzu UV-240). EI–MS: Finnigan-Mat SSQ 20 column (35 × 2.5 cm) using MeOH to yield compound 3 (23 mg).
7000 spectrometer. Oxidative stress biomarkers were determined Fraction C (40% MeOH/H2 O; 10.5 g) was subjected to Sephadex CC
using double beam spectrophotometer (Schimadzu, Japan). Col- (35 × 2.5 cm) using H2 O as eluent. It was purified by PPC using
umn chromatography; Polyamide S6 (Riedel-De-Haen AG, Seelze BAW (double solvent) followed by 15% HOAc, then on a Sephadex
Haen AG, Seelze Hanver, Germany) using MeOH/H2 O as elu- LH-20 column (35 × 2.5 cm) using MeOH to yield compounds 4
ent. Paper chromatography (descending); Whatman No. 1 and (39 mg), 5 (52 mg), and 6 (47 mg). Fraction D (60% MeOH/H2 O;
3 MM papers. Sephadex LH-20 (Pharmacia). Authentic samples 14 g) was subjected to PPC using BAW as eluent. Subsequently, it
were obtained from the Phytochemistry and Plant Systematics was applied to Sephadex CC (35 × 2.5 cm) using methanol/water
Department, NRC. EOS Canon camera. Olympus Microscope, CX 41. (1:1 v/v) as eluent. The isolated compounds were further purified
Olympus digital camera, E-330. OK glucometer (Lifescan, Milpitas, by re-chromatography on Sephadex LH-20 column (60 × 1.5 cm)
CA). using methanol as eluent yielding compounds 7 (8 mg) and 8
(11 mg). Fraction E (80% MeOH/H2 O; 11.5 g) yielded compounds
9 (48 mg), 10 (18 mg), 11 (11.5 mg) and 12 (19 mg) by applying it
to a Sephadex CC (35 × 2.5 cm) using methanol/water (1:1 v/v) as
Plant material eluent. Fraction F (100% MeOH; 13 g) was also chromatographed
on PPC using BAW followed by 50% HOAc then on Sephadex LH-20
Fresh leafy branches of Brachychiton populneus (Schott & Endl.) column (35 × 2.5 cm) using MeOH to yield compounds 13 (7.5 mg),
R.Br., Malvaceae, were collected in March, 2012 from Orman Botan- 14 (18 mg), 15 (11 mg), 16 (16 mg) and 17 (6 mg).
ical Garden, Giza, Egypt. The plant was kindly identified by Dr. M. The structures of the compounds were elucidated using Rf val-
El Gibali, former researcher of botany, National Research Centre ues, colour reactions, chemical methods (acid hydrolysis and FeCl3
(NRC), Egypt. A voucher specimen was kept at the Herbarium of oxidative hydrolysis) as well as spectral analysis (UV, MS and
the Department of Pharmacognosy No. (21-12-2016 II), Faculty of NMR). Further confirmation was performed through comparing
Pharmacy, Cairo University. with authentic samples and/or published data. Acid hydrolysis for

Please cite this article in press as: Ragheb, A.Y., et al. Morphological, phytochemical and anti-hyperglycemic evaluation of Brachychiton
populneus. Revista Brasileira de Farmacognosia (2019), https://doi.org/10.1016/j.bjp.2019.05.001
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Fig. 1. Photographs of Brachychiton populneus (Schott & Endle.) R. Br. (A) Whole tree in its natural habitat (X = 0.0058), a close up view to flowers (X = 0.2), (B) alternate
arrangement of the leaves (X = 0.135), (C) the upper surface of the leaf (X = 0.39), (D) the lower surface of the leaf (X = 0.39), (E) flowers; 1, male flower, 2, female flower
(X = 0.14), F) fruits in star-like cluster (X = 0.25), (G) opened fruit (X = 0.47), (H) seeds (X = 0.75).

O-glycosides (2 N HCl, 2 h, 100 ◦ C) were carried out and followed by Research Centre (Dokki, Giza, Egypt), and acclimatised with free
co-PC with authentic samples to identify the aglycones and sugar access to food (standard laboratory pellets of 20% protein, 5% fats,
moieties. The sugar units of C-glycosyl flavonoids were determined and 1% vitamins) and tap water for at least one week at room
using ferric chloride oxidative hydrolysis (20% FeCl3 , 6 h) followed temperature at 23–25 ◦ C. The animals were fasted for 24 h before
by co-PC with a standard sugar mixture using BBPW (benzene, induction of hyperglycemia but allowed free access to water. All
n-butane, pyridine, water (1:5:3:3)) as eluent (Mabry et al., 1970). animal procedures were performed after approval by the National
Research Centre Medical Ethics Committee (09085) and in accor-
dance with the recommendations of the proper care and use of
Biological assay laboratory animals.

Animals
Ten albino mice (weighing 25–30 g) were used for determina- Experimental design
tion of LD50 and forty male albino rats, eight weeks old (weighing Ten healthy rats were treated with saline and served as a nor-
120–150 g) obtained from the breeding colonies of the National moglycemic group (Control). Diabetes was induced by a single

Please cite this article in press as: Ragheb, A.Y., et al. Morphological, phytochemical and anti-hyperglycemic evaluation of Brachychiton
populneus. Revista Brasileira de Farmacognosia (2019), https://doi.org/10.1016/j.bjp.2019.05.001
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Fig. 2. Micromorphology of the lamina of Brachychiton populneus (Schott & Endle.) R. Br. (A) T.S. of the lamina (X = 100), B) detailed T.S. in the leaf showing midrib region
(X = 240), C) detailed T.S. in the leaf showing lamina region (X = 160), c.b.; central bundles, col.; colenchyma, l.ep.; lower epidermis, lam.; lamina, m.c.; mucilage cavity, m.r.;
medullary rays, mid.; midrib, pal.; palisade cells, par.; parenchyma, par. t.; parenchyma with tannin content, per.f.; pericyclic fibers, ph.; phloem, sp.t.; spongy tissue, th.cu.;
thick cuticle, u.ep.; upper epidermis, v.b.; vascular bundles, v.t.; vascular tissue, x.v.; xylem vessel.

injection of a freshly prepared alloxan monohydrate (Sigma, No. Oxidative stress biomarkers
242-646-8) according to rat weight at dose (150 mg/kg, i.p.) to Serum was separated from blood samples (at 24 h) by cen-
overnight-fasted rats (Rao et al., 2001). After a period of three days trifugation at 1008g-force for 10 min at 4 ◦ C. The clear sera were
the rats which did not develop more than 200 mg/dl glucose lev- immediately stored at −70 ◦ C in a clean plastic eppendorf for the
els, were rejected. The alloxan-induced diabetic rats were further subsequent determination of glutathione content (GSH), malondi-
classified into three groups with ten rats in each group. Group aldehyde level (MDA) and nitric oxide content (NO).
1 received saline and served as hyperglycemic group. Group 2
received Diamicron (5 mg/kg, p.o.) and was given as standard group.
Group 3 received BPE (500 mg/kg, i.p.). After injection of BPE, blood Determination of serum reduced glutathione content. Reduced glu-
samples were withdrawn from the tail vein of the rats at zero, 4 tathione content of serum was measured according to method
and 24 h. The collected samples were kept into covered test tubes. of Bulaj et al. (2002). In centrifuge tubes containing 0.5 ml of
precipitating solution (10% trichloroacetic acid-6 mM Na2 EDTA),
50 ␮l of serum was added, vortexed and centrifuged at 448g-force
Determination of blood glucose level (BGL) for 5 min. 0.1 ml of the resulting clear supernatant was added
The blood glucose level was determined by the glucose-oxidase to 1.85 ml of potassium phosphate buffer (100 mM, pH 8) and
principle (Beach and Turner, 1958) using the OK glucometer and 0.1 ml of Ellman’s reagent [5,5’ -dithiobis (2-nitrobenzoic acid)]
results were reported as mg/dl (Rheney and Kirk, 2000). and mixed thoroughly. After 5 min, the absorbance was measured

Please cite this article in press as: Ragheb, A.Y., et al. Morphological, phytochemical and anti-hyperglycemic evaluation of Brachychiton
populneus. Revista Brasileira de Farmacognosia (2019), https://doi.org/10.1016/j.bjp.2019.05.001
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Fig. 3. Micromorphology of the petiole of Brachychiton populneus (Schott & Endle.) R. Br. (A) T.S. of the petiole (X = 70), (B) detailed T.S. in the petiole (X = 400), cam;
cambium, c.b.; central bundles, cl.; clusters of calcium oxalate, col.; collenchyma, ep.; epidermis, m.r.; medullary rays, m.c.; mucilage cavity, p.; pith, par.; parenchyma, par.
t.; parenchyma with tannin content, per.f.; pericyclic fibers, ph.; phloem, th.cu.; thick cuticle, x.v.; xylem vessel.

at 412 nm using a double beam spectrophotometer (Schimadzu, deproteinize it. Samples were then centrifuged at 16,128g-force
Japan) against reagent blank. The GSH level in serum was expressed for 20 min using cooling centrifuge (Hermle, Germany). 250 ␮l
as mmole/l and calculated from the following formula: GSH content of NEDD reagent [N-(1-naphthyl) ethylenediaminedihyrochloride
At
(mmole/l) = At/As × Cs × dilution factor As × Cs × dilutionfactor, 0.1% (w/v) in distilled water] was added to 250 ␮l of the obtained
Where: At = absorbance of test sample, As = absorbance of standard supernatant, and incubated at 37 ◦ C for 10–15 min afterward 250 ␮l
and Cs = concentration of standard. of sulphanilamide solution [2% (v/v) in distilled water] was added
and incubated at 37 ◦ C for 10–15 min. The mixture was cooled and
Determination of thiobarbituric acid reactive substances the absorbance of the pink coloured chromophore was measured
Lipid peroxide levels were measured in serum as thiobarbi- at 540 nm using a double beam spectrophotometer (Schimadzu,
turic acid reactive substances (TBARS). Malondialdehyde (MDA), Japan) against a reagent blank where 250 ␮l distilled water was
one of the degradation products of lipid peroxides, was used used instead of the sample. The level of total nitrite/nitrate (NOx )
as standard (Uchiyama and Mihara, 1978). In centrifuge tubes was expressed as ␮mol/l and was calculated by: NOx (␮mol/l) =
containing 0.5 ml of precipitating solution (10% trichloroacetic At/As × Cs × dilution factor, where At: absorbance of the test sam-
acid-6 mM Na2 EDTA), 50 ␮l of serum was added, vortexed and ple, As: absorbance of the standard sample and Cs: concentration
centrifuged at 448g-force for 5 min. 0.5 ml of the resulting clear of standard.
supernatant was added to 3 ml of ortho-phosphoric acid (1%, v/v)
and 1 ml of thiobarbituric acid (0.67%, w/v) then heating for 20 min Statistical analysis
at 100 ◦ C, the mixture was cooled and 4 ml of n-butanol were All values are presented as means ± SEM (standard error of the
added and mixed vigorously, then separated by centrifugation at means) for ten rats in each group. Comparison between groups
1008 × g-force for 10 min. Optical density was measured against was carried out using the non-parametric one-way analysis of vari-
reagent blank at 532 nm using a double beam spectrophotometer ance (ANOVA) followed by Tukey-HSD multiple comparisons test to
(Schimadzu, Japan). TBARS concentration in serum was expressed judge the difference between various groups. Difference was con-
as nmole MDA/l and calculated from the following equation: sidered significant when p < 0.05. Graph pad software (version 7.00)
TBARS (nmole/l) = ODt/ODs × Cs × dilution factor, Where: OD: Opti- and Excel 10 were used to carry out these statistical tests and plot
cal density, Cs: Concentration of standard malondialdehyde (MDA) graphs.
solution.
Results and discussion
Determination of nitric oxide content. Nitric oxide (NO), an unsta-
ble reactive nitrogen free radical, was determined in the present Botanical study
study using biochemical method of Montgomery and Dymock
(1961) where the production of NO is expressed by endogenous Macro-morphological study
nitrate/nitrite metabolites. In a test tube, 0.25 ml of zinc sul- Brachychiton populneus is a bottle-shaped taper like tree, with
phate (10%) was added to 0.25 ml serum and left for 15 min to dark brown bark and crowded leaves at the end of branches mainly

Please cite this article in press as: Ragheb, A.Y., et al. Morphological, phytochemical and anti-hyperglycemic evaluation of Brachychiton
populneus. Revista Brasileira de Farmacognosia (2019), https://doi.org/10.1016/j.bjp.2019.05.001
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Fig. 4. Powdered leaf of Brachychiton populneus (Schott & Endle.) R. Br. (A) Fragment of upper epidermis (X = 400), (B) fragment of lower epidermis showing anomocytic
stomata (X = 280), C) fragment of neural epidermal cells (X = 160), an; annular xylem vessel, ano.st.; anomocytic stomata, cl.; clusters of calcium oxalate (X = 350), pal., palisade
cells (X = 400), per.f.; pericyclic fiber (X = 260,700), pt.; pitted xylem vessel, s.cr.; solitary crystal (X = 133), sc.; scalariform xylem vessel, sp.x.v.; spiral xylem vessel (X = 300),
tr.; tracheid, w.f.; wood fiber (X = 180, 533), x.v.; xylem vessel (X = 180).

Table 1
Effect of Brachychiton populneus extract on blood glucose level (BGL) and body weight of alloxan-induced diabetic rats.

Treatments BGL (mgdl) BW (g)

0h 4h 24 h 0h 24 h % changes

Normoglycemic 97.8 ± 4.49 94.60 ± 8.16 95.00 ± 6.78 128.33 ± 3.15 147 ± 2.23 14.39 ± 2.09
Hyperglycemic 551.10 ± 22.90b 488.33 ± 46.12b 448.50 ± 43.07b 127.5 ± 0.92 108.33 ± 5.57a,c 15.07 ± 4.15
Standard control (Diamicron) 502 ± 20.08b 405.42 ± 24.08b 161.42 ± 11.15a 126.33 ± 3.62 138.925 ± 1.7b 9.97 ± 3.70
BPE 387.5 ± 42.11b,a 227.3 ± 30.28 157.6 ± 16.57b,a 121 ± 3.26 122.73 ± 4.67a,b 1.43 ± 2.25

Values are expressed as mean ± SEM, n = 10.Values are statistically significant at p < 0.05.
a
Significant from saline normoglycemic control.
b
Significant from Alloxan hyperglycemic control
c
Significant from diamicron standard control.

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distributed in coastal and sub-coastal areas (Fig. 1A). Leaves are 2–3 rows of parenchyma cells. The collenchymatous cells are small
simple, grayish-green in colour, glabrous and glossy, cauline, alter- rounded cells with thick cellulosic walls and no intercellular spaces
nate, exstipulate, petiolate (2–5 cm). It is ovate to broadly ovate while the parenchymatous cells appeared large, rounded with thin
with acuminate apex (5–11 cm in length and 2–6 cm in width), cellulosic walls and narrow intercellular spaces. The endodermis
sinuate margine and cuneate base, showing pinnate reticulate is parenchymatous and indistinct. Pericycle is sclerenchymatous;
venations and having papery to coriaceous texture, no odour and formed of patches of lignified and non-lignified fibers encircling the
a characteristic taste (Fig. 1B–D). Inflorescence is terminal or short vascular tissue. The fibers are long showing wide lumina and blunt
axillary panicles, 12–15 cm long, 10–15 flowered (Fig. 1A). Flow- apices (Fig. 2B, 4). Vascular stele (Fig. 2A, B) appeared as crescent
ers are pedicellate (0.3–1.5 cm), epicalyx absent, calyx consists of shaped arch extended to the lower side and another two smaller
five lanceolate sepals with entire margin and acuminate apex, off- upper inverted ones of collateral type showing the phloem towards
white with reddish dots inside in colour, slightly pubescent outside, outside and the xylem inside. 2–3 Small central collateral bundles
glabrous inside. The sepals united and form bell shape (campan- surrounded by strands of non-lignified sclerenchymatous fibers are
ulate calyx), corolla absent. Androecium is formed of ten yellow, present. Phloem consists of sieve elements, companion cells and
glabrous anthers, showed as a capitate-globose cluster on a white, phloem parenchyma. Cambium is undifferentiated. The xylem is
glabrous androphore (male flower). Gynoecium is consisting of formed of vessels, wood parenchyma and wood fibers. The ves-
ovoid, sessile, tomentose ovary, with a ring of ten vestigial anthers sels have annular, spiral, scalariform and pitted thickenings (Fig. 4).
at its base, 5-carpels, axial placentation with long and tomen- The wood parenchyma is rectangular, slightly elongated cells, with
tose style and curved and lobed stigma (female flower) (Fig. 1E). slightly thin pitted walls (Fig. 4). The wood fibers are fusiform,
Fruits are dark brown woody follicles (5–12) in star-like clusters, having slightly thick, lignified walls, wide lumina and blunt apices
pubescent, dehiscent, boat-shaped, with a size of (4–7) × (1–2.5) (Fig. 4). The medullary rays are uni-, bi- and multi-seriate of rect-
cm and (3–6) cm woody stalk (Fig. 1F, G). Seeds are ovoid and angular thin walled cellulosic parenchyma cells (Fig. 4). Calcium
angular in shape with hairy surface that may cause skin and eye oxalate clusters and prisms are few, scattered in the outer and
irritation, brownish yellow in colour, ranged from 10 to 15 with inner ground tissues and phloem. Parenchymatous cells contain-
dimensions of (0.5–0.8) × (0.2–0.5) cm (Fig. 1H). ing tannins (FeCl3 test) are present. Mucilage cavities (idioblast)
are present in the upper outer ground tissue and lamina (Fig. 2A,
Micromorphological study B).
Lamina. The transverse section of lamina appeared biconvex
slightly more prominent in the lower (Fig. 2A). The upper and lower
epidermises are devoid from the glandular and non-glandular tri-
chomes. The leaf displayed a dorsiventral mesophyll interrupted in
the midrib region with collenchymatous cells. Idioplasts contain-
ing calcium oxalate clusters and prisms, mucilage (cavities) and Petiole. The petiole transverse section is nearly circular in outline
tannin (cells) are present. Upper epidermis is formed of one row of (Fig. 3A). Epidermis followed by a relatively narrow cortex sur-
radially elongated cells with thick cuticle, as seen in the transverse rounding a closed ring of collateral vascular bundles capped by
section (Fig. 2A, C). In surface view, the cells appeared polygo- patches of pericycle fibers. The pith is relatively narrow with 5–6
nal, nearly isodiametric with straight to slightly wavy anticlinal fine central vascular bundles. Epidermis resemble that of neural
walls, covered with faintly striated cuticle and showing no stomata region, composed of radially elongated cells with straight anticlinal
(Fig. 4A). Lower epidermis is formed of one row of radially elon- walls, covered with faintly striated cuticle and showing no stomata.
gated cells with thick cuticle, as observed in the transverse section Cortical tissue consists of 2–4 rows of small rounded collenchyma-
(Fig. 2A, C). In surface view, the cells appeared polygonal, nearly tous cells with thick cellulosic walls and no intercellular spaces
isodiametric with straight to slightly wavy and beaded anticlinal followed by 3–7 large polygonal to oval parenchymatous cells with
walls, covered with faintly striated cuticle and showing anomocytic thin cellulosic walls and narrow intercellular spaces. Mucilage cav-
stomata. The stomata are with wide osteoles and surrounded by ities (5–7) are scattered. Calcium oxalate clusters and prisms are
3–5 subsidiary cells (Fig. 4B). Neural epidermis (upper and lower) Frequent. Parenchymatous cells containing tannins are present
composed of radially elongated cells; the lower neural epidermal (Fig. 3). The Endodermis is parenchymatous and indistinct. Peri-
cells are smaller in size compared to the upper ones covered with cycle appeared as incomplete ring interrupted with parenchyma
thick cuticle, as viewed in the transverse section (Fig. 2A, B). In sur- cells, sclerenchymatous; formed of strands of long lignified fibers
face view, it showed polygonal, axially elongated cells with straight encircling the vascular tissue with wide lumina and blunt apices
and beaded anticlinal walls. They are covered with faintly striated (Fig. 4). Vascular stele appeared as an interrupted closed ring of col-
cuticle and devoid of stomata (Fig. 4C). Mucilage was detected in lateral vascular bundles, showing the phloem directed to the outer
most of the epidermal cells (ruthenium red test). Minute clusters side and the xylem extended towards inner one. 5–6 Small collat-
and prisms of calcium oxalate crystals were rarely seen. Mesophyll eral central bundles were scattered (Fig. 3A). The phloem is formed
is heterogeneous and dorsiventral composed of 2–3 layers of pal- of sieve elements, companion cells and phloem parenchyma. The
isade cells discontinuous in the midrib region, occupying half of cambium is represented by 3–4 rows of tangentially elongated, thin
the leaf thickness. The cells are closely packed, radially elongated walled meristematic cells (Fig. 3B). The xylem is formed of ves-
columnar, containing choloroplasts, with thin and straight anticli- sels, tracheids, wood parenchyma and wood fibers. The vessels are
nal walls. Lower layers are shorter than the upper ones abutting annular, spiral, pitted and scalar form. The wood parenchyma is
the upper epidermis. The spongy tissue is formed of 3–5 layers of polygonal elongated cells, having wide lumina with slightly pitted
rounded, thin walled and slightly irregular chlorenchyma cells with lignified walls. The wood fibers are fusiform having slightly thick,
intercellular spaces, occupying half of leaf thickness. Few calcium lignified walls, wide lumina and blunt to pointed apices (Fig. 4). The
oxalate clusters and prisms are dispersed through. Small collateral medullary rays are uni-, bi- and multi-seriate of rectangular thin
vascular bundles are scattered in the mesophyll in the small lateral walled cellulosic parenchyma cells. Pith is formed of 5–7 rows of
veins (Fig. 2C). Cortical tissue of the midrib region (Fig. 2B) in the large thin walled rounded parenchyma cells and frequent calcium
upper region shows 2–3 rows of subepidermal collenchyma cells oxalate clusters and prisms. Parenchymatous cells containing tan-
followed by 8–12 rows of parenchyma cells. The lower layer con- nins are present. 8–10 scattered mucilage cavities are also present
sists of 1–2 rows of subepidermal collenchyma cells followed by (Fig. 3A).

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Phytochemical investigation Youssef, 1997), while quercetin 3-O-glucoside is characteristic for

B. australis only (Kassem et al., 2002) and quercetin 3-O-galactoside
Isolation and structural elucidation of flavonoid compounds for B. acerifolius (De Laurentis et al., 2003). Moreover, quercetin
Seventeen flavonoid compounds were isolated and identified 3-O-rhamnoside was reported in B. discolor (Kassem, 2007).
from the leafy branches of B. populneus. They were identified The 3-O-rutinoside as well as 3-O-(2 -rhamnosyl-rutinoside) of
as apigenin 8-C-␤-glucopyranoside (vitexin 1) (Marzouk et al., kaempferol and isorhamnetin were isolated from B. populneus and
2008), apigenin 6-C-␤-glucopyranoside (isovitexin 2) (Peng et al., B. rupestris, while the 3-O-(2 -rhamnosyl robinoside) of kaempferol
2008), apigenin 6,8-di-C-␤-glucopyranoside (vicenin 3) (Hussein and isorhamnetin were characteristic for B. rupestris (Desoky
et al., 2009), isorhamnetin 3-O-␤-rutinoside (4) (Bragg et al., and Youssef, 1997). Furthermore, quercetin 3-O-rutinoside was
1978), quercetin 3-O-␤-rutinoside (rutin 5) (Marzouk et al., 2016), detected in B. populneus, B. acerifolius and B. australis while isorham-
kaempferol 3-O-␤-rutinoside (6) (Marzouk et al., 2016), lute- netin 3-O-rutinoside was characteristic for B. populneus, B. rupestris
olin 7-O-␤-glucuronide (7) (El Sherei et al., 2018), apigenin and B. australis (Kassem et al., 2002; De Laurentis et al., 2003; Farag
7-O-␤-glucuronide (8) (El Sherei et al., 2018), quercetin 3-O-␣- et al., 2015).
rhamnopyranoside (quercetrin 9) (Kassem et al., 2013), kaempferol C-glycosyl flavones were common in B. populneus and repre-
3-O-␤-glucopyranoside (astralagin 10) (Marzouk et al., 2012), lute- sented as vitexin, isovitexin and vicenin, the later compound was
olin 7-O-␤-glucopyranoside (11) (Marzouk et al., 2008), apigenin also found in B. acerifolius along with two anthocyanin glycosides
7-O-␤-glucopyranoside (12) (El Sherei et al., 2018), isorhamnetin (Farag et al., 2015).
(13) (Hussein et al., 2009), quercetin (14) (Marzouk et al., 2010), Hence, the flavonoid profiles of the five Brachychiton species
apigenin (15) (El Sherei et al., 2018), luteolin (16) (El Sherei et al., growing in Egypt may provide useful taxonomic differences at the
2018), and kaempferol (17) (Kawashty et al., 2012). Ten compounds infraspecific level, where B. populneus and B. acerifolius sharing
(1-3, 7-9, 11, 12, 15 and 16) were isolated for the first time from three flavonoid groups (flavones, flavonols and C-glycosyl flavones)
the plant species B. populneus.

Chemosystematic significance
Chemotaxonomic studies constitute one of the most impor- which differ from the other species by sharing a flavonol group only.
tant methods of determining the taxonomic positions of taxa. B. acerifolius is also characterized by the synthesis of anthocyanin
From the interspecific point of view, five of approximately 31 nuclei.
accepted species of Brachychiton, including the present one, are At the intergeneric level, no report regarding the flavonoid
known to produce around 27 flavonoid compounds belonging to constituents of Pterocymbium and the survey of the other genera
four classes containing seven flavones, fifteen flavonols, three C- (Brachychiton, Firmiana, Hildegardia, Pterygota, Scaphium and Ster-
glycosyl flavones and two anthocyanins (Table 3). The flavones culia) showed a wide range of flavonoid compounds (Table S1).
are characteristic of B. populneus and B. acerifolius; they are repre- They occurred commonly as glycosides of flavones and flavonols.
sented as apigenin and luteolin along with their 7-O-glucoside and The flavone glycosides are present as 7-O-glucoside and/or 7-O-
7-O-glucuronide (De Laurentis et al., 2003 and Farag et al., 2015). glucuronide of apigenin and luteolin distributed among the species
Only one flavone diglycosides (apigenin 7-O-(2 -␣-rhamnoside)- of Brachychiton, Pterygota and Sterculia, while those of chrysoe-
O-␤-glucuronide) was recently reported for B. acerifolius (Abou Zeid riol are characteristic for genus Sterculia (Xia, 2009; Hossain et al.,
et al., 2017). 2012). The 6- or 8-hydroxyflavones (scutellarein, isoscutellarein,
The flavonols are recognised in the five species and are repre- 6-hydroxyluteolin and hypolaetin) were also detected and charac-
sented as four aglycones (kaempferol, quercetin, rhamnetin and teristic for S. foetida.
isorhamnetin), and eleven glycoside derivatives of kaempferol, Glycosylation of flavonols were often substituted on the OH
quercetin and isorhamnetin. Quercetin aglycone is common among group at position 3 for kaempferol, quercetin and/or isorham-
the five Brachychiton species (Desoky and Youssef, 1997; Kassem netin. Members of the genus Firmiana are characterized by mono
et al., 2002; De Laurentis et al., 2003; Kassem, 2007; Farag et al., and/or di-glycosides of kaempferol and/or quercetin, but the 7-
2015), while kaempferol and isorhamnetin were reported for B. O-glucoside of kaempferol and isorhamnetin were reported for
populneus, B. acerifolius and B. rupestris (Desoky and Youssef, 1997; Scaphium scaphigerum (G. Don) Guib. and Planch. (Petchlert et al.,
De Laurentis et al., 2003; Farag et al., 2015) as well as rhamnetin 2012). The only flavonol-O-acyl glycoside derivative; kaempferol 3-
which is characteristic for B. discolor (Kassem, 2007). The glycosy- O-␤-(4 -p-coumaryl)-glucopyranoside was recorded in P. alata (El
lation patterns of flavonols were generally represented at position Sherei et al., 2018). On the other hand, two tetra methylated deriva-
3. They are reported as 3-O-monoglycoside of kaempferol and tives of quercetin were reported in S. foetida only (Anjaneyulu and
quercetin, 3-O-diglycosides (rutinoside) of kaempferol, quercetin Murty, 1981).
and isorhamnetin as well as 3-O-triglycosides of kaempferol and C-glycosylflavones occurred as mono- and di-glycosides. Two
isorhamnetin (Table 3). Kaempferol 3-O-glucoside and quercetin mono-C-glucosides of apigenin were recorded: vitexin in Sterculia
3-O-arabinoside are characteristic for B. populneus (Desoky and colorata Roxb. (Rajasekharreddy and Pathipati, 2014) and B. pop-

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Table 2 Therefore, the variability of the flavonoid pattern of the

Effect of Brachychiton populneus extract on serum reduced glutathione content
six related genera (Brachychiton, Firmiana, Hildegardia, Ptery-
(GSH), malondialdehyde level (MDA) and nitric oxide content (NO) of Alloxan-
induced diabetic rats. gota, Scaphium and Sterculia) suggested that flavones, 6- or
8-hydroxyflavones, flavonols, isoflavones, flavans, isoflavans, C-
Treatments Parameters
glycosyl flavonoids and anthocyanins could be used as chemosys-
GSH (mmol/l) MDA (nmol/l) NO (␮mol/l) tematic markers to differentiate between them (Table S1). These
Normal saline 2.44 ± 0.02 70.35 ± 1.05 2.47 ± 0.17 related genera are not only chemically different, but are also
BPE 2.35 ± 0.03b 21.31± 1.88b 1.98 ± 0.15b morphologically dissimilar. The macro-morphological characters
Alloxan 1.01 ± 0.12a 118.9 ± 5.29a 4.69 ± 0.48a displayed considerable variation in for example leaf (type, phyl-
Values are expressed as mean ± SEM, n = 10.Values are statistically significant at p < lotaxis, shape, size, apex, margin, base, venation, texture and
0.05. petiole), inflorescence, fruit (dehiscence, surface, shape, size and
a
Significant from saline normoglycemic control. color), as well as, seed (number, color, shape, surface and dimen-
b
Significant from Alloxan hyperglycemic control. sions). The main micro-morphological characters differentiating
the genera are the absence or presence of non-glandular and glan-
dular trichomes, their features and the epidermal cells characters
ulneus, while isovitexin was reported in B. populneus. Apigenin (Ragheb, 2017).
6,8-di-C-glucoside was reported in S. foetida (Xia et al., 2009), B.
acerifolius (Farag et al., 2015), P. alata (El Sherei et al., 2018) and Biological investigation
B. populneus, while other reported di-C-glycosides of apigenin and Acute toxicity estimation of BPE
luteolin were characteristic for P. alata (El Sherei et al., 2018). Acute lethal toxicity estimation of BPE; using the graphical
Finally, the 6-C-␤-glucopyranoside-7-O-␤-glucopyranoside of api- method in mice (Lorke, 1983), showed 50% mortality of mice up
genin and luteolin were also characterized for P. alata (El Sherei to 7.211 g/kg with no adverse effects. Referring to conversion table
et al., 2018). of Paget and Barnes (1964), the LD50 of mice was altered to rat dose
A single isoflavone structure with a C-glucosyl substitution at and calculated to be found 3.1 g/kg. Therefore, a dose of 500 mg/kg
position 8 (puerarin) had been reported in S. foetida (Xia et al., bw of the BPE was selected as an average dosing schedule for the
2009). The only flavans and isoflavans were reported in Hilde- biochemical studies.
gardia barteri (Mast.) Kosterm. (Meragelman et al., 2005). Finally,
anthocyanins were mainly reported as 3-O-glycoside derivatives of Anti-hyperglycemic effect of BPE
pelargonidin and cyanidin in Sterculia parviflora Roxb. and Sterculia Effect of BPE on BGL and BW. The effect of i.p. injection of the
kunstleri King. (Lowry, 1971), B. acerifolius (Farag et al., 2015). BPE (500 mg/kg) on blood glucose level (BGL) and body weight

Table 3
Flavonoid constituents of the five Brachychiton species growing cultivated in Egypt.

Compound B. populneus B. acerifolius B. rupestris B. discolor B. australis

Flavones
Apigenin +a +c – – –
Apigenin 7-O-␤-D-glucoside +a +c – – –
Apigenin 7-O-␤-D-glucuronide +a +c – – –
Apigenin 7-O-(2-̈␣-rhamnoside)-␤- glucuronide – +c – – –
Luteolin +a +c – – –
Luteolin 7-O-␤-glucoside +a – – – –
Luteolin 7-O-␤-glucuronide +a +c – – –
Flavonols
Kaempferol +a,b +c +b – –
Kaempferol 3-O-␤-glucoside +a,b – – – –
Kaempferol 3-O-rutinoside +a,b – +b –
Kaempferol 3-O-(2¨,6-̈dirhamnosyl)-␤-glucoside [K 3-O-(2-̈rhamnosylrutinoside)] +b – +b – –
Kaempferol 3-O-(2¨,6-̈dirhamnosyl)-␤-galactoside [K 3-O-(2-̈rhamnosylrobinoside)] – – +b – –
Quercetin +a,b +c +b +d +e
Quercetin 3-O-arabinoside +b – – – –
Quercetin 3-O-rhamnoside (quercitrin) +a – – +d –
Quercetin 3-O-␤-glucoside – – – – +e
Quercetin 3-O-galactoside (hyperoside) – +c – – –
Quercetin 3-O-(6-̈␣-rhamnosyl)-␤-glucoside (rutin) +a +c – – +e
Quercetin 7-methyl ether (rhamnetin) – – – +d –
Quercetin 3’-methyl ether (isorhamnetin) +a,b +c +b – –
Isorhamnetin 3-O-rutinoside +a,b – +b – +e
Isorhamnetin 3-O -(2¨,6-̈dirhamnosyl)-␤-D-galactoside [I 3-O-(2-̈rhamnosylrobinoside)] – – +b – –
C-Glycosylflavonoids
Apigenin 6-C-␤-glucopyranoside (isovitexin) +a – – – –
Apigenin 8-C-␤-glucoside (vitexin) +a – – – –
Apigenin 6,8-di-C-␤-glucoside(vicenin) +a +c – – –
Anthocyanins
Pelargonidin 3-O-glucoside – +c – – –
Cyanidin 3-O-rutinoside – +c – – –

(+); Present, (−); absent.

a
Compounds isolated and detected in the present study.
b
Compounds reported by Desoky and Youssef (1997).
c
Farag et al. (2015); Abou Zeid et al. (2017).
d
Kassem (2007).
e
Kassem et al. (2002).

Please cite this article in press as: Ragheb, A.Y., et al. Morphological, phytochemical and anti-hyperglycemic evaluation of Brachychiton
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(BW) of alloxan-induced diabetic rats was determined at 0, 4 and Acknowledgments

24 h time intervals. Results are reported as mg/dl (Table 1). At
zero hour, no change in blood glucose level detected. After 4 h The authors are grateful for Dr. Shaimaa Mohamed El-Shebiney,
of treatment, BPE exhibited a decrease in blood glucose levels up Department of Toxicology and Narcotics, Medical Research Divi-
to 227.3 ± 30.28 and throughout 24 h; a significant reduction was sion, NRC, Cairo, Egypt, for hosting the biological part of this study.
achieved (157.6 ± 16.57) at p < 0.05 compared to hyperglycemic
control (488.33 mg/dl after 4 h and 448.50 mg/dl after 24 h). This
Appendix A. Supplementary data
result is comparable to that of the standard drug Diamicrone;
161.42 ± 11.15, after 24 h. Alloxan caused weight reduction by
Supplementary data associated with this article can be found, in
about 15.07%, which was nearly reserved by BPE (1.43%) at the end
the online version, at doi:10.1016/j.bjp.2019.05.001.
of 24 h. Diamicron restored the weight reduction by 9.97%.

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Please cite this article in press as: Ragheb, A.Y., et al. Morphological, phytochemical and anti-hyperglycemic evaluation of Brachychiton
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