Hepatoprotective, cytotoxic, antimicrobial and antioxidant activities of Dioon spinulosum leaves Dyer Ex Eichler and its isolated secondary metabolites

Article abstract of DOI:10.1080/14786419.2020.1789636

Given the lack of adequate research on Dioon spinulosum (D. spinulosum) Dyer Ex Eichler, this study was conducted focusing on different biological activities and phytochemical investigation of D. spinulosum for the first time. D. spinulosum showed strong

Full text of DOI:10.1080/14786419.2020.1789636

Natural Product Research
Formerly Natural Product Letters
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Hepatoprotective, cytotoxic, antimicrobial
and antioxidant activities of Dioon spinulosum
leaves Dyer Ex Eichler and its isolated secondary
metabolites
Walaa A. Negm , Kamilia A. Abo El-Seoud , Amal Kabbash , Amira A. Kassab &
Mona El-Aasr
To cite this article: Walaa A. Negm , Kamilia A. Abo El-Seoud , Amal Kabbash , Amira A. Kassab
& Mona El-Aasr (2020): Hepatoprotective, cytotoxic, antimicrobial and antioxidant activities of
Dioonꢀspinulosum leaves Dyer Ex Eichler and its isolated secondary metabolites, Natural Product
Research, DOI: 10.1080/14786419.2020.1789636
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1789636
Hepatoprotective, cytotoxic, antimicrobial and
antioxidant activities of Dioon spinulosum leaves Dyer Ex
Eichler and its isolated secondary metabolites
Walaa A. Negma, Kamilia A. Abo El-Seouda, Amal Kabbasha, Amira A. Kassabb
and Mona El-Aasra
b
aDepartment of Pharmacognosy, Faculty of Pharmacy, Tanta University, Tanta, Egypt; Department
of Histology and Cell biology, Faculty of Medicine, Tanta University, Tanta, Egypt
ABSTRACT
ARTICLE HISTORY
Received 19 March 2020
Accepted 21 June 2020
Given the lack of adequate research on Dioon spinulosum (D. spi-
nulosum) Dyer Ex Eichler, this study was conducted focusing on
different biological activities and phytochemical investigation of
D. spinulosum for the first time. D. spinulosum showed strong pro-
tective activity against DNA damage and potent activity against
VERO cell line. It also presented antimicrobial and hepatoprotec-
tive activity. Phytochemical investigation of the leaves resulted in
isolation of two new flavonoids, apigenin 7-O-a-D-glucopyranoside
(15) and amentoflavone 4’-O-a-L-rhamnopyranoside (16), in add-
ition to fifteen known compounds: phytone (1), trans-phytol (2),
b-sitosterol (3), stigmasterol (4), oliveriflavone (5), 7,40,700,4000-tetra-
methylamentoflavone (6), 7,4’,7’’-trimethylamentoflavone (7), scai-
dopitysin (8), bilobetin (9), isoginkgetin (10), aromadendrin (11),
sotusflavone (12), engeletin (14) and eriocitrin (17) for the first
time together with amentoflavone (13). Compounds (11) and (13)
displayed very strong cytotoxic activity and showed the highest
protective activity against DNA damage.
KEYWORDS
Apigenin 7-O-a-D-
glucopyranoside; amentofla-
vone 4’-O-a-L-
rhamnopyranoside; biflavo-
noids; cytotoxicity; Dioon
spinulosum;
hepatoprotective
1. Introduction
Gymnosperms are still a dark area in scientific research. Cycadales order as living rep-
resentative of gymnosperms is classified into 11 genera in three families; Cycadaceae,
CONTACT Mona El-Aasr
moelaasar@pharm.tanta.edu.eg
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1789636.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
W. A. NEGM ET AL.
Zamiaceae and Stangeriaceae, and distributed all over the world (Walters and Osborne
2004). It is regrettable that cycads are approaching the extinction line (Agrawal 2017).
Zamiaceae was chosen to be the target of this study. Zamiaceae is considered a rich
family of flavonoids, biflavonoids, their glycosides and methoxylated biflavonoids
(Negm et al. 2016). Dioon spinulosum (D. spinulosum) is the largest Cycad in the
Americas and the second largest in the world. D. spinulosum gains its Latin name from
the spiny margins of its leaflets (Whitelock 2002). The lack of biological and phyto-
chemical research motivates us to undertake the present study.
Liver diseases are one of the most challenging health care problems worldwide
(Gaafar et al. 2019). Baking and frying food at high temperatures cause acrylamide for-
mation at high levels. Acrylamide is a dangerous chemical substance having genotoxic
and carcinogenic effects. The mechanism involved in acrylamide induced liver damage
is attributed to the significant reduction in the glutathione concentration in liver cells
inducing oxidative stress. This results in oxidative damage to various cell components,
which affects liver cell function and leads to liver cell damage (Ansar et al. 2016). The
aim of this study was to investigate the hepatoprotective effect of D. spinulosum total
extract using acrylamide induced liver cell damage in albino rats by assessment of the
morphological changes. We also investigated the cytotoxic, antimicrobial and antioxi-
dant activities.
2. Result and discussion
2.1. Phytochemical investigation
Seventeen compounds were isolated from petroleum ether, methylene chloride, ethyl
acetate and n-butanol fractions of the MeOH extract of the leaves of Dioon spinulosum
Dyer Ex Eichler family Zamiaceae using extensive column chromatography. Their struc-
tures were identified by spectroscopic analysis (1D, 2D NMR and HRMS) and compari-
son with the literature data. Among them from ethyl acetate fraction compounds 15
and 16 were new compounds (Figure 1).
Compound 15 is a yellowish white amorphous powder, HR-ESI-MS of 15 showed a
pseudo molecular ion at m/z 433.335 for [M þ H]þ with a molecular formula
C21H20O10. UV spectrum showed kmax at 267 and 329 nm which suggested a flavon-
1
oid structure. H NMR spectrum indicated meta-coupled doublets at d 6.43 and 6.82
(J ¼ 2.5 Hz) assigned to H-6 and H-8 protons in ring A. The two protons resonating
at d 7.95 (d, J ¼ 9.0 Hz) can be assigned as ring B protons at 20, 60 positions ortho-
coupled to the signal at d 6.92 assigned to H-30 and H-50 protons. A singlet signal at
1
d 6.86 corresponding to H-3 proton. H NMR spectrum is characteristic for a 5,7,40-tri-
substituted flavone. The presence of sugar protons resonating at d 3.16–3.74 (m) and
one anomeric proton at d 5.40 (d, J ¼ 4.0 Hz) suggested the presence of a-D-glucose.
13C NMR spectrum showed 21 carbons, 15 were assigned to apigenin and 6 signals
at d 99.5, 73.1, 77.1, 69.5, 76.4 and 60.5 confirmed the presence of D-glucose
(Agrawal 1989). The site of the attachment of the D-glucose moiety was confirmed
by HMBC correlations between the D-glucose anomeric proton at d 5.40 (d,
J ¼ 4.0 Hz) to C-7 at d 164.2, to C-6 at d 99.6 and also to C-8 at d 94.8, respectively
(Supporting Information Figure S3). Acid hydrolysis of compound 15 yielded apigenin

NATURAL PRODUCT RESEARCH
3
Figure 1. Chemical structures of isolated compounds.
the aglycone part which was confirmed by direct comparison with the authentic
sample by TLC. Paper chromatography of the sugar part after acid hydrolysis showed
that D-glucose is the sugar moiety. Compound 15 was identified as apigenin 7-O-a-D-

4
W. A. NEGM ET AL.
glucopyranoside. This new compound is isolated for the first time from Dioon species
leaves.
Compound 16 is a yellowish white amorphous powder, HR-ESI-MS of 16 showed a
pseudo molecular ion at m/z 684.578 for [M þ H]þ with a molecular formula
C36H28O14. UV spectrum showed kmax at 282 and 333 nm which suggested a flavonoid
1
structure. The H NMR spectrum showed an AA’BB’ systems for ring E protons as indi-
cated by d 7.56 (2H, d, 9.5 Hz) for H-20’’, 60’’ and d 6.62 (2H, d, 9.5 Hz) for H-30’’, 50’’. The
1H NMR spectrum showed signals for meta-coupled protons at d 6.18 (d, J ¼ 1.5 Hz)
and 6.41 (d, J ¼ 1.5 Hz) for H-6 and H-8 in ring A protons, respectively, the only signal
at d 6.38 was assigned to H-60’. Additionally an ABX system was also shown by the sig-
nals at d 7.95 (d, J ¼ 2.5 Hz) for H-20, d 7.14 (d, J ¼ 9.5 Hz) for H-50 and d 7.97 (dd,
J ¼ 9.5, 2.5 Hz) for H-60 in ring B protons. These data indicated that C-80’ and C-30 are
involved in the biflavonoid linkage. The presence of one anomeric proton at d 4.60
singlet and doublet methyl protons at d 1.20 (d, J ¼ 6.0 Hz) confirmed the L-rhamnose
sugar moiety. 13C NMR spectrum showed 36 carbons and confirmed the conjugation
linkage between C-80’ at d 105.5 and C-30 at d 123.2 due to the downfield shift of C-80’
by 10 ppm and of C-30’ by 6.00 ppm, respectively (Harborne et al. 1975; Markham et al.
1978; Markham et al. 1987; Andersen and Markham 2006). The sugar was identified as
L-rhamnose and the anomeric carbon was at d 100.1. HMBC correlations confirmed the
involvement of C-30 and C-80’ in the interflavonoid linkage via the correlation between
H-50 at d 7.14 to C-30 at d 123.2 and H-20 at d 7.95 to C-80’ at d 105.5. The attachment
site of L-rhamnose was confirmed through HMBC correlation between the anomeric
proton at d 4.60 to C-7 at d 166.1 (Supporting Information Figure S7). Acid hydrolysis
of compound 16 yielded amentoflavone as the aglycone part which was confirmed by
direct comparison with the isolated amentoflavone (13) by TLC. Paper chromatography
of the sugar part after acid hydrolysis showed that it is a-L-rhamnopyranoside.
Compound 16 was identified as amentoflavone 40-O-a-L-rhamnopyranoside. This is the
first report of this compound from plants.
Other isolated known compounds were identified as 6,10,14-trimethylpentadecan-2-
one, also known as hexahydrofarnesylacetone or phytone (1) (Duke 2004), trans
3a,7a,11a,15a-tetramethyl-2-hexadecen-1-ol (trans-phytol) (2) (Wong and Brown 2002),
b-sitosterol (3) (Sen et al. 2012), stigmasta-5,22-dien-3b-ol (stigmasterol) (4) (Habib
et al. 2007), 5,7,40,70’,40’’-pentamethylamentoflavone, oliveriflavone (5) (Wu et al. 1986),
7,40,70’,40’’-tetramethylamentoflavone (6) (Markham et al. 1987; Moawad and El Amir
2016), 7,40,70’-trimethylamentoflavone (7) (Markham et al. 1987), 7,40,4000-trimethylamen-
toflavone (scaidopitysin) (8) (Markham et al. 1987; Moawad and El Amir 2016), bilobe-
tin (9) (Markham et al. 1987; Moawad and El Amir 2016), isoginkgetin (10) (Markham
et al. 1987; Moawad et al. 2010; Moawad and El Amir 2016), aromadendrin (dihydro-
kaempferol) (11) (Xiang et al. 2011; Zhang et al. 2016), sotusflavone (12) (Lopez-saez
et al. 1994), amentoflavone (13) (Markham et al. 1987; Negm et al. 2016), dihydroka-
mepherol 3-O-a-L-rhamnopyranoside or engeletin (14) (Huang et al. 2011; Ali et al.
2014; Gulluce et al. 2015) and eriocitrin or eriodictyol glycoside (17) (Miyake et al.
1997). Compounds (1, 2, 4, 5, 7, 11, 14 and 17) were isolated for the first time from
Dioon species leaves. Compounds (3, 6, 8, 9, 10 and 12) were isolated for the first time
from D. spinulosum. (Supporting Information).

NATURAL PRODUCT RESEARCH
5
2.2. Biological investigation
2.2.1. Histopathological effects of acrylamide and D. spinulosum methanol extract
on liver cells (Aschner et al. 2005; Chen et al. 2009; Molina et al. 2013)
Examination of hematoxylin and eosin H & E stained sections of liver of control group
(Group I) as well as D. spinulosum extract-treated rats (Group II) revealed normal histo-
logical architecture of the hepatocytes. The cells were arranged in anastomosing cords
and were separated by the blood sinusoids. They were polyhedral shaped cells and
had large rounded vesicular nuclei with prominent nucleoli (Supporting Information
Figure S10(a)).
Examination of liver sections of acrylamide-treated group (Group III) showed distor-
tion of the normal hepatocyte architecture. Many hepatocytes showed severe histo-
logical changes in the form of markedly vacuolated cytoplasm and deeply stained
shrunken nuclei (pyknotic nuclei) as presented in (Supporting Information Figure
S10(b)). Conversely, liver sections of rats in Group IV (D. spinulosum and acrylamide-
treated group) showed apparently more or less normal hepatocyte architecture nearly
that comparable to the control group (Supporting Information Figure S10(c)).
2.2.2. Immunohistochemical results
P53 immunostaining: p53-immunostained liver sections of the control (Group I) as well
as D. spinulosum extract-treated rats (Group II) revealed negative immunoreaction for
p53 in the liver cells (Supporting Information Figure S10(d)). In acrylamide-treated
group (Group III), strong positive cytoplasmic and/or nuclear p53 immunoreactions
were observed in many liver cells (Supporting Information Figure S10(e)), while in D.
spinulosum extract and acrylamide-treated group (Group IV), moderate positive immu-
noreaction for p53 was detected only in some liver cells (Supporting Information
Figure S10(f)).
PCNA immnunostaining: PCNA-immunostained liver sections of the control (Group
I) and D. spinulosum extract-treated rats (Group II) revealed weak positive nuclear
immunoreaction for PCNA in a few liver cells (Supporting Information Figure S10(g)).
In acrylamide-treated group (Group III), strong positive nuclear PCNA immunoreaction
was observed in many liver cells (Supporting Information Figure S10(h)), while in D.
spinulosum extract and acrylamide-treated group (Group IV), moderate positive nuclear
immunoreaction for PCNA was detected only in some liver cells (Supporting
Information Figure S10(i)).
2.2.3. Morphometric and statistical results
The mean p53% (the percentage number of p53 positive liver cells) in the acrylamide-
treated group (Group III) showed a significant increase (38.51 1.15) compared to the
control group (1.11 1.22), while D. spinulosum extract and acrylamide-treated group
(Group IV) showed a significant increase (9.62 1.15) compared to control group and a
highly significant decrease compared to Group III.
The mean PCNA labeling index showed a significant increase (68.44 4.78) in the
acrylamide-treated group (Group III) compared to the control group (9.55 1.83).
Moreover, D. spinulosum and acrylamide-treated group (Group IV) showed a

6
W. A. NEGM ET AL.
nonsignificant increase (11.11 1.48) compared to the control group. (Supporting
Information Table S3)
D. spinulosum was introduced to examine its probable role in alleviating the liver
cell damage induced by acrylamide. Results from this study clearly demonstrated that
D. spinulosum offered an evident improvement in the hepatocyte structural changes.
In Conclusion: Concomitant treatment with D. spinulosum ameliorated the liver cell
damage induced by acrylamide.
2.2.4. Cytotoxic activity of D. spinulosum methanol extract
Cytotoxic effect was carried out using MTT assay (Skehan et al. 1990). Breast cancer
(MCF7), Liver cancer (HepG2), kidney cancer African green monkey (VERO), Colorectal
carcinoma (HCT-116) and Normal cell (WI-48) lines are used. The results revealed that
D. spinulosum methanol extract showed very strong activity against MCF7, HCT-116,
VERO and HepG2 cell line with IC50 of 9.55 0.8, 7.53 0.9, 8.35 0.7
and11.27 1.0 lg/mL, respectively. The cytotoxic activity of D. spinulosum methanol
extract (IC50 ¼ 8.35 0.7) was comparable to that of the positive control
(IC50 ¼ 9.01 0.9) against VERO cell line. D. spinulosum methanol extract showed mod-
erate cytotoxic activity against Normal cell line (WI-38) with IC50 of 26.73 1.7 lg/mL
which showed safer effect comparable to that of positive control (IC50 of 9.68 0.3).
Different fractions of D. spinulosum were tested against Kidney (VERO) and Colorectal
carcinoma (HCT-116) cell lines. All the tested extract and different fractions of D. spinu-
losum showed different inhibition percentages against Colorectal carcinoma (HCT-116)
and Kidney carcinoma (VERO) cell line. Inhibition percentage against Kidney carcinoma
(VERO) was higher than that in Colorectal carcinoma cell line (HCT-116). n-Butanol frac-
tion showed very strong cytotoxic activity with IC50 of 8.55 0.9, 6.67 0.7 lg/mL that
exceeds 5-Fluorouracil (IC50 of 9.01 0.9 and 7.19 0.8) against both VERO and HCT-
116 cell lines, respectively, according to Ayyad et al. (2012) classification. Ethyl acetate
fraction presented strong cytotoxic activity with IC50 of 12.61 1.4 and 11.86 1.1 lg/
mL against both VERO and HCT-116 cell lines, respectively. Methylene chloride fraction
presented moderate cytotoxic activity with IC50 of 35.59 1.9 and 44.10 2.3 lg/mL
against both VERO and HCT-116 cell lines, respectively (Supporting Information Table
S4 and Figure S11). These results are in accordance to that reported by (Rejon et al.
2009), concerning the cytotoxic activity of petioles of D. spinulosum on Hep-2 (laryn-
geal carcinoma). Our results are in agreement with that reported by (Moawad et al.
2010; Negm et al. 2016) on other Cycadales plants.
2.2.5. Antioxidant activity
Antioxidant activity of D. spinulosum extract and different fractions are carried out
using 2,20-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) ABTS method (Lissi et al.
1999). D. spinulosum n-butanol fraction and total methanol extract presented the high-
est antioxidant activity (87.8%, 84.2% inhibition) followed by ethyl acetate fraction
(81.6% inhibition), that comparable to ascorbic acid which used as a positive control
(89.3% inhibition; Supporting Information Figure S12).
The DNA protective activity of D. spinulosum extract and different fractions was car-
ried out using (Aeschbach et al. 1994) method. The results are presented in

NATURAL PRODUCT RESEARCH
7
(Supporting Information Figure S13). The absorbance of D. spinulosum total methanol
extract and n-butanol fraction was 0.058 and 0.073 for sample concentration of
0.1 mg/mL, respectively. It showed the highest protective activity against DNA damage
which exceeded the positive control (absorbance ¼ 0.075).
2.2.6. Antimicrobial activity
D. spinulosum n-butanol fraction showed high activity against Gram-negative bacteria,
e.g. Escherichia coli and Pseudomonas aeruginosa, than Gram-positive bacteria, e.g.
Staphylococcus aureus and Bacillus subtilis. n-Butanol fraction presented high antifungal
activity against Aspergillus flavus more than Candida albicans. Ethyl acetate fraction
possessed also high effect against Candida albicans, followed by methylene chloride
fraction. Both fractions showed activity against Aspergillus flavus. Pet-ether fraction
showed weak activity against all tested organisms. These results are in accordance
with that reported by Moawad et al. 2010, concerning the antimicrobial activity of
other Cycadals plants. The results are presented in (Supporting Information Table S5).
2.2.7. Biological activity of isolated pure compounds
According to Ayyad et al., aromadendrin and amentoflavone showed very strong cyto-
toxic activity (IC50¼ 5.87 0.6, 7.49 0.8, respectively) exceeds 5-Fluorouracil (IC50¼
9.01 0.9) against VERO cell lines, sotusflavone, bilobetin and isoginkgetin showed
strong cytotoxic activity with IC50¼ 10.20 1.1, 14.17 1.9 and 15.94 1.9, respectively.
While 7,40,700-trimethylamentoflavone, scaidopitysin, 7,40,700,4000-tetramethylamentofla-
vone and oliveriflavoneexhibited moderate cytotoxic activity with IC50¼ 26.04 2.8,
26.87 3.7, 46.49 3.1, respectively. (Supporting Information Figure S14)
There is a reverse relationship between the number of methoxy groups in biflavo-
noids and cytotoxic activity. Increase methoxylation decrease activity and vice versa.
Amentoflavone possess no methoxy group showed higher cytotoxic activity that
exceeds 5-Fluorouracil while oliveriflavone possess five methoxy group presented the
lowest cytotoxic activity. Flavanol as aromadendrin showed the highest activity in
comparison with biflavonoid isolated compounds.
ABTS method was carried out to evaluate antioxidant activity. Aromadendrin pos-
sessed the highest antioxidant activity (89.6% inhibition) which was comparable to the
positive control (89.3% inhibition) followed by amentoflavone and sotusflavone
(79.1%, 64.3% inhibition, respectively) (Supporting Information Figure S15(a)).
The results of DNA protective activity are presented in (Supporting Information
Figure S15(b)). Aromadendrin presented the highest protective activity against DNA
damage followed by amentoflavone as the absorbance of aromadendrin and amento-
flavone was 0.085 and 0.092, respectively at a concentration of 0.1 mg/mL comparable
to that of ascorbic acid (positive control) (absorbance ¼ 0.075). Isoginkgetin possessed
protective activity (absorbance ¼ 0.104) but lower than amentoflavone. Oliveriflavone
has the lowest protective activity (absorbance ¼ 0.173). There is also reverse relation-
ship between methoxylation level of biflavonoids and antioxidant activity. Biflavonoids
have diverse biological activities; include antibacterial, antifungal, anticancer, antiviral,
anti-hepatotoxic, anti-allergic and immune suppressive activities (Saponara and Bosisio

8
W. A. NEGM ET AL.
1998). Our results are in accordance to that reported concerning the activity of
biflavonoids.
3. Experimental
3.1. Plant material
Leaves of Dioon spinulosum Dyer Ex Eichler family Zamiaceae were collected from El
Abd garden in Giza city in January 2017. It was kindly identified by Dr. Esraa Ammar
plant Ecology lecturer, Botany Department, Faculty of Science, Tanta University and
researcher Rabea Sharawy Agronomist and palm researcher. A voucher sample No.
PGG-002 was deposited at the Herbarium of Pharmacognosy Department, Faculty of
Pharmacy, Tanta University.
3.2. Extraction and isolation (Supporting Information)
3.3. Spectroscopic data
Compound 15 (apigenin 7-O-a-D-glucopyranoside)Yellowish white powder; [a]25.4
¼
D
þ 19.98ꢀ (c ¼ 0.5, CH3OH); UV kmax (MeOH) 221, 267 and 329. IR (KBr) 3858, 3423,
2958, 2854, 1726, 1622, 1450, 1282, 1165, 1113, 1076, 877, 785, 468, 323. Positive HR-
ESI-MS (m/z) 433.335: [M þ H]þ with a molecular formula C21H20O10
1H NMR [DMSO-d6, 500 MHz] d: 6.86 (1H, s, H-3), 6.43 (1H, d, J ¼ 2.5 Hz, H-6), 6.82 (1H, d,
J ¼ 2.5 Hz, H-8), 7.95 (2H, d, J ¼ 9.0 Hz, H-20, 60), 6.92 (2H, d, J ¼ 9.0 Hz, H-30, 50), 5.40 (1H, d,
J ¼ 4.0 Hz, Glc H-10’), 3.87 (1H, d, J ¼ 10.0 Hz, Glc H-20’), 3.16-3.74 (5H, m, H-30’, 40’, 50’, 60’),
5.15 (20’ OH), 5.07 (30’ OH), 5.05 (40’ OH), 4.6 (60’ OH). 13C NMR [DMSO-d6, 125 MHz] d: 162.9
(C-2), 103.1 (C-3), 182.0 (C-4), 156.9 (C-5), 99.6 (C-6), 164.2 (C-7), 94.8 (C-8), 161.4 (C-9), 105.3
(C-10), 121.0 (C-10), 128.6 (C-20), 116.0 (C-30), 161.1 (C-40), 116.0 (C-50), 128.6 (C-60). Glucose
moiety: 99.5 (C-10’), 73.1 (C-20’), 77.1 (C-30’), 69.5 (C-40’), 76.4 (C-50’), 60.5 (C-60’).
Compound 16 (amentoflavone- 4’-O-a-L-rhamnopyranoside)
Yellow amorphous powder; [a]25.4 ¼ ꢁ19.98ꢀ (c ¼ 0.5, CH3OH); UV kmax (MeOH)
D
333, 282 and 221 nm. IR (KBr) 3928, 3782, 3420, 2926, 2860, 2305, 1723, 1650, 1609,
1498, 1429, 1362, 1285, 1245, 1172, 1106, 948, 836, 744, 637, 560, 513. Positive HR-ESI-
MS (m/z) 684.578: [M þ H]þ with a molecular formula C36H28O14.
1H NMR [CD3OD, 500 MHz] d: 6.71 (1H, s H-3), 6.18 (1H, d, J ¼ 1.5 Hz, H-6), 6.41 (1H,
d, J ¼ 1.5 Hz, H-8), 7.95 (1H, d, J ¼ 2.5 Hz, H-20), 7.14 (1H, d, J ¼ 9.5 Hz, H-50), 7.93 (1H,
dd, J ¼ 9.5, 2.5 Hz, H-60), 6.73 (1H, s, H-30’), 6.38 (1H, s, H-60’) , 7.56 (2H, d, J ¼ 9.5 Hz, H-
20’’, 60’’), 6.62 (2H, d, J ¼ 9.5 Hz, H-30’’, 50’’), 4.60 (1H, s, Rha H-1), 3.3-3.65 (m, Rha H-
2–Rha H-5), 1.20 (3H, d, J ¼ 6.0 Hz, Rha C-5-CH3).13C NMR [CD3OD, 125 MHz] d: 166.0
(C-2), 103.4 (C-3), 184.3 (C-4), 163.6 (C-5), 95.9 (C-6), 166.1 (C-7), 95.1 (C-8), 159.4 (C-9),
103.9 (C-10), 123.2 (C-10), 128.9 (C-20), 123.2 (C-30), 161.4 (C-40), 117.5 (C-50), 132.8 (C-60),
166.0 (C-20’), 103.3 (C-30’), 183.9 (C-40’), 162.2 (C-50’), 99.9 (C-60’), 162.5 (C-70’), 105.5 (C-
80’), 154.9 (C-90’), 104.0 (C-100’), 123.1 (C-10’’), 129.3 (C-20’’), 116.8 (C-30’’), 163.6 (C-40’’),
116.8 (C-50’’), 129.3 (C-60’’), Rhamnose moiety: 100.1 (Rha C-1), 69.5 (Rha C-2), 70.1 (Rha
C-3), 73.9 (Rha C-4), 69.5 (Rha C-5), 17.3 (Rha C-6).
Spectroscopic data of known compounds (Supporting Information).

NATURAL PRODUCT RESEARCH
9
3.4. Acid hydrolysis of the isolated glycosides (Trendafilova et al. 2011)
(Supporting Information)
3.5. Biological activity
For in vivo hepatoprotective effect of extract (Hall et al. 1990; Bancroft and Gamble
2008; Gaertner et al. 2008; Saleh et al. 2017; Bera et al. 2018), cytotoxic, antioxidant,
antimicrobial and biological activities of isolated compounds (See Supporting
Information).
3.6. Statistical analysis
This analysis was carried out by Graph Pad Prism 5 Program. All measurements were
performed in triplicate (n ¼ 3), shown as mean standard deviation (SD) and significant
difference at p ꢂ .05 by Student’s test.
4. Conclusions
D. spinulosum showed promising hepatoprotective activity. D. spinulosum displayed
potent activity against MCF-7, HepG2, VERO and HCT-116 cells. Secondary metabolites
isolated from D. spinulosum showed the highest protective activity against DNA dam-
age, higher than the positive control. Two new flavonoids apigenin 7-O-a-D-glucopyra-
noside (15) and amentoflavone 40-O-a-L-rhamnopyranoside (16) were isolated from D.
spinulosum leaves, in addition to fifteen known compounds. A promising future awaits
D. spinulosum in drug discovery.
Acknowledgement
The authors would like to thank Mr. Rabea Sharawy for providing the necessary plants for this
study and to make a lot of his valuable time in helping to identify the plants.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Mona El-Aasr
http://orcid.org/0000-0002-3656-6423
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