Synthesis and antioxygenic activities of seabuckthorn flavone-3-ols and analogs

Article abstract of DOI:10.1016/j.bmcl.2011.07.008

A practical synthesis of polyhydroxy- and regiospecifically methylated flavone-3-ols which are components of commercial 'seabuckthorn flavone' has been achieved by modified Algar-Flynn-Oyamada method. Antioxidant activities of seabuckthorn extracts, isolated products and a number of flavone-3-ols have been determined. Structure-activity relationships have been discussed. Amongst the compounds tested, gallic acid, which is also present in seabuckthorn, was found to be the most effective antioxidant and radioprotectant.

Full text of DOI:10.1016/j.bmcl.2011.07.008

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5328–5330
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antioxygenic activities of seabuckthorn flavone-3-ols and analogs
⇑
N. Pandurangan, Chinchu Bose, A. Banerji
Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri, Kollam 690525, Kerala, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A practical synthesis of polyhydroxy- and regiospecifically methylated flavone-3-ols which are compo-
nents of commercial ‘seabuckthorn flavone’ has been achieved by modified Algar–Flynn–Oyamada
method. Antioxidant activities of seabuckthorn extracts, isolated products and a number of flavone-3-
ols have been determined. Structure–activity relationships have been discussed. Amongst the compounds
tested, gallic acid, which is also present in seabuckthorn, was found to be the most effective antioxidant
and radioprotectant.
Received 4 May 2011
Revised 18 June 2011
Accepted 6 July 2011
Available online 14 July 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Hippophae sp.
Seabuckthorn flavone
Synthesis
Flavone-3-ols
AFO reaction
Antioxidant
Radioprotectant
Many of the remarkable biological activities of the plant, sea-
buckthorn (Hippophae sp., SBT) have been attributed to the pres-
Flavonoids, one of the oldest group of natural products, are
attracting considerable attention for their recently discovered bio-
activities, such as radio-protective,5 kinase inhibitory6 and apopto-
tic activities related to cancer.7 They show antioxygenic and metal
chelating activities8 and have also been implicated in the condi-
tions related to degenerative diseases (aging), wound healing and
cardio-vascular problems. Flavonoids occur sporadically in nature.
Procurement of desired flavonoids in sufficient quantities from the
natural sources often poses logistic problems. The insights derived
from the studies on bioactivities of SBT extracts made it imperative
to locate the active molecule(s). Recent reports suggest that the
polyhydroxy flavonoids are metabolized in the biological systems
into more bioavailable partially protected forms (e.g., methyl
ethers) which may be the actual active entity. For example, plasma
of rats fed with quercetin 6c were devoid of it, but contained its
30-methyl ether namely isorhamnetin 6.9 Thus preparation of
partially protected compounds assumes special importance in
metabolic studies.
Unlike flavones, only few methods are available for the synthe-
sis of flavonols (flavone-3-ols).10 Allan-Robinson synthesis has
been the method of choice for flavone-3-ols. But it employs very
harsh experimental conditions and requires ingenious selective
protection and deprotection of the free hydroxyl groups with ben-
zyl and/or benzoyl groups.11
Alternate method using Algar–Flynn–Oyamada (AFO) reaction
though offers flavone-3-ols directly, the yields of the reaction are
variable and other side products are also formed.12 Of the ‘SBT flav-
ones’, isorhamnetin 6 seems to the most important for it shows a
multitude of bioactivities.13 Therefore we chose the synthesis of
ence of flavonoids.
A
commercial product, so called
‘seabuckthorn flavone’ (SBT flavone)1 is reported to have a variety
of bioactivities, although its composition was not clearly defined.
Though wide-ranging bioactivities on Indian seabuckthorn (Hip-
pophae rhamnoides turkestanika ssp.) have been reported,2 not
much attention has been paid to the isolation and molecular char-
acterization of the active constituents. This attracted our attention
to analyze ‘SBT flavone’ for flavonoids and to synthesize the flavo-
nols (flavone-3-ols). Our analysis of commercial ‘SBT flavone’ sam-
ples of different origin revealed that they mainly contained a
mixture of isorhamnetin 6, quercetin 6c and kaempferol 6b with
traces of myrcetin in highly variable proportions and yields (15–
30%)3 with more than 50% intractable materials. No evidence for
presence of glycosides or tannins was found in ‘SBT flavone’ sam-
ples. Considerable variations in phytochemicals of SBT have been
observed in related species. Thus though bioactive compounds,
such as isorhamnetin 6 and ursolic acid are the major bioactive
components in H. rhamnoides from Leh (Ladakh, India), they are
only minor components in closely related H. salicifolia from Lachen
(Sikkim, India).4
Ambiguous reported bioactivities and composition of ‘SBT fla-
vone’ prompted us to synthesize individual flavone-3-ols, so that
pure compounds are available in sufficient amounts for studies
on structure–activity relationship.
⇑
Corresponding author. Tel.: +91 476 2801280; fax: +91 476 2899722.
E-mail address: banerjiasoke@gmail.com (A. Banerji).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.008

N. Pandurangan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5328–5330
5329
isorhamnetin 6 as a model candidate (Scheme 1). Isorhamnetin 6
has been synthesized earlier by Allan-Robinson method11 or by
selective methylation of quercetin 6c.9
constituents of ‘SBT flavone’. Identity of the compounds (Table 1)
were established by comparison of the properties with authentic
samples and literature data.19
In our hands the conventional AFO reaction with protected
chalcones resulted in the formation of mixture of compounds with
low and variable yields. In the present synthesis, partial methyl
ethers such as isorhamnetin 6, tamarixetin 6a and kaempferide
6d were described. Methoxymethyl (MOM) ether was chosen be-
cause they not only offer necessary protection but are also easily
deprotected.14 The chalcone 3 was obtained by the condensation
of 2,4,6-tri-MOM phloracetophenone 1 with 4-MOM vanillin 2.15
The chalcone 3 was obtained as a pale yellow oily product with
UV kmax at 324 nm. It did not respond Shinoda test (Mg–HCl); no
distinct color change was observed on addition of alcoholic ferric
chloride suggesting that MOM protections were intact.
Antioxygenic activities of flavonoids of seabuckthorn extracts
have been associated with many bioactivities, such as radioprotec-
tion, cancer prevention, anti-aging and prevention of cardiovascu-
lar diseases.20 Therefore it was of interest to carry out
a
comparative study of antioxygenic activities of SBT extracts, ‘SBT
flavone’ and other common flavonoids (Table 2, a–y). For this the
free radical scavenging activities were determined using quench-
ing of DPPH. EC50 were calculated as lg required for inhibition of
50% of activity.21 Trolox (g, 6-hydroxy-2,5,7,8-tetramethylchro-
man-2-carboxylic acid) was used as a reference antioxidant.
Amongst the SBT extracts, 70% aqueous acetone extract of SBT
leaves was found to be the most active; the seed and pulp oils
showed lower activities. A sample enriched in phenolic PC (h)
was isolated from 70% aqueous acetone extract was found to have
high antioxygenic activity (EC50, 12.8). Analysis of PC (h) showed
the presence of quercetin 6c (i), isorhamnetin 6 (p) and gallic acid
(a). Amongst isolated pure compounds, gallic acid (a) showed the
maximum activity followed by ellagic acid (b). Both these may
be artifact of hydrolysable tannins present in SBT leaves. Quercetin
6c (i) and kaempferol 6b (j) showed much better activity compared
to the major flavonoid, isorhamnetin 6 (p) of ‘SBT flavone’. Isorh-
amnetin 6 (p, 30-methyl ether), tamarixetin 6a (u, 40-methyl ether),
kaempferide 6d (r, 40-methyl ether) are less active than the parent
compounds viz. quercetin 6c (i) and kaempferol 6b (j). Gossypetin
(c, 8-hydroxyquercetin) and quercetagetin (e, 6-hydroxyquercetin)
showed high activities. Patuletin (f, 6-methyl quercetagetin) also
showed similar trend in activities. Blocking of hydroxyl by glyco-
sylation decreases the bioactivities considerably as can be seen
for rutin (k, quercetin-3-rhamnoglucoside) and gossypin (o, gos-
sypetin-8-glucoside). 8-Glucronide of gossypetin (l, hibifolin)
showed good activity. Dihydroflavonols (v–y) did not show signif-
icant activity except dihydroquercetin 5c (n). Thus it may be in-
ferred that radical scavenging activities are dependent on
number and location of hydroxyl groups. Presence of o-dihydroxy
group acts as metal chelating systems also.
The chalcone 3 was converted to epoxide 4 using alkaline H2O2
in 70–85% yield.16 The formation of the epoxide 4 was confirmed
with UV (kmax 285 nm) and IR (m
883 cmꢀ1) spectral data.12
Treatment of epoxide 4 with methanolic HCl resulted into re-
gio-specific opening of the epoxide ring with concurrent removal
of MOM protections to give dihydroflavonol 5 in 55–60% yield.
The dihydroflavonol 5 showed expected spectroscopic data (UV
kmax 285 nm, IR
m
1644 cmꢀ1). It responded to Shinoda’s (Mg–
HCl) and Pew’s (Zn–HCl) color reactions (pink color).
Conversions of 5 to isorhamnetin 6 was achieved by treating 5
with potassium metabisulfite solutions for 5 h at 100 °C.17 After
the completion of reaction, monitored by TLC (methanol 0.8: for-
mic acid 0.2: toluene 3: ethyl acetate 3), the reaction mixture
was poured into crushed ice when 6 precipitated as a pale solid
and collected by centrifugation (55% yield). Isorhamnetin 6 was
purified by column chromatography (SiO2). It was characterized
by UV, IR, NMR and HPLC and direct comparison with an authentic
sample isolated from Pluchea lanceolata.18
Two other partial methyl ethers, tamarixetin 6a and kaempfe-
ride 6d were synthesized by the same sequence of reactions using
1 and MOM isovanillin 2a and p-anisaldehyde 2d, respectively.
This protocol was also useful for the synthesis of polyhydroxy fla-
vone-3-ols such as quercetin 6c and kaempferol 6b, which are the
R1
RO
OR
O
R1
R2
RO
OR
O
a
H
CH3
R2
O
OR
OR
3
1
2
3) R1=MOM, R2=OCH3.
3a) R1=OCH3, R2=MOM.
3b) R1=MOM, R2=H.
2) R1=MOM, R2=OCH3.
2a) R1=OCH3, R2=MOM.
2b) R1=MOM, R2=H.
R = MOM = CH2OCH3
b
3c) R1=MOM, R2=MOM.
3d) R1=OCH3, R2=H.
2c) R1=MOM, R2=MOM.
2d) R1=OCH3, R2=H.
R1
R2
RO
OR
O
R1
R2
R1
R2
O
c
HO
O
HO
O
d
OR
OH
OH
4
OH
O
OH
O
4) R1=MOM, R2=OCH3.
4a) R1=OCH3, R2=MOM.
4b) R1=MOM, R2=H.
6) R1=OH,
R2=OCH3.
5) R1=OH,
R2=OCH3.
6
5
6a) R1=OCH3, R2=OH.
5a) R1=OCH3, R2=OH.
6b) R1=OH,
6c) R1=OH,
R2=H.
5b) R1=OH,
5c) R1=OH,
R2=H.
4c) R1=MOM, R2=MOM.
4d) R1=OCH3, R2=H.
R2=OH.
R2=OH.
6d) R1=OCH3, R2=H.
5d) R1=OCH3, R2=H.
Scheme 1. Synthesis of flavone-3-ols: reagents and conditions: (a) KOH, DMF and 0 °C; (b) 30% H2O2, NaOH and methanol; (c) HCl, methanol and reflux; (d) K2S2O5 soln,
100 °C.

5330
N. Pandurangan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5328–5330
Table 1
Yields, UV and IR spectral data of compounds
Chalcones Yield
(%)
UV
nm
IR (CO)
cmꢀ1
Epoxides Yield
(%)
UVnm IR (CO)
cmꢀ1
Dihydroflavonols Yield
(%)
UV
nm
IR (CO)
cmꢀ1
Flavonols Yield
(%)
UV nm
3
80–85 327
78–82 305
85–88 322
80–83 330
80–88 328
1677
1676
1647
1676
1676
4
85–90 285
1700,883.3*
5
56–59 288
52–56 286
56–60 292
55–60 293
53–58 283
1644
1635
1636
1640
1634
6
50–55 254,370
40–50 256,372
55–60 266,366
45–50 255,371
50–55 268,364
3a
3b
3c
3d
4a
4b
4c
4d
73–79 283
79–85 276
70–75 282
75–80 275
1696,882.5* 5a
1700,884.8* 5b
1699,884.3* 5c
1700,884.5* 5d
6a
6b
6c
6d
*
Epoxide ring.
2. Geetha, S.; Basu, M.; Jayamurthy, A. S.; Malhotra, A. S.; Pal, K.; Prasad, R.;
Kumar, R.; Sawhney, R. C. In Seabuckthorn (Hippophae L.) Advances in Research
and Development; Singh, Virendra, Ed.; Daya Publishing House: Delhi, 2008;
Vol. III, pp 245–253.
Table 2
Antioxidant properties
Products
EC50 value (lg)
3. Korovina, M. A.; Fefelov, V. A. In Seabuckthorn (Hippophae L.) Biochemistry and
Pharmacology; Singh, Virendra, Ed.; Daya Publishing House: Delhi, 2006; Vol. II,
pp 108–132.
4. Bose, Chinchu; Pandurangan, N.; Banerji, A.; Pradhan, S.; Basnett, R. K.;
Basishtha, B. C. In Presented at the National Conference on Seabucthorn; DIPAS:
Delhi, 2010.
5. Goel, H. C.; Kumar, I. P.; Samanta, N.; Rana, S. V. S. Mol. Cell Biochem. 2003, 254,
57.
6. Ya-ming, X.; Jeffrey, A.; Smith, J. A.; Deborah, A.; Lannigan, D. A.; Sidney, M.;
Hecht, S. M. Bioorg. Med. Chem. 2006, 14, 3974.
(a) Gallic acid
(b) Ellagic acid
(c) Gossypetin
(d) Tannic acid
(e) Quercetagetin
(f) Patuletin
(g) Trolox
(h) PC
(i) Quercetin (6c)
(j) Kaempferol (6b)
(k) Rutin
3.8
4.8
5.5
8.8
10.5
10.6
12.5
12.8
14.4
14.6
15.5
16
18.5
24
25.5
26.2
40
7. Brusselmans, K.; Vrolix, R.; Verhoeven, G.; Swinnen, J. V. J. Biol. Chem. 2005, 280,
5636.
8. Gao, X. In Seabuckthorn (Hippophae L.) Biochemistry and Pharmacology; Singh,
Virendra, Ed.; Daya Publishing House: Delhi, 2006; Vol. II, pp 390–401.
9. Mohamed, B.; Stephane, L.; Aziz, A.; Christian, R. Tetrahedron 2002, 58, 10001.
10. Ganguly, A. K.; Kaur, S.; Mahata, P. K.; Biswas, D.; Pramanik, B. N.; Chan, T. M.
Tetrahedron Lett. 2005, 46, 4119.
11. Tom, H.; Robinson, R. J. Chem. Soc. 1926, 2336.
12. (a) Oyamada, T.; Hajime, B. Bull. Chem. Soc. 1966, 39, 507; (b) Benett, M.; Burke,
A. J.; O’Sullivan, W. I. Tetrahedron 1996, 52, 7163.
13. (a) Jongsung, L.; Eunsun, J.; Jienny, L.; Saebom, K.; Sungran, H.; Youngsoo, K.;
Yongwoo, K.; Sang, Y. B.; Yeong-Shik, K.; Deokhoon, P. Obesity 2009, 172, 226;
(b) Manuel, S.; Federica, L.; Rocio, V.; Inmaculada, C. V.; Angel, C.; Rosario, J.;
Laura, M.; Miguel, R.; Juwan, T.; Francisco, P. V.; Juan, D. J. Nutr. 2005, 137, 910;
(c) Bao-song, T.; Yan-Hua, L.; Zheng-Tao, W.; Xin-Yi, T.; Dong-Zhi, W. Pharm.
Res. 2006, 54, 186; (d) Ma, G.; Yang, C.; Qu, Y.; Wei, H.; Zhang, T.; Zhang, N.
Chem. Biol. Int. 2007, 167, 153; (e) Manuel, I.; Francisco, P. V.; Angel, C.; Juan, D.;
Francisco, Z. A.; José, G. L. L.; Juan, T. Planta Med. 2002, 68, 307.
14. Wuts, P. G. M.; Greene, T. W. Protecting Groups in Organic Synthesis; John Wiley
& Sons: New York, 2007.
(l) Hibifolin
(m) SBT flavone
(n) Dihydroquercetin (5c)
(o) Gossypin
(p) Isorhamnetin (6)
(q) Seabucthorn leaf 70% acetone ext
(r) Kaempferide (6d)
(s) SBT seed oil
(t) SBT pulp oil
(u) Tamarixetin (6a)
(v) Dihydroisorhamnetin (5)
(w) Dihydrotamarixetin (5a)
(x) Dihydrokaempferol (5b)
(y) Dihydrokaempferide (5d)
97
>100
>100
>100
>100
>100
>100
>100
15. Ya-ming, X.; Jeffrey, A.; Smith, J. A.; Deborah, A.; Lanningan, D. A.; Sidney, M.;
Hecht, S. M. Bioorg. Med. Chem. 2006, 14, 1599.
In conclusion, a synthetic route to polyhydroxy- or partial
methyl ethers of flavone-3-ols of biological importance has been
achieved using readily accessible starting materials and mild reac-
tion conditions. Amongst the compounds tested, gallic and ellagic
acids were found to have higher antioxygenic activities compared
to flavone-3-ols. Also we have found anti-diabetic and radio-pro-
tective activity of gallic acid.22 Therefore due importance should
be given to the presence of gallic and ellagic acids for bioactivity
studies of SBT extracts. Since many flavone-3-ols show better
activities when compared to flavones from SBT, it should be feasi-
ble to develop improved bioactive formulations compared to SBT
products.
16. General synthetic procedure: (a) 3, 3a, 3b, 3c and 3d; The chalcone (3) was
obtained by the condensation of 2,4,6-tri-MOM phloracetophenone (1,
1 mmol) with 4-MOM vanillin (2, 1 mmol) in DMF (5 ml) using powdered
KOH (1 mmol) under anhydrous conditions at 0–4 °C for 4 h. Water (20 ml)
was added and extracted with dichloromethane (30 ml ꢁ 2), dried with
anhydrous sodium sulfate and solvent removed under reduced pressure. The
product was purified by column (SiO2) chromatography.
(b) Epoxides 4, 4a, 4b, 4c and 4d: An aqueous solution of NaOH (4.8 ml, 5%) and
H2O2 (5.0 ml, 30%) were added to a stirred solution of chalcone (3, 1 mmol) in
methanol (20 ml) at rt (4 h). Water (100 ml) was added and extracted with
ethyl acetate (30 ml ꢁ 2), dried with anhydrous sodium sulfate and solvent
removed under reduced pressure. The products were purified by column (SiO2)
chromatography.
(c) Dihydroflavonols 5, 5a, 5b, 5c and 5d: Epoxide (4, 0.9 mmol) in methanol
(44 ml) and concentrated HCl (2 ml) were refluxed for 20 (min) and poured
into ice-water. The product was collected by centrifugation.
(d) Flavonols 6, 6a, 6b, 6c and 6d: Dihydroflavonols (5, 0.3 mmol) in ethanol
(2.5 ml) was added to potassium metabisulphite, (5.0 ml, 20%) and heated at
100 °C (5–8 h). The reaction mixture is poured into crushed ice. The
centrifuged product was purified by column (SiO2) chromatography. The
purity of final products were analyzed by HPLC and NMR (Supplementary
data).
Acknowledgments
Partial grant from Department of Biotechnology, Government of
India is gratefully acknowledged. We are thankful to the Dean,
School of Biotechnology for his keen interest in this work.
17. Masa, A.; Vilanova, M.; Pomar, F. J. Chromatogr. A 2007, 1164, 291.
18. Bahl, C. P.; Banerji, A.; Seshadri, T. R. Curr. Sci. 1968, 37, 1.
19. (a) Jae, S. C.; Won, S. W.; Jong, H. P. Arch. Pharm. Res. 1990, 13, 374; (b) Nian, G.
L.; Zhi, H. S.; Yu, P. T.; Jian, P. Y.; Jin, A. D. Beilstein J. Org. Chem. 2009, 5, 60.
20. Singh, Virendra In Seabuckthorn (Hippophae L.) Biochemistry and Pharmacology;
Singh, Virendra, Ed.; Daya Publishing House: Delhi, 2006; Vol. II, pp 1–69.
21. Banerjee, D.; Chakrabarti, S.; Hazra, A. K.; Banerjee, S.; Ray, J.; Mukherjee, B. Afr.
J. Biotechnol. 2008, 7, 805.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.008.
22. (a) Vishnuprasad, C. N.; Anjana, T.; Banerji, A.; Anilkumar, G. FEBS Lett. 2010,
584, 531; Amitava, K.; Dipesh, D.; Mahuya, S.; Krishnendu, M.; Rishikesh, B.;
Sanjit, D. In Presented at the National Conference on Seabucthorn; DIPAS: Delhi,
2010.
References and notes
1. Gupta, A.; Kumar, R.; Pal, K.; Singh, V.; Banerjee, P. K.; Sawhney, R. C. Mol. Cell
Biochem. 2006, 290, 193.