Synthesis of cytotoxic and antioxidant Schiff's base analogs of aloin

Article abstract of DOI:10.1080/10286021003775327

Aloin (10-glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-9(10H)-anthracenone) , a bioactive compound in Aloe vera, although known to have an anticancer effect, has not been used in current drug research. Optimization of the lead structure could enhance the utility of this compound. Hence, aloin was modified using natural amino acids to produce Schiff's base, a potential pharmacophore, and its corresponding aglycones. The synthetic derivatives exhibited significant enhancement in their efficacy toward antioxidant (DPPH radical scavenging) and cytotoxic activities than those of the parent compound, aloin showing promise for application in cancer treatment.

Full text of DOI:10.1080/10286021003775327

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Synthesis of cytotoxic and antioxidant
Schiff's base analogs of aloin
S. Kumar a , D. Priya Matharasi a , Sreeraj Gopi a , S. Sivakumar a
S. Narasimhan a
&
a Asthagiri Herbal Research Foundation , 7/1, Thirumazhisai
Street, Sundaram Colony, East Tambaram, Chennai, Tamil Nadu,
600059, India
Published online: 20 May 2010.
To cite this article: S. Kumar , D. Priya Matharasi , Sreeraj Gopi , S. Sivakumar & S. Narasimhan
(2010) Synthesis of cytotoxic and antioxidant Schiff's base analogs of aloin, Journal of Asian Natural
Products Research, 12:5, 360-370, DOI: 10.1080/10286021003775327
To link to this article: http://dx.doi.org/10.1080/10286021003775327
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Journal of Asian Natural Products Research
Vol. 12, No. 5, May 2010, 360–370
ORIGINAL ARTICLE
Synthesis of cytotoxic and antioxidant Schiff’s base analogs of aloin
S. Kumar, D. Priya Matharasi, Sreeraj Gopi, S. Sivakumar and S. Narasimhan*
Asthagiri Herbal Research Foundation, 7/1, Thirumazhisai Street, Sundaram Colony, East
Tambaram, Chennai, Tamil Nadu 600059, India
(Received 4 June 2009; final version received 15 March 2010)
Aloin (10-glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-9(10H)-anthracenone), a
bioactive compound in Aloe vera, although known to have an anticancer effect, has not
been used in current drug research. Optimization of the lead structure could enhance
the utility of this compound. Hence, aloin was modified using natural amino acids to
produce Schiff’s base, a potential pharmacophore, and its corresponding aglycones.
The synthetic derivatives exhibited significant enhancement in their efficacy toward
antioxidant (DPPH radical scavenging) and cytotoxic activities than those of the parent
compound, aloin showing promise for application in cancer treatment.
Keywords: Schiff’s base; aloin; semi-synthetic modification; activity studies
1. Introduction
logical activities including analgesic, anti-
viral, antifungal, antibacterial, and anti-
inflammatory effects have been attributed
to this plant. Aloin (10-glucopyranosyl-
1,8-dihydroxy-3-hydroxymethyl-9(10H)-
anthracenone), an active compound
obtained from A. vera species (Figure 1),
was confirmed to exhibit an anticancer
effect. However, scientifically proven
[10,11] information available is limited
for the in vivo supplements of aloin-based
drugs in cancer research. Hence, synthetic
modifications are necessary to project their
efficacy and bioavailability for antitumor
studies. Of the possible modifications of
the functional groups present in the lead
molecule aloin, products derived mainly
from the reaction of the keto group and
amino acids are of interest since azo-
methine linkage is a necessary unit in all
active drugs [12,13]. Also, the glucose unit,
which contributes to the hydrophilicity, can
Cancer is the second leading cause of
death in the world. Almost all synthetic
agents used in cancer therapy are known to
be toxic and produce severe damages to
normal cells. Hence, chemoprevention of
these toxic agents is one approach to
minimize the effect of the cancerous cells.
Naturally occurring antioxidants found in
medicinal plants are good alternatives to
these chemical anticancer agents. Many
studies reveal that the naturally occurring
polyphenolic compounds can be utilized
for the prevention and treatment of cancer
[1–6]. One such naturally occurring
medicinal plant is Aloe vera.
A. vera (Asphodelaceae) is a well-
known, widely distributed herb found all
over the Asian countries [7]. This plant has
been used from time immemorial for
various applications in medical, cosmetic,
and nutraceutical areas [8,9]. Many bio-
*Corresponding author. Email: narasimhan_s@yahoo.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286021003775327
http://www.informaworld.com

Journal of Asian Natural Products Research
361
hours of refluxing (12 h). Therefore, in
order to prepare the glycosides from
these amino acids, the reaction rate
has to be enhanced. Interestingly, this
was done by employing microwave
irradiation for 30–45 min when these
Schiff’s bases could be synthesized with-
out the cleavage of glucoside. In other
cases, the deglycosylated Schiff’s bases
were made from the corresponding glyco-
sylated derivatives by oxidative hydrolysis
using Jones reagent (Figure 3).
OH
O
OH
OH
OH
H
O
H
OH
H
HO
H
OH
Figure 1. The structure of aloin.
It is interesting to note that the
structures (Figure 4) represent systematic
variation of the alkyl substituent from
methyl to sterically hindering isopropyl
and isobutyl groups, and polar substituents
such as hydroxyl and thiol groups.
be cleaved to produce the aglycones of the
corresponding Schiff’s bases.
In this study, we report the synthesis of
18 Schiff’s bases (glycone and aglycone
products) and their antioxidant and cyto-
toxic activities.
The cytotoxic assay using brine shrimp
lethality test was carried out for the parent
compound aloin and its aglycone and all
Schiff’s bases at different concentrations
(100–400 mM) to obtain the MIC. From
the MIC, the ED50 values were obtained
using Finney’s Probit–Logit analysis [14].
The application of the structure–activity
relationship indicated two distinct fea-
tures: (1) the presence of the glycoside unit
and (2) the alkyl chain variation. Interest-
ingly, the glycosides show better cyto-
toxicity than the corresponding aglycones.
The results are shown in Table 1. The
presence of branching in the alkyl groups
enhances the activity in the following
2. Results and discussion
The Schiff’s bases were obtained by
refluxing aloin with the corresponding
amino acids in a methanol medium, using
sulfuric acid as the catalyst (Figure 2). The
glycoside linkage was intact in most of the
cases except in the case of amino acids
such as glycine, alanine, and phenyl
alanine, wherein the reaction resulted in
the cleavage of the glucose moiety. These
reactions were sluggish requiring long
R
O
OH
OH
OH
N
OH
O
OH
R
O
6–8 hours, reflux
MeOH, Con.H2SO4
OH
OH
+
OH
OH
OH
NH2
O
H
O
H
H
OH
H
H
OH
H
H
HO
H
HO
H
OH
H
OH
Figure 2. Synthesis of glycosylated Schiff’s bases.

362
S. Kumar et al.
R
O
R
O
OH
OH
OH
OH
OH
N
OH
N
Con.H2SO4
K2Cr2O7
OH
OH
OH
O
H
H
H
OH
H
H
HO
OH
Figure 3. Synthesis of deglycosylated Schiff’s bases.
O
R2
OH
OH
N
OH
OH
H
R1
Corresponding
Compound
Glycones (1)
Aglycones (2)
amino acids used
R1 = glu, R2 = H
R1 = glu, R2 = CH3
R1 = glu, R2 = C6H5CH2
R1 = H, R2 = H
A
B
C
D
E
Glycine
R1 = H, R2 = CH3
Alanine
R1 = H, R2 = C6H5CH2
R1 = H, R2 = CH2CH(CH3)2
Phenyl alanine
Leucine
R1 = glu, R2 = CH2CH(CH3)2
R1 = glu, R2 = CH(CH3)
CH2CH3
R1 = H, R2 = CH(CH3)CH2CH3 Isoleucine
R1 = glu, R2 = CH2SH
R1 = H, R2 = CH2SH
F
Cysteine
R1 = glu, R2 = CH2CH2SCH3
R1 = H, R2 = CH2CH2SCH3
G
Methionine
R1 = glu, R2 = CH(CH3)2
R1 = H, R2 = CH(CH3)2
H
I
Valine
R1 = glu, R2 = CH–OH–CH3
R1 = H, R2 = CH–OH–CH3
Threonine
Figure 4. List of amino acids used.

Journal of Asian Natural Products Research
363
band which is proportional to the number
of electrons taken up.
Table 1. The comparison of cytotoxicities of
synthesized compounds.
The activity was studied at different
concentrations (0.1–0.4 mmol) for each
compound. The variations in activity for
glycones and aglycones are represented in
graphs (Figures 5 and 6). All the molecules
showed a typical scavenging activity with
little variation at higher concentrations.
From the data, the antioxidant activity was
calculated as a percentage with reference
to gallic acid as the standard. The values
are given in Table 2. The IC50 values for
each of the Schiff’s bases are calculated
and presented in Table 3.
ED50 (mM)
Compound
Glycones (I)
Aglycones (II)
Aloin
A
B
C
D
E
F
G
H
I
95.17
407.84
130.02
311.64
32.06
35.24
10.16
403.93
19.13
27.60
143.21
1028.33
338.64
334.67
72.4
245.62
423.01
835.46
145.11
338.64
The striking feature observed from this
study is that the glycosylated Schiff’s
bases possess slightly increased activity
than the aglycones. However, the variation
is not significant, indicating that the
glucose unit does not contribute to the
antioxidant effect. The alkyl substituents
have shown strikingly the same gradation
in the activity as observed for the
cytotoxicity as follows:
order:
isopropyl . secondary butyl . isobutyl
. benzyl:
The comparison points out that most
polar groups have significant improvement
in the cytotoxic activity. This is also
reflected in the polar substituent in the
alkyl chain: SH and OH.
isopropyl ¼ secondary butyl . isobutyl
. benzyl:
The free radical scavenging activity of
Schiff’s bases including both glycones and
aglycones was tested by their ability to
bleach the stable radical DPPH. The
activity was monitored by following the
absorption at 517 nm in a visible spectro-
photometer. In the presence of any free
radical scavenger, this odd electron pairs up
and causes the diminishing of absorption
The presence of polar groupsin the alkyl
chain also showed a similar activity trend.
In conclusion, we find that the
presence of the functional groups such as
glycoside, isopropyl, and thiol signifi-
cantly contributes to the bioactivity of
Antioxidant activity (1A–1I)
1A
1B
1C
1D
1E
1F
1G
1H
1I
120
100
80
60
40
20
0
0
0.1
0.2
0.3
0.4
0.5
Con (mM)
Figure 5. Antioxidant activities of glycone Schiff’s bases.

364
S. Kumar et al.
Antioxidant activity (2A–2I)
2A
2B
2C
2D
2E
2F
2G
2H
2I
120
100
80
60
40
20
0
0.5
0
0.1
0.2
0.3
0.4
Con (mM)
Figure 6. Antioxidant activities of deglycosylated Schiff’s bases.
the lead molecule, aloin. In view of their
antioxidant and cytotoxic activities, the
synthetic Schiff’s base analogs have
potential in cancer treatment.
flow rate of 0.5 ml/min. The purity was
confirmed by HPLC and NMR. The NMR
spectra were recorded at 278C with a
Bruker 500 MHz spectrometer using TMS
as the internal standard and CD3OD as the
solvent. The synthesized compounds were
evaluated for biological activities such as
cytotoxicity and antioxidant activity.
3. Experimental
Fresh A. vera was collected from a farm in
Tirupathi hills and processed as per the
literature method to obtain aloin as a
yellow solid [15]. Melting points were
determined using a Raaga Industries
melting point apparatus and are uncor-
rected. Mass spectrometry was performed
in JEOL GC mate in IITM. HPLC were
performed on a Shimadzu LC-10 ATVP
high-pressure pump and a C18 Luna 5
column (250 £ 4.60 mm) and the peaks
were detected at 215 nm (SPD-10 AVP
UV–vis detector), the mobile phase being
the methanol–water system (95:5) at a
3.1 Synthesis of glycosylated Schiff’s
bases
In a typical procedure, weighed quantities
of aloin and amino acid (Sigma Aldrich’s
Standard, Buchs, Germany) in the ratio of
1:1 were allowed to reflux in a methanol
medium with a catalytic amount of
concentrated sulfuric acid. The reaction
was monitored by TLC (60–120 mesh;
Sigma Aldrich). After 6–8 h, methanol
was distilled off in a rotary evaporator and
the residue was subjected to column
chromatography. Pure Schiff’s base com-
pound was eluted in an 8:2 methanol–
chloroform mobile phase. The purity,
yield, and melting point of the synthesized
glycones and aglycones are given in
Tables 4 and 5.
Table 2. The comparison of antioxidant
potential of synthesized compounds.
Antioxidant potential (%)
Compound
Glycones (I)
Aglycones (II)
A
B
C
D
E
F
G
H
I
62.58
77.55
63.2
81.63
96.59
87.07
64.62
96.59
85.71
73.13
80.17
68.9
80.43
97.13
89.35
67.31
95.12
87.1
3.1.1 Compound 1A
IR (KBr) nmax: 3200, 1673, 699.3 cm21
;
1H NMR (CH3OH-d4) d in ppm: 11.00 (br,
1, acidic proton), 7.23, 6.78, 6.71, 6.65,
6.58 (br m, 5H, aromatic protons), 5.00 (br
s, 2H, phenolic), 4.79 (s, 2, CH2 of primary

Journal of Asian Natural Products Research
365
s, 2H, phenolic), 4.79 (s, 2, CH2 of primary
OH), 4.37 (s, 2, H on 10-C), 4.18 (d, 3,
J ¼ 11 Hz, methyl in alanine), 2.00 (s, 1
primary OH), 1.48 (q, 1, J ¼ 13 Hz, CH in
alanine), 4.50, 3.38, 3.49, 3.76, 3.79 (br m,
5H, glucose unit), 3.54 (br s, 2H, CH2 of
Table 3. Radical scavenging activities of
synthesized products.
IC50 (mM)
Compound
Glycones (I)
Aglycones (II)
Aloin
A
132
95
145
85
glucose), 2.40 (s, 4H, OH of glucose); 13
C
NMR (CH3OH-d4) d in ppm: 177.5 (acidic
C of amino acid), 162.7 (8-C), 161.8 (1-C),
150.2 (9-C), 148.6 (3-C), 135.4 (6-C),
120.3 (5-C), 119.6 (4-C), 112.9 (7-C),
112.5 (2-C), 64.1 (11-C), 56.5 (C attached
to N and carbonyl group), 39.2 (10-C),
18.1 (methyl C of alanine), 85.0, 72.7,
78.2, 70.4, 82.1, 63.0 (glucose C); MS: m/z
489 [M]þ, 249.
B
C
D
E
205
256
258
96
98
235
95
98
268
151
88
F
98
G
H
I
271
110
179
240
OH), 4.51 (s, 2, CH2 in glycine), 4.37 (s, 2,
H on 10-C), 2.00 (s, 1, primary OH), 4.44,
3.40, 3.49, 3.76, 3.79 (br m, 5H, glucose
unit), 3.54 (br s, 2H, CH2 of glucose), 2.00
(s, 4H, OH of glucose); 13C NMR
(CH3OH-d4) d in ppm: 178.1 (acidic C of
amino acid), 162.7 (8-C), 162.2 (1-C),
152.9 (9-C), 146.5 (3-C), 135.5 (6-C),
121.6 (5-C), 119.7 (4-C), 114.1 (7-C),
112.6 (2-C), 63.5 (11-C), 51.4 (C attached
to N and carbonyl group), 40.5 (10-C),
85.3, 72.1, 78.2, 70.5, 82.1, 63.0 (glucose
C); MS: m/z 473.6 [M]þ, 244.6.
3.1.3 Compound 1C
IR (KBr) nmax: 3433, 2492, 1655,
1014 cm21 1H NMR (CH3OH-d4) d in
;
ppm: 11.00 (br s, 1, acidic proton), 7.40,
7.23, 6.78, 6.71, 6.65, 6.58 (br m, 5H,
aromatic protons), 5.00 (br s, 2H, pheno-
lic), 4.79 (s, 1, CH2 of primary OH), 4.37
(s, 2, H on 10-C), 4.35 (t, 1, J ¼ 13.0 Hz, H
linked to C via N and carbonyl C), 3.38,
3.13 (CH2 of phenyl alanine), 2.00 (s, 1,
primary OH), 4.40, 3.30, 3.49, 3.77, 3.79
(br m, 5H, glucose unit), 3.54 (br s, 2H,
CH2 of glucose), 2.10 (s, 4H, OH of
glucose); 13C NMR (CH3OH-d4) d in ppm:
176.7 (acidic C of amino acid), 171.2 (1-
C), 171.1 (8-C), 138.2 (9-C), 130.4 (7-C),
130.3 (6-C), 129.91 (4-C), 129.5 (5-C),
128.3 (3-C), 127.8 (2-C), 68.6 (11-C), 56.6
(C attached to N and carbonyl group), 38.5
(10-C), 38.5 (CH of phenyl alanine),
127.8, 126.0, 128.7, 129.7, 139.5 (aro-
matic carbons, phenyl group of alanine),
85.1, 72.3, 78.1, 70.1, 82.3, 63.0 (glucose
C); MS: m/z 415 [M]þ, 245, 204.
3.1.2 Compound 1B
IR (KBr) nmax: 3333, 1603, 1021 cm21; 1H
NMR (CH3OH-d4) d in ppm: 11.00 (br s,
1, acidic proton), 7.23, 6.78, 6.71, 6.65,
6.58 (br m, 5H, aromatic protons), 5.00 (br
Table 4. Synthesis of the glycosylated
product.
Compound 1 Yield (%) Melting point (8C)
A
B
C
D
E
F
G
H
I
55
52
67
58
48
57
48
55
44
128
121
174
117
144
140
157
148
3.1.4 Compound 1D
IR (KBr) nmax: 3200, 2927, 1603 cm21; 1H
NMR (CH3OH-d4) d in ppm: 11.00 (br s,
1, acidic proton), 10.70 (s, 1, acidic
proton), 7.23, 6.78, 6.71, 6.65, 6.58 (br
Decomposed

366
S. Kumar et al.
(acidic C of amino acid), 162.5 (8-C),
161.2 (1-C), 152.2 (9-C), 143.3 (3-C),
135.8 (6-C), 128.7 (5-C), 121.5 (4-C),
115.8 (7-C), 111.2 (2-C), 68.7 (11-C), 66.0
(CH of isoleucine), 56.1 (C attached to N
and carbonyl group), 43.9 (d, 1, H linked to
C via N and carbonyl C), 36.8 (10-C), 25.6
(CH2 of isoleucine), 24.3 (CH of leucine),
16.8, 11.8 (methyl C of leucine), 83.1,
82.6, 79.1, 74.6, 73.1, 62.8 (glucose C);
MS: m/z 519 [M]þ, 245.
Table 5. Synthesis of the deglycosylated
product.
Compound 2 Yield (%) Melting point (8C)
A
B
C
D
E
F
G
H
I
43
48
68
49
65
62
57
55
68
139
147
189
154
162
157
154
144
138
3.1.6 Compound 1F
m, 5H, aromatic protons), 5.01 (br s, 2,
phenolic), 4.80 (s, 1, CH2 of primary OH),
4.00 (t, 1, J ¼ 14 Hz, H linked to C via N
and carbonyl C), 3.80 (s, 1, H on 10-C),
2.30 (s, 1, primary OH), 1.84 (m, 4, CH2 of
leucine), 1.83 (m, 1, CH on leucine), 1.01
(d, J ¼ 11.0 Hz, 6, methyl H on leucine),
4.40, 3.40, 3.49, 3.76, 3.79 (br m, 5H,
glucose unit), 3.50 (br s, 2H, CH2 of
IR (KBr) nmax: 3200, 2922, 1619,
1024 cm21 1H NMR (CH3OH-d4) d in
;
ppm: 10.80 (br s, 1, acidic proton), 7.30,
6.80, 6.71, 6.65, 6.58 (br m, 5H, aromatic
protons), 5.40 (br s, 2, phenolic), 5.01 (t,
J ¼ 13.0 Hz, 2, H linked to C via N and
carbonyl C), 4.70 (s, 1, CH2 of primary
OH), 3.80 (s, 1, H on 10-C), 2.60 (s, 1,
primary OH), 1.50 (s, 1, H attached to S),
4.30, 3.79, 3.76, 3.54, 3.50 (br m, 5H,
glucose unit), 3.49 (br s, 2H, CH2 of
glucose), 2.00 (s, 4H, OH of glucose); 13
C
NMR (CH3OH-d4) d in ppm: 176.7 (acidic
C of amino acid), 161.2 (1-C), 161.2 (8-C),
153.6 (9-C), 143.5 (3-C), 135.8 (6-C),
127.8 (4-C), 127.4 (5-C), 115.4 (7-C),
111.1 (2-C), 63.6 (11-C), 55.0 (C attached
to N and carbonyl group), 36.8 (10-C),
26.7 (CH2 of leucine), 23.1 (CH of
leucine), 21.2 (methyl C of leucine),
85.3, 72.1, 78.2, 70.1, 82.0, 63.0 (glucose
C); MS: m/z 505 [M]þ, 496, 245.5
glucose), 2.30 (s, 4H, OH of glucose); 13
C
NMR (CH3OH-d4) d in ppm: 179.0 (acidic
C of amino acid), 161.7 (1-C), 161.2 (8-C),
152.1 (9-C), 144.6 (3-C), 125.2 (6-C),
120.7 (5-C), 120.2 (4-C), 115.7 (7-C),
112.4 (2-C), 70.7 (C attached to N and
carbonyl group), 68.2 (11-C), 37.8 (10-C),
23.4 (CH2 attached to SH), 84.1, 80.7,
79.1, 70.1, 70.3, 63.1 (glucose C); MS: m/z
529 [M]þ, 245.5.
3.1.5 Compound 1E
3.1.7 Compound 1G
IR (KBr) nmax: 3200, 2922, 1640,
1023 cm21
;
1H NMR (CH3OH-d4) d in
IR (KBr) nmax: 3200, 2922, 1636,
1129 cm21 1H NMR (CH3OH-d4) d in
;
ppm: 10.60 (br s, 1, acidic proton), 7.25,
6.78, 6.71, 6.54, 6.53 (br m, 5H, aromatic
protons), 4.90 (br s, 2, phenolic), 4.80 (s, 2,
CH2 of primary OH), 3.80 (s, 1, H on 10-
C), 2.50 (s, 1, primary OH), 2.00 (m, 1, CH
on isoleucine), 1.00, 0.96 (methyl H on
isoleucine), 4.44, 3.79, 3.76, 3.49, 3.54 (br
m, 5H, glucose unit), 3.40 (br s, 2H, CH2
of glucose), 2.00 (s, 4H, OH of glucose);
13C NMR (CH3OH-d4) d in ppm: 178.9
ppm: 10.60 (br s, 1, acidic proton), 7.23,
6.78, 6.71, 6.65, 6.58 (br m, 5H, aromatic
protons), 5.00 (br s, 2, phenolic), 4.79 (s, 2,
CH2 of primary OH), 4.00 (t, 1, J ¼ 14 Hz,
H linked to C via N and carbonyl C), 3.80
(s, 1, H on 10-C), 2.70 (s, 1, primary OH),
2.44 (CH2 of methionine), 2.25, 2.09 (s, 3,
methyl H on methionine), 4.44, 3.79, 3.76,
3.54, 3.49 (br m, 5H, glucose unit), 3.40

Journal of Asian Natural Products Research
367
(br s, 2H, CH2 of glucose), 2.00 (s, 4H, OH
of glucose); 13C NMR (CH3OH-d4) d in
ppm: 178.9 (acidic C of amino acid), 161.2
(8-C), 161.2 (1-C), 152.6 (9-C), 144.3
(3-C), 134.6 (6-C), 120.3 (5-C), 119.6
(4-C), 110.1 (2-C), 111.6 (7-C), 69.3
(11-C), 60.3 (C attached to N and carbonyl
group), 41.6 (10-C), 31.1 (CH2 of meth-
ionine), 30.1, 18.1 (methyl C of methion-
ine), 84.4, 81.2, 78.5, 72.2, 71.1, 63.0
(glucose C); MS: m/z 529 [M]þ, 245.5.
3.76, 3.54, 3.49 (br m, 5H, glucose unit),
3.40 (br s, 2H, CH2 of glucose), 2.00 (s, 4H,
OH of glucose); 13C NMR (CH3OH-d4) d
in ppm: 177.5 (acidic C), 161.8 (1-C),
161.6 (8-C), 154.4 (9-C), 145.4 (3-C),
123.5 (6-C), 121.2 (5-C), 120.9 (4-C),
113.5 (7-C), 110.4 (2-C), 68.7 (11-C), 67.0
(CH of threonine), 62.7 (CH attached to N),
37.1 (10-C), 25.4 (CH of threonine), 19.8
(methyl C of threonine), 85.2, 82.3, 78.7,
72.3, 70.0, 63.0 (glucose C); MS: m/z 517.4
[M]þ, 245.5, 204.
3.1.8 Compound 1H
3.2 Synthesis of deglycosylated Schiff’s
bases
IR (KBr) nmax: 3200, 2921, 1635,
1083 cm21 1H NMR (CH3OH-d4) d in
;
ppm: 10.90 (br s, 1, acidic proton), 7.23,
6.78, 6.71, 6.65, 6.58 (br m, 5H, aromatic
protons), 5.00 (br s, 2, phenolic), 4.65
(s, 2, CH2 of primary OH), 4.35 (d, 1,
J ¼ 11.0 Hz, H linked to C via N and
carbonyl C), 3.80 (s, 1, H on 10-C), 2.91 (s,
1, primary OH), 2.20 (m, 1, CH on valine),
1.01 (d, 6, methyl H on valine), 4.53, 3.79,
3.76, 3.49, 3.32 (br m, 5H, glucose unit),
3.30 (br s, 2H, CH2 of glucose), 2.20 (s,
4H, OH of glucose); 13C NMR (CH3OH-
d4) d in ppm: 178.3 (acidic C of amino
acid), 162.3 (8-C), 162.1 (1-C), 152.4 (9-
C), 145.4 (3-C), 120.4 (4-C), 119.4 (5-C),
115.1 (6-C), 114.4 (7-C), 110.4 (2-C), 69.3
(11-C), 56.5 (C attached to N and carbonyl
group), 38.2 (10-C), 38.2 (CH of valine),
20.1, 20.7 (methyl C of valine), 83.2, 80.6,
79.7, 71.1, 70.6, 64.3 (glucose C); MS: m/z
547 [M]þ, 245.
The glycosylated Schiff’s bases of aloin
are taken in methanol and refluxed with
stoichiometric quantities of Jones reagent
(concentrated sulfuric acid and potassium
dichromate). The reaction was monitored
by TLC (60–120 mesh; Sigma Aldrich).
After 3–4 h, methanol was distilled off
and the product was obtained as aglycones.
3.2.1 Compound 2A
IR (KBr) nmax: 3200, 1637 cm21; 1H NMR
(CH3OH-d4) d in ppm: 11.0 (br s, 1, acidic
proton), 7.23, 6.78, 6.71, 6.65, 6.58 (br m,
5H, aromatic protons), 5.00 (br s, 2,
phenolic), 4.79 (s, 2, CH2 of primary
OH), 4.51 (s, 2, CH2 in glycine), 4.37 (s, 2,
H on 10-C), 2.00 (s, 1, primary OH); 13C
NMR (CH3OH-d4) d in ppm: 178.3 (acidic
C of amino acid), 162.1 (8-C), 162.3 (1-C),
152.0 (9-C), 146.7 (3-C), 135.1 (6-C),
121.7 (5-C), 119.9 (4-C), 112.1 (2-C),
114.8 (7-C), 63.7 (11-C), 51.7 (C attached
to N and carbonyl group), 40.3 (10-C);
MS: m/z 310 [M]þ, 245.
3.1.9 Compound 1I
IR (KBr) nmax: 3200, 2922, 163,
1029 cm21 1H NMR (CH3OH-d4) d in
;
ppm: 10.70 (br s, 1, acidic proton), 7.23,
6.78, 6.71, 6.65, 6.58 (br m, 5H, aromatic
protons), 5.00 (br s, 2, phenolic), 4.79 (s, 2,
CH2 of primary OH), 3.80 (s, 1, H on 10-
C), 3.70 (d, 1, J ¼ 11.0 Hz, H linked to C
via N and carbonyl C), 3.20 (s, 1, secondary
OH on threonine), 2.80 (s, 1, primary OH),
1.21 (methyl H on threonine), 4.40, 3.79,
3.2.2 Compound 2B
IR (KBr) nmax: 3233, 2237, 1618 cm21; 1H
NMR (CH3OH-d4) d in ppm: 11.0 (br s, 1,
acidic proton), 7.23, 6.78, 6.71, 6.65, 6.58
(br m, 5H, aromatic protons), 5.00 (br s, 2,
phenolic), 4.79 (s, 2, CH2 of primary OH),

368
S. Kumar et al.
4.37 (s, 2, H on 10-C), 4.18 (d, 3,
J ¼ 11.0 Hz, methyl in alanine), 2.00 (s,
1, primary OH), 1.48 (q, J ¼ 15.0 Hz, 1,
CH in alanine); 13C NMR (CH3OH-d4) d
in ppm: 177.5 (acidic C of amino acid),
162.8 (8-C), 161.8 (1-C), 150.2 (9-C),
148.5 (3-C), 135.5 (6-C), 120.3 (5-C),
119.6 (4-C), 112.2 (2-C), 112.9 (7-C), 64.9
(11-C), 56.5 (C attached to N and carbonyl
group), 39.3 (10-C), 18 (methyl C of
alanine); MS: m/z 323 [M]þ, 247, 227.
leucine), 1.01 (d, J ¼ 11.0 Hz, 6, methyl H
on leucine); 13C NMR (CH3OH-d4) d in
ppm: 176.7 (acidic C of amino acid), 161.5
(1-C), 161.2 (8-C), 153.3 (9-C), 143.7
(3-C), 135.8 (6-C), 127.4 (5-C), 127.2
(4-C), 115.7 (7-C), 111.8 (2-C), 63.9
(11-C), 55.2 (C attached to N and carbonyl
group), 36.8 (10-C), 26.7 (CH2 of leucine),
23.8 (CH of leucine), 21.2 (methyl C of
leucine); MS: m/z 334 [M]þ, 245, 204.
3.2.5 Compound 2E
IR (KBr) nmax: 3300, 2963, 1636 cm21; 1H
NMR (CH3OH-d4) d in ppm: 10.60 (br s,
1, acidic proton), 7.25, 6.78, 6.71, 6.54,
6.53 (br m, 5H, aromatic protons), 4.90 (br
s, 2, phenolic), 4.80 (s, 2, CH2 of primary
OH), 3.80 (s, 1, H on 10-C), 2.50 (s, 1,
primary OH), 2.00 (m, 1, CH on
isoleucine), 1.00, 0.96 (methyl H on
isoleucine); 13C NMR (CH3OH-d4) d in
ppm: 178.9 (acidic C of amino acid), 162.5
(8-C), 161.2 (1-C), 152.8 (9-C), 143.3 (3-
C), 135.8 (6-C), 128.7 (5-C), 121.2 (4-C),
115.7 (7-C), 111.2 (2-C), 68.7 (11-C), 66.0
(CH of isoleucine), 56.2 (C attached to N
and carbonyl group), 44.0 (C of isoleucine
attached to N), 36.8 (10-C), 25.6 (CH2 of
isoleucine), 24.0 (CH of leucine), 16.8
(methyl C of leucine), 11.8 (methyl
attached to CH2 of isoleucine); MS: m/z
336.3 [M]þ, 245.9, 177.7.
3.2.3 Compound 2C
IR (KBr) nmax: 3333, 2237, 1618 cm21; 1H
NMR (CH3OH-d4) d in ppm: 11.0 (br s, 1,
acidic proton), 7.23, 6.78, 6.71, 6.65, 6.58
(br m, 5H, aromatic protons), 5.00 (br s, 2,
phenolic), 7.07, 7.07, 7.07, 7.23, 7.23, (br
m, 5H, aromatic protons of phenyl
alanine), 4.79 (s, 1, CH2 of primary OH),
4.37 (s, 2, H on 10-C), 4.35 (t, 1,
J ¼ 13.0 Hz, H linked to C via N and
carbonyl C), 3.38, 3.13 (CH2 of phenyl
alanine), 2.00 (s, 1, primary OH); 13C
NMR (CH3OH-d4) d in ppm: 176.7 (acidic
C of amino acid), 171.8 (8-C), 171.2 (1-C),
129.2, 129.2, 125.6, 121.6, 121.6 (aro-
matic carbons of the phenyl group of
alanine), 151.4 (C attached to O of the
phenyl alanine ring), 138.2 (9-C), 130.4
(7-C), 130.3 (6-C), 129.9 (4-C), 129.7,
129.5 (5-C), 128.3 (3-C), 128.0, 127.8 (2-
C), 127.5, 126.0, 38.5 (10-C), 68.6 (11-C),
56.6 (C attached to N and carbonyl group),
38.5 (CH2 of phenyl alanine); MS: m/z 388
[M]þ, 245, 204.
3.2.6 Compound 2F
IR (KBr) nmax: 3300, 2963, 1636 cm21; 1H
NMR (CH3OH-d4) d in ppm: 10.80 (br s,
1, acidic proton), 7.30, 6.80, 6.71, 6.65,
6.58 (br m, 5H, aromatic protons), 5.40 (br
s, 2, phenolic), 5.01 (t, J ¼ 14.0 Hz, 2, H
linked to C via N and carbonyl C), 4.70 (s,
1, CH2 of primary OH), 3.80 (s, 1, H on
10-C), 2.60 (s, 1, primary OH), 1.50 (s, 1,
H attached to S); 13C NMR (CH3OH-d4) d
in ppm: 179.1 (acidic C of amino acid),
161.4 (8-C), 161.3 (1-C), 152.8 (9-C),
144.8 (3-C), 125.8 (6-C), 120.2 (4-C),
120.1 (5-C), 115.7 (7-C), 112.5 (2-C), 70.4
(C attached to N and carbonyl group), 68.1
3.2.4 Compound 2D
IR (KBr) nmax: 3333, 2237, 1618 cm21; 1H
NMR (CH3OH-d4) d in ppm: 10.70 (br s,
1, acidic proton), 7.23, 6.78, 6.71, 6.65,
6.58 (br m, 5H, aromatic protons), 5.00 (br
s, 2, phenolic), 4.80 (s, 1, CH2 of primary
OH), 4.00 (t, 1, J ¼ 14.0 Hz, H linked to C
via N and carbonyl C), 3.80 (s, 1, H on
10-C), 2.30 (s, 1, primary OH), 1.84 (m, 4,
CH2 of leucine), 1.83 (m, 1, CH on

Journal of Asian Natural Products Research
369
(11-C), 37.3 (10-C), 23.4 (CH2 attached to
SH); MS: m/z 366.3 [M]þ, 245.9, 177.7.
(br m, 5H, aromatic protons), 5.00 (br s, 2,
phenolic), 4.79 (s, 2, CH2 of primary OH),
3.80 (s, 1, H on 10-C), 3.70 (d, 1,
J ¼ 11 Hz, H linked to C via N and
carbonyl C), 3.20 (s, 1, secondary OH on
threonine), 2.80 (s, 1, primary OH), 1.21
(methyl H on threonine); 13C NMR
(CH3OH-d4) d in ppm: 177.5 (acidic C),
161.8 (1-C), 161.6 (8-C), 154.4 (9-C),
145.4 (3-C), 123.2 (6-C), 121.0 (5-C),
120.9 (4-C), 113.5 (7-C), 110.4 (2-C), 68.7
(11-C), 67.0 (CH of threonine), 62.7 (CH
attached to N), 37.1 (10-C), 19.8 (methyl C
of threonine); MS: m/z 356 [M]þ, 245,
213.
3.2.7 Compound 2G
IR (KBr) nmax: 3324, 2922, 1648 cm21; 1H
NMR (CH3OH-d4) d in ppm: 10.6 (br s, 1,
acidic proton), 7.23, 6.78, 6.71, 6.65, 6.58
(br m, 5H, aromatic protons), 5.00 (br s, 2,
phenolic), 4.79 (s, 2, CH2 of primary OH),
4.00 (t, 1, J ¼ 13.0 Hz, H linked to C via N
and carbonyl C), 3.80 (s, 1, H on 10-C),
2.70 (s, 1, primary OH), 2.44 (CH2 of
methionine), 2.25, 2.09 (s, 3, methyl H on
methionine); 13C NMR (CH3OH-d4) d in
ppm: 178.9 (acidic C of amino acid), 161.7
(1-C), 161.2 (8-C), 152.7 (9-C), 144.2
(3-C), 134.1 (6-C), 120.2 (5-C), 119.8
(4-C), 110.3 (2-C), 111.0 (7-C), 69.5
(11-C), 60.6 (C attached to N and carbonyl
group), 41.9 (10-C), 31.0 (CH of meth-
ionine), 30.0, 18.0 (methyl C of methion-
ine); MS: m/z 356 [M]þ, 245, 220.
3.3 Cytotoxic assay
The nauplii of brine shrimp Artemia salina
were hatched from cysts that were
obtained from the National Institute of
Ocean Technology, Chennai. Cyst hatch-
ing was initiated 24 h prior to the start of
the toxicity test. Typically, the cyst was
taken in a standard seawater with a salinity
of 35 g/l prepared in the laboratory and
exposed to a light source (1000–4000 lux)
for 24 h at 258C. The toxicity experiment
was performed in a multiwell plate, each
well containing 5 ml of test solution
prepared with the respective aloin analog
and 10 larvae [16]. A control was
maintained in which the larvae were
taken in a standard seawater. The multi-
well plate was maintained at 258C for 24 h
for the toxicity tests. After the incubation
was completed, the number of dead
Artemia species for each compound at
different concentrations was counted and
the percentage mortality was evaluated.
The experiments were done at different
concentrations. All toxicity tests were
done in duplicate.
3.2.8 Compound 2H
IR (KBr) nmax: 3324, 2922, 1648 cm21; 1H
NMR (CH3OH-d4) d in ppm: 10.90 (br s,
1, acidic proton), 7.23, 6.78, 6.71, 6.65,
6.58 (br m, 5H, aromatic protons), 5.00 (br
s, 2, phenolic), 4.65 (s, 2, CH2 of primary
OH), 4.35 (d, 1, J ¼ 11.0 Hz, H linked to C
via N and carbonyl C), 3.80 (s, 1, H on 10-
C), 2.90 (s, 1, primary OH), 2.20 (m, 1, CH
on valine), 1.01 (d, J ¼ 11.0 Hz, 6, methyl
H on valine); 13C NMR (CH3OH-d4) d in
ppm: 178.3 (acidic C of amino acid), 162.4
(8-C), 162 (1-C), 152.1 (9-C), 145.3 (3-C),
120.8 (4-C), 119.2 (5-C), 115.7 (6-C),
114.6 (7-C), 110.1 (2-C), 69.5 (11-C), 56.4
(C attached to N and carbonyl group), 38.9
(CH of valine), 38.3 (10-C), 20.0, 20.0
(methyl C of valine); MS: m/z 385 [M]þ,
245, 220.
3.2.9 Compound 2I
3.4 Antioxidant activity using DPPH
radical
IR (KBr) nmax: 3339, 2973, 1637 cm21; 1H
NMR (CH3OH-d4) d in ppm: 10.7 (br s, 1,
acidic proton), 7.23, 6.78, 6.71, 6.65, 6.58
To measure the antioxidant potential,
experiments were carried out according

370
S. Kumar et al.
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to the standard method. Solutions (1 mM)
of DPPH radical in methanol were
prepared and 1 ml of it was mixed with
3 ml of the sample solution in ethanol.
After 30 min, the absorbance was
measured at 517 nm [17]. A decrease in
the absorption of the DPPH solution
indicates an increase in the DPPH radical
scavenging activity. The results were
compared with respect to gallic acid as
the standard.
[9] H.R. Denius and P. Homm, Plant Physiol.
49, 873 (1972).
[10] T. Pecere, M.V. Gazzola, C. Mucignat,
C. Parolin, F.D. Vecchia, A. Cavaggioni,
G. Basso, A. Diaspro, B. Salvato, M. Carli,
3.5 Statistical analysis
Determination of equivalent dosage for
cytotoxic studies was calculated using
Finney’s Probit–Logit analysis.
`
and G. Palu, Cancer Res. 60, 2800 (2000).
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[13] M.F. Esua and J.-W. Rauwaid, Carbo-
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Acknowledgement
The authors thank Dr Latha Narasimhan for
statistical analysis.
[14] D.J. Finney, Probit Analysis (Cambridge
University Press, Cambridge, 1947).
[15] J.T. Chavez, L. Jacobs, C. Munger,
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[16] A. Nemeikaite-Ceniene, A. Imbrasaite,
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[17] A.A. Ordoudi, M.Z. Tsimidou, A.P.
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