Utility of dipyrromethane in the synthesis of some new A2B2 porphyrin and their related porphyrin like derivatives with their evaluation as antimicrobial and antioxidant agents

Article abstract of DOI:10.1016/j.dyepig.2020.109008

In this work, the dipyrromethane derivatives 2a,b were synthesized from an aldehyde and excess of pyrrole at room temperature in solvent-free conditions. The reaction of dipyrromethanes 2a,b with different aromatic aldehydes in the presence of hydrochlori

Full text of DOI:10.1016/j.dyepig.2020.109008

Dyes and Pigments 191 (2021) 109008
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Utility of dipyrromethane in the synthesis of some new A2B2 porphyrin and
their related porphyrin like derivatives with their evaluation as
antimicrobial and antioxidant agents
,
,c,**
Ahmed A. Faddaa, Eman El-Gendya *, Hala M. Refatb, Eman H. Tawfika
a Chemistry Department, Faculty of Science, Mansoura University, 35516, Mansoura, Egypt
b Chemistry Department, Faculty of Science, Al-Arish University, Egypt
c Chemistry Department, Faculty of Science and Arts, Taibah University, Ulla, Saudi Arabia
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this work, the dipyrromethane derivatives 2a,b were synthesized from an aldehyde and excess of pyrrole at
room temperature in solvent-free conditions. The reaction of dipyrromethanes 2a,b with different aromatic al-
dehydes in the presence of hydrochloric acid or p-toluenesulfonic acid as a catalyst afforded some new porphyrin
like and A2B2 porphyrin derivatives 4a-g and 5a-d, respectively. Biological evaluation of the newly synthesized
compounds 4a-g and 5a-d showed promising activity as antimicrobial and antioxidant agents. Compounds
4c,4e,4g,5c, and 5d exhibited the highest antimicrobial activity, whereas, compounds 4a,4f,5a,5b, and 5d
recorded the highest antioxidant activity.
Dipyrromethane
Porphyrin like and A2B2 Porphyrins
Antimicrobial
DNA damage
Hemolysis activities
Antioxidant
1. Introduction
treatment using photodynamic therapy and boron neutron capture
therapy [18–21].
Porphyrins have received great attention as they serve as biomimetic
material and are used in material chemistry [1–5]. Most synthesized
porphyrins have potential applications in medicinal chemistry [6–8].
Many reported methods for the synthesis of porphyrin derivatives have
previously been reported [9,10]. Traditional strategies described by
Rothemund and Alder for porphyrin synthesis facilitated the synthesis of
porphyrins with poor yields and long reaction times under drastic con-
ditions [11]. In 1978, T. J. Doupherty was the first to utilize a hema-
toporphyrin derivative (HpD) for photodynamic cancer therapy [12,13].
Extensive research has been conducted to develop photosensitizer-based
drugs in the last three decades [14–16]. It was reported a photosensi-
tivity and photo-toxicity in a mice’s skin that was administrated with
hematoporphyrin [14]. The characteristic red fluorescence property of
HpD is one essential indication of tumors and provides a means for
endoscopic investigations [17]. Ultimately, partially purified heamato-
porphyrin derivatives were approved in 1993 under the trade name
Photofrin (porfimer sodium) as a first-generation photosensitizer for the
treatment of bladder cancer [12]. Porphyrin derivatives have been
tested as synthesizing drugs for application in tumor diagnosis and
Dipyrromethanes are important building blocks for the synthesis of
different porphyrins, calixpyrrols, and corroles [22,23]. Although the
stability of dipyrromethanes to oxidation is always a cause for concern
during the synthetic procedure, isolation, and storage of such com-
pounds, diversity conditions have been established allowing good to
excellent yields of dipyrromethanes to be obtained in the case where
adequate substituents are present on the pyrrole rings. For this, we are
looking for a solvent-free conditions system for the preparation of
dipyrromethanes which are then used to synthesize porphyrin. Different
methods have been used in green chemistry including manual and me-
chanical grinding which able to induce mechanochemical trans-
formation [24], and solvent-free synthesis [25,26]. The application of
grinding had been assisted in the development of many pharmaceutical
compounds [23,25,27].
2. Results and discussion
Most of the reported methodologies for the synthesis of porphyrin
derivatives had been afforded symmetrical porphyrin (A4) which is
* Corresponding author.
** Corresponding author. Chemistry Department, Faculty of Science, Mansoura University, 35516, Mansoura, Egypt.
E-mail addresses: afadda50@yahoo.com (A.A. Fadda), emanorganic@gmail.com (E. El-Gendy), hala7223530@hotmail.com (H.M. Refat), emanhelmy4@yahoo.
com (E.H. Tawfik).
https://doi.org/10.1016/j.dyepig.2020.109008
Received 23 May 2020; Received in revised form 7 October 2020; Accepted 12 November 2020
Available online 29 March 2021
0143-7208/© 2020 Elsevier Ltd. All rights reserved.

A.A. Fadda et al.
Dyes and Pigments 191 (2021) 109008
difficult to achieve excitation when irradiated to study energy transfer.
So, our aim in the present work has been directed to synthesize un-
symmetrical porphyrins like 4a-g and A2B2 porphyrin derivatives 5a-d,
which can be easily achieved the selective excitation (Schemes 2 and
3). Many synthetic routes have been pointed out in the preparation of
dipyrromethane [28,29] and porphyrin derivatives [30,31]. Substan-
tially, all latest porphyrin synthesis depends on starting with pyrrole and
all of them have the prospect to be used in combinatorial chemistry [32].
Compounds 4a-g are defined as porphyrin like derivatives due to the loss
of aromaticity of the porphyrin ring.
These porphyrins like and/or porphyrin derivatives 4a-g and 5a-
d were achieved by two pathways: A) dipyrromethanes 2a,b have
been firstly dissolved in HCl acid and reacted with the proper aldehyde
derivatives 3a-j under nitrogen gas condition and heated in a silicon oil
bath for 15–35 min. The obtainable porphyrin like and/or porphyrin
derivatives 4a-g and 5a-d have been established their structures based
on their correct elemental and spectral analyses, B) dipyrromethanes 2a,
b were dissolved in DMF (as solvent and capping agent [29]) and added
to a solution of the proper aldehydes 3a-j in DMF. To this reaction
mixture, p-toluenesulfonic acid in DMF was added. The reaction mixture
was then heated in a silicon oil bath for 15–40 min, which then chro-
matographed on silica gel to afford the corresponding porphyrin like
and/or porphyrin derivatives 4a-g and 5a-d, respectively in good yield
[29]. The newly obtainable porphyrins like and/or porphyrin de-
rivatives 4a-g and 5a-d, in this case, showed identical spectral data to
that acquired by method A. The reaction product for porphyrins 5a-d has
been monitored by UV spectra; which showed after 5–10 min in the first
cut of the reaction mixture to absorb at 504 nm due to the reacting
compounds. On continuing heating at 150 ◦C the λmax of porphyrins 5a-d
peak continued to grow in the range of 424–429 nm with the corre-
sponding decrease in the reacting compounds having λmax at 504 nm. On
the other hand, the UV–vis spectra of porphyrin like compounds 4a-g in
different solvents, in general, displayed two λmax at 276–294 and
Firstly, the desired dipyrromethanes 2a,b have been achieved by the
reaction of pyrrole and carbonyl compounds (acetophenone and/or
3,4,5-trimethoxybenzaldehyde) by grinding at room temperature cata-
lyzed by iodine (Scheme 1). By this method, we improved the previously
reported methods to minimize the issues associated with the formation
of by-products as N-confused dipyrromethane isomer and tripyrro-
methane [28]. On the other hand, we concerned with the environmen-
tally harmless catalytic system. All previously reported works have
many disadvantages, such as high price reagents, the use of excess
pyrrole aldehyde ratio, and prolonged reaction time. Thus, in our
method, we overcome all these problems and obtained the dipyrro-
methanes in high yield. 1HNMR and TLC control of the formed dipyr-
romethanes showed very high purity during the reaction mentioned
time (10–15 min), while the precipitate which formed after longer re-
action times showed a gradual increase in tripyrromethane content. The
obtainable dipyrromethanes 2a,b have been purified chromatographi-
cally in basic medium cyclohexane/chloroform 0.5% triethylamine (1:2)
as eluent to prevent the decomposition of dipyrromethane on silica gel.
It was preferred to store the resulted dipyrromethanes in the refrigerator
in absence of light. In this method, we present simple and efficient
procedures for the synthesis of dipyrromethane, mild reaction condition,
absence of solvent, shorter reaction time, easy and quick purification of
the product, and excellent yield are the main advantages of this pro-
cedure which make this method economically and environmentally
attractive. The obtained dipyrromethanes 2a,b have been subjected to
react with different aromatic aldehydes using HCl acid as an acid cata-
lyst or p-toluenesulfonic acid to afford their corresponding new
porphyrin like and/or A2B2 porphyrin derivatives 4a-g and 5a-d,
respectively (Schemes 2 and 3).
389–398 nm due to π-π* and n-π* as broad bands.
The infrared spectra of porphyrin like 4a-g and porphyrin derivatives
5a-d, in general, showed absorption bands at 3315-3317 cmꢀ 1 indicates
the presence of NH. Also, the band at 3504 cmꢀ 1 in compounds 4a and 4f
indicates the presence of OH groups. IR spectra showed also absorption
bands of 2925 cmꢀ 1 due to the C–H group. 1HNMR exhibited, in general,
two doublets for pyrrolic C–H proton in the region at δ 5.0–7.0 ppm.
In conclusion, our reported results expand the scoop of the synthesis
of porphyrin like and A2B2 porphyrin derivatives using dipyrromethane
and aldehydes. Our developed methods increase the yield of the resulted
products, in general, in comparison with the binary mixed aldehyde
condensation method. P-Toluenesulfonicacid, an alternative catalyst of
these reactions instead of many other acid catalysts, could decrease the
acidolysis of dipyrromethanes and the number of oligomers formed.
Scheme 1. Synthesis of dipyrromethane derivatives 2a,b.
2

A.A. Fadda et al.
Dyes and Pigments 191 (2021) 109008
Scheme 2. Synthesis of porphyrin like derivatives 4a-g from dipyrromethane 2a.
vitro antifungal activity. For the report, both antibacterial and antifungal
activity agar-diffusion method [33] has been used. We choose Cepha-
lothin, Chloramphenicol, and Cycloheximide to indicate the antimicro-
bial activity of the tested porphyrins. The antibacterial and antifungal
activity of the synthesized porphyrins has been listed as rated inhibition
zone diameter (IZ) of the development of bacteria or fungi. The lowest
concentrations of tested compounds that suppress the development of
bacteria or fungi were recorded as minimum inhibitory concentration
(MIC) and IZ in Table 2. The outcomes detect that most porphyrins have
satisfied activity against Gram-positive and Gram-negative bacterial and
fungal strains. Compounds 4c, 4e and 4g showed suppressor activity to
Gram-positive bacteria (B. subtilis) with the MICs in the range of
3.125–6.25
against B. thuringienesis with MICs 6.25
Gram-negative bacteria (E. coli and P. aeruginosa) was found to be
12.5–50.0 g/mL. Among the observed results of Gram-negative bacte-
ria, the highest activity of compounds 4c, 4e and 4g have been noted for
E.coli with MIC values of 25.0 g/mL, followed by P. aeruginosa with
MICs range 25.0–50.0 g/mL. Additionally, the F. oxyporum and B. fabae
were highly inhibited by the compounds 4c, 4e and 4g with the MICs
25.0 g/mL. The halogenated porphyrins 4c,4g, 5c and 5d inhibited
Gram-positive bacteria with the MIC range of 3.125–25.00 g/mL, and
μ
g/mL, while compounds 4c and 4e displayed high activity
μ
g/mL. The activity against
μ
μ
μ
μ
μ
inhibition zone range of 29–44 mm. Whereas, compounds 5a-c showed
moderate antimicrobial activity (MIC≤ 50 μg/mL) against most of the
bacterial strains.
Scheme 3. Synthesis of porphyrin derivatives 5a-d from dipyrromethane 2b
The consequences of antimicrobial activity of the tested compounds
exhibited the following presumptions for the structure-activity re-
lationships (SAR,s): (1) it noticed that all compounds (4c,4e,4g,5c, and
5d) having electron-withdrawing groups such as NO2, Cl and Br recor-
ded higher antibacterial activity. (2) The substitution of benzene ring by
methoxy groups (compounds 5a-d) showed the lowest antimicrobial
activity than compounds 4a-g. (3) it is clear from the results that the
tested compounds 4a-g and 5a-d are more active against Gram-positive
than Gram-negative bacteria, these results can be illustrated due to the
differences in the cell wall thickness of both type of bacteria due to the
peptidoglycan layer in which the Gram-positive bacteria have a thick
peptidoglycan layer and no outer lipid membrane, on the other hand,
the negative Gram bacteria have thin peptidoglycan and outer lipid
membrane. These distinctions in the cell wall structure can create con-
trasts in the antibacterial sensibility of synthesized compounds 4a-g and
2.1. Biological investigation
I) Antimicrobial evaluation:
A series of the newly unsymmetric synthesized targeted porphyrin
like and porphyrin derivatives 4a-g, 5a-d has been prepared to expand
the spectrum of activity of this class of antibiotics to include Gram-
negative and positive organisms and have been estimated versus Gram-
positive bacteria (Bacillus subtilis and Bacillus thuringienesis) and Gram-
negative bacteria (Escherichia coli and Pseudomonas aeruginosa), to detect
the in vitro antimicrobial activity of the synthesized porphyrins (see
Table 1). The newly synthesized compounds were evaluated against
Fusarium oxysporum and Botrytis fabae fungal strains to estimate the in
3

A.A. Fadda et al.
Dyes and Pigments 191 (2021) 109008
Table 1
Minimum inhibitory concentration (MIC,
μ
g/mL) and inhibition zone (mm) of some new compounds.
Compound No.
MICa in
μg/mL, and inhibition zone (mm)
Bacteria
Fungi
Gram-positive Bacteria
Gram-negative Bacteria
B. Subtilis
B. Thuringiensis
E. Coli
P. Aeruginosa
F. Oxysporum
B. Fabae
4a
6.25 (40)
6.25 (44)
3.125 (57)
50.0 (18)
3.125 (61)
25.0 (29)
3.125 (52)
25.0 (32)
50.0 (18)
12.5 (32)
12.5 (36)
3.125 (44)
6.25 (36)
B
6.25 (34)
12.5 (29)
6.25 (29)
50.0 (18)
6.25 (39)
12.5 (22)
6.25 (33)
25.0 (35)
50.0 (21)
25.0 (38)
25.0 (26)
3.125 (44)
6.25 (37)
B
50.0 (15)
50.0 (16)
25.0 (22)
50.0 (19)
25.0 (24)
25.0 (26)
25.0 (30)
25.0 (32)
50.0 (19)
50.0 (21)
25.0 (26)
6.25 (37)
6.25 (38)
b
50.0 (14)
50.0 (17)
50.0 (20)
50.0 (19)
50.0 (16)
50.0 (19)
25.0 (24)
50.0 (17)
50.0 (23)
25.0 (32)
50.0 (24)
6.25 (38)
6.25 (37)
b
50.0 (15)
50.0 (12)
50.0 (19)
100.0 (13)
100.0 (11)
50.0 (19)
50.0 (18)
50.5 (21)
50.0 (19)
50.5 (22)
25.0 (24)
B
50.0 (22)
50.0 (26)
25.0 (32)
50.0 (20)
25.0 (26)
50.0 (15)
25.0 (36)
25.0 (34)
100.0 (10)
50.0 (17)
50.0 (14)
B
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
Chloramphenil
Cephalothin
Cycloheximide
B
B
3.125 (43)
3.125 (42)
MICa: Minimum inhibitory concentration values with SEM = 0.02 (The lowest concentration that inhibited the bacterial growth). bNT: Not tested.
II) Antioxidant evaluation:
a) Bleomycin-dependent DNA damage
such diverse applications as pharmaceuticals, oxidation inhibitors, and
metal complexing agents. Pyrrole derivatives have antimicrobial, anti-
inflammatory, anti-cancer, and many other biological activities. From
the obtained results, it can be concluded that pyrrole moiety is essential
for the protection activity against DNA damage induced by bleomycin-
iron complex, and pyrrole ring is also required for the activity and an
unsubstituted benzene ring (compounds 4a-g) exhibited better activity
than those substituted derivatives (compounds 5a-d). The pro-oxidant
activities of the aforementioned derivatives have been assisted by
their effects on the bleomycin-induced DNA damage and their corre-
sponding absorbencies. From Table 2 we noticed that as λmax decreased
the activity of the tested compounds increased. The positive control was
vitamin C and showed 88.61%, absorbance 0.00881 at the same con-
centration of the tested compounds.
Table 2
Results of bleomycin-dependent DNA damage assay of isolated compounds and
antioxidant assay for the newly prepared compounds.
Compound no.
Absorbance
ABTS inhibition (%)
Erythrocyte hemolysis (%)
4a
0.00882
0.00899
0.00913
0.00895
0.00911
0.00884
0.00981
0.00886
0.00888
0.00890
0.00893
0.00881
86.25
73.70
60.25
74.38
60.18
83.24
53.28
81.44
79.22
73.54
74.24
88.61
0.84
0.88
0.94
0.92
0.91
0.80
0.94
0.85
0.86
0.87
0.89
0.85
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
L-Ascorbic acid
b) ABTS inhibition and erythrocyte hemolysis to evaluate Antioxidant
activity
ABTS + scavenging activity (%) = [(Ac ꢀ As)/Ac] × 100, where Ac is the
absorbance value of the control and As is the absorbance value of the added
samples test solution. The concentration of the pure compounds was 2 mM. The
concentration shows 50% inhibition is expressed in mM. The positive control
was vitamin C and showed 88.61%, absorbance 0.00881 at the same concen-
tration of the tested compounds.
The antioxidant activity has been evaluated for the synthesized
porphyrins using ABTS assay which mirrors the capacity of peroxidation
inhibition in rat brain and specific kidney enzyme and the rate of he-
molysis of erythrocyte. From Table 2 it was found that porphyrin de-
rivatives 4a, 4f, 5a and 5b exhibited antioxidant activity, where
derivatives 4d, 5d, 4b and 5c displayed moderate activity, while de-
rivatives 4c, 4e and 4g displayed weak activity.
5a-d [34].
One of the important members of the huge family of antitumor an-
tibiotics is bleomycin. The role of bleomycin is to evaluate the pro-
oxidant of antioxidants and the restriction of iron and DNA. The
formed bleomycin-iron complex decays DNA by heating with thio-
barbituric acid (TBA) due to the formation of a pink chromogen. Addi-
tions of antioxidants create a competition with DNA which leads to
minify the formation of chromogen. Between different synthetic com-
pounds 4a-g and 5a-d, compounds 4a, 4f, 5a, and 5b exhibited elevated
protection level against DNA damage stimulated by the complex.
Whereas, compounds 5c, 5d, 4d and 4b showed low to moderate ac-
tivity, and compound 4e, 4c and 4g displayed low activity, these results
summarized in Table 2.
c) Induction of RBC hemolysis by AAPH assay
Decomposition of AAPH thermally in aq. suspension of RBCs leads to
•
the formation of starting free radical (R ) which reacts with the lipid in
RBC membranes to give lipid peroxidation (Eqs. 1-6). The starting rate of
AAPH is 1.3 × 10‾6 [AAPH]/s. The free radical chain reaction of lipid
peroxidation and the starting radical create reaction up to 50 propaga-
tion reactions which lead to fast damage of RBCs membrane leading to
hemolysis. From the other side, if antioxidant (AH) is present or added to
RBCs, it was reacting with propagation radical to stop peroxidation and
inhibit hemolysis (Eq. 7).
All compounds have been tested to bleomycin DNA damage. The
results indicated that they may have some protective activity to DNA by
a certain mechanism. A series of compounds 4a, 4f, 5a, 5b, 4d, and 5d
exhibited a high antioxidant activity. On the other hand, all compounds
show a potent protective effect on the DNA from the induced damage by
bleomycin concerning ascorbic acid (Table 2). Synthetic compounds 4a-
g and 5a-d which have pyrrole ring systems have found their way into
4

A.A. Fadda et al.
Dyes and Pigments 191 (2021) 109008
4.5. Synthesis of unsymmetric porphyrin derivatives (4a-g and 5a-d)
4.5.1. General procedure
4.5.1.1. Method A. Dipyrromethanes 2a,b (0.01 mol), was dissolved in
dil. HCl acid (2 mL), aldehydes (3a-j) (0.1 mol) were combined in a
three-neck round bottom flask equipped with a reflux condenser and N2
(g) inlet. The mixture was flushed with N2 gas for 5 min, then heated to
100◦c and kept at this temperature for 10 min and then heated was
continued to 150◦c for 15–35 min. The color changed to deep blood-red,
greenish and reddish black color within a few minutes. The ultraviolet
spectra were used to follow up the reaction product via the reaction
progress. So, when heated the reaction mixture up to 150◦c, we found
λmax in compounds 5a-d 424–429 nm due to the soret band is appeared
with continuous disappearing of λmax at 504 nm due to Q band whereas
the UV–vis spectra of compounds 4a-g exhibited two λmax at 276–294
and 389–398 nm due to π-π* and n-π* transitions as broad bands. The
solid material that was obtained when the reaction mixture was poured
onto ice cold water was collected by filtration and purified chromatog-
raphy (silica gel, chloroform/hexane: 1.5/1 eluent).
3. Conclusions
The porphyrin like 4a-g and porphyrin derivatives 5a-d (A2B2) are
novel, they have been synthesized from the reaction of their corre-
sponding dipyrromethanes 2a,b with different aldehydes. With signifi-
cant high yield and screened for antimicrobial and antioxidant activity
in which all compounds 4c,4e,4g, 5c and 5d having electron-
withdrawing groups have been exhibited markedly higher antimicro-
bial activity, whereas compounds 4a,4f,5a,5b, 4d and 5d have been
recorded higher antioxidant activity than the other derivatives.
4.5.1.2. Method B. This method was carried out according to the pre-
viously reported work [31].
4.6. Synthesis of 4,4’-((4,6,15,19)-10,20-dimethyl-10,20-diphenyl-
1H,9H,10H,20H-porphyrin-5,15-diyl)diphenol (4a)
Yield (86%); m.p. 234 C; IR (KBr): νmax (cmꢀ 1): 3410 (OH), 3352
̊
(NH); 1H NMR (DMSO‑d6): δ (ppm): 1.44 (s, 3H, CH3), 1.85 (s, 3H, CH3),
2.95 (d, 1H, pyrrolic-H), 3.78 (d, 1H, pyrrolic-H), 5.11 (d, 1H, pyrrolic-
H), 5.29 (d, 1H, pyrrolic-H), 5.31 (d, 1H, pyrrolic-H), 5.59 (d, 1H,
pyrrolic-H), 6.24 (d, 1H, pyrrolic-H), 6.25 (d, 1H, pyrrolic-H), 6.54 (s,
1H, NH), 6.68–7.84 (m, 18H, Ar–H), 7.84 (d, 1H, pyrrolic-H), 9.55 (s,
2H, 2OH), 12.00 (s, 1H, NH); 13CNMR (DMSO‑d6): δ (ppm): 19.4, 25.4,
46.9, 51.3, 66.5, 76.4, 108.2, 112.4, 115.6 (4C), 117.6, 119.9 (2C),
122.2, 125.8 (2C), 126.4 (4C), 128.6 (5C), 130.0 (3C), 131.1 (2C), 134.7
(2C), 135.2, 142.5, 143.7, 144.8 (2C), 147.8 (2C), 148.9, 151.7, 157.6
(2C), 164.5; UV–Vis spectrum: (λmax), 283,398 nm. Anal. calcd. For
C46H38N4O2 (678.84): C, 81.39; H, 5.64; N, 8.25%. Found: C, 81.46; H,
5.71; N, 8.36%.
4. Experimental
4.1. Instruments
All of the spectroscopic data and elemental analysis were executed as
formerly reported [31].
4.2. Synthesis of dipyrromethane derivatives 2a,b
General procedure: Grinding a mixture of pyrrole (0.02 mol), appro-
priate aldehydes 1a,b (0.01 mol) and iodine crystals (0.001 mol) in a
morter was continued for 10–15 min afforded dipyrromethanes 2a,b.
The crude reaction product was washed with water and dried (Na2SO4).
The obtained dipyrromethanes 2a,b was purified by flash chromatog-
raphy silica gel using (2:1) chloroform/cyclohexane as eluent.
4.7. Synthesis of (4,6,15,19)-5,15-bis(4-methoxyphenyl)-10,20-
dimethyl-10,20-diphenyl-1H,9H,10H,20H-porphyrin (4b)
Yield (95%); m.p. 321 C; IR (KBr): νmax (cmꢀ 1): 3347 (NH); 1H NMR
̊
(DMSO‑d6): δ (ppm): 1.45 (s, 3H, CH3), 1.86 (s, 3H, CH3), 2.95 (d, 1H,
pyrrolic-H), 3.62 (d, 1H, pyrrolic-H), 3.90 (s, 6H, 2OCH3), 5.13 (d, 1H,
pyrrolic-H), 5.59 (d, 1H, pyrrolic-H), 5.66 (t, 1H, pyrrolic-H), 5.81 (t,
1H, pyrrolic-H), 6.27 (d, 1H, pyrrolic-H), 6.29 (d, 1H, pyrrolic-H), 6.66
(s, 1H, NH), 6.68 (d, 1H, pyrrolic-H), 7.08–7.47 (m, 18H, Ar–H), 7.86 (d,
1H, pyrrolic-H), 12.04 (s, 1H, NH); 13CNMR (DMSO‑d6): δ (ppm): 19.7,
25.1, 46.2, 50.3, 56.1 (2C), 67.5, 76.6, 107.9, 112.2, 114.4 (4C), 117.3,
119.1, 119.8, 121.8, 125.8 (2C), 126.3 (4C), 127.9, 128.5 (4C), 130.1,
134.7 (2C), 136.2 (5C), 142.5, 144.3, 145.1, 146.2, 146.8, 149.0, 150.9,
159.6 (2C), 164.4 (2C); UV–Vis spectrum: (λmax), 288,389 nm. Anal.
calcd. For C48H42N4O2 (706.89): C, 81.56; H, 5.99; N, 7.93%. Found: C,
81.47; H, 6.09; N, 7.81%.
4.3. Synthesis of 2-(1-phenyl-1-(2H-pyrrol-2-yl)ethyl)-1H-pyrrole (2a)
Yield (86%); m.p. 250 C; IR (KBr): νmax (cmꢀ 1): 3213 (NH stretch-
̊
1
–
–
ing), 1610 (C C). H NMR (DMSO‑d ): δ (ppm): 1.83 (s, 3H, CH ), 2.94
6
3
(d, J = 9.5 Hz, 1H, pyrrolic-H), 5.17 (d, J = 10 Hz, 1H, pyrrolic-H), 5.56
(t, J = 9 Hz, 1H, pyrrolic-H), 5.71 (d, 1H, pyrrolic-H), 6.25 (t, 1H, Ar–H),
6.54 (d, 1H, pyrrolic-H), 7.25–7.58 (m, 5H, Ar–H), 7.79 (d, 1H, pyrrolic-
H), 12.03 (s, 1H, NH); Anal. calcd. For C16H16N2 (236.32): C, 81.32; H,
6.82; N, 11.85%. Found: C, 81.11; H, 6.69; N, 11.99%.
4.4. Synthesis of 2-((2H-pyrrol-2-ylidene) (3,4,5-trimethoxyphenyl)
methyl)-1H-pyrrole (2b)
Yield (79%); m.p. 238 C; IR (KBr): νmax (cmꢀ 1): 3223 (NH), 1600
4.8. Synthesis of (4,6,15,19)-5,15-bis(4-chlorophenyl)-10,20-dimethyl-
10,20-diphenyl-1H,9H,10H,20H-porphyrin (4c)
̊
(C C), 1256 (ph-O-C); 1H NMR (DMSO‑d6): δ (ppm): 3.61 (s, 3H,
–
–
OCH3), 3.85 (d, 6H, 2OCH3), 5.82 (d, 1H, pyrrolic-H), 6.20 (t, 1H,
pyrrolic-H), 6.39 (d, 1H, pyrrolic-H), 6.54 (s, 2H, Ar–H), 7.12 (d, 1H,
pyrrolic-H), 7.85 (d, 1H, pyrrolic-H), 8.81 (d, 1H, pyrrolic-H), 11.52 (s,
1H, NH); Anal. calcd. For C18H18N2O3 (310.35): C, 69.66; H, 5.85; N,
9.03%. Found: C, 69.53; H, 5.79; N, 8.91%.
Yield (88%); m.p. 281 C; IR (KBr): νmax (cmꢀ 1): 3338 (NH), 750
̊
(C–Cl); 1H NMR (DMSO‑d6): δ (ppm): 1.44 (s, 3H, CH3), 1.89 (s, 3H,
CH3), 2.95 (d, 1H, pyrrolic-H), 3.78 (d, 1H, pyrrolic-H), 5.12 (d, 1H,
pyrrolic-H), 5.29 (t, 1H, pyrrolic-H), 5.34 (d, 1H, pyrrolic-H), 5.55 (t,
5

A.A. Fadda et al.
Dyes and Pigments 191 (2021) 109008
1H, pyrrolic-H), 6.25 (d, 1H, pyrrolic-H), 6.26 (d, 1H, pyrrolic-H), 6.43
(d, 1H, pyrrolic-H), 6.54 (s, 1H, NH), 7.06–7.62 (m, 18H, Ar–H), 7.84 (d,
1H, pyrrolic-H), 12.02 (s, 1H, NH). 13CNMR (DMSO‑d6): δ (ppm): 19.3,
24.7, 46.4, 50.6, 67.5, 76.6, 107.5, 112.1, 117.5, 119.3, 119.9, 122.2,
126.2 (6C), 127.9 (2C), 128.2, 128.5 (4C), 128.9 (4C), 129.7, 130.2
(2C), 133.6 (2C), 135.6, 140.6 (2C), 142.8, 144.3, 145.2, 146.5 (2C),
149.1, 151.6, 164.7 (2C); UV–Vis spectrum: (λmax), 294,390 nm. Anal.
calcd. For C46H36Cl2N4 (715.72): C, 77.20; H, 5.07; N, 7.83%. Found: C,
77.27; H, 5.19; N, 7.71%.
4.12. Synthesis of (4,6,15,19)-5,15-bis(4-bromophenyl)-10,20-
dimethyl-10,20-diphenyl-1H,9H,10H,20H-porphyrin (4g)
Yield (88%); m.p. 281 C; IR (KBr): νmax (cmꢀ 1): 3338 (NH), 759
̊
(C–Br); 1H NMR (DMSO‑d6): δ (ppm): 1.42 (s, 3H, CH3), 1.81 (s, 3H,
CH3), 2.92 (d, 1H, pyrrolic-H), 3.71 (d, 1H, pyrrolic-H), 5.12 (d, 1H,
pyrrolic-H), 5.29 (t, 1H, pyrrolic-H), 5.34 (t, 1H, pyrrolic-H), 5.57 (d,
1H, pyrrolic-H), 6.25 (d, 1H, pyrrolic-H), 6.29 (d, 1H, pyrrolic-H), 6.43
(d, 1H, pyrrolic-H), 6.54 (s, 1H, NH), 7.15–7.67 (m, 18H, Ar–H), 7.79 (d,
1H, pyrrolic-H), 12.01 (s, 1H, NH). 13CNMR (DMSO‑d6): δ (ppm): 19.3,
24.7, 46.6, 50.7, 67.5, 76.6, 107.5, 112.2, 117.3, 119.3, 119.6, 122.2,
122.4 (2C), 126.1 (2C), 126.3 (4C), 128.2, 128.5 (4C), 128.7 (2C),
129.9, 130.2 (2C), 131.3 (4C), 135.3, 141.2 (2C), 142.5, 144.2, 145.3,
146.4, 146.5, 148.7, 151.7, 164.6 (2C); UV–Vis spectrum: (λmax),
288,392 nm. Anal. calcd. For C46H36Br2N4 (804.63): C, 68.67; H, 4.51;
N, 6.96%. Found: C, 68.46; H, 4.39; N, 6.75%.
4.9. Synthesis of (4,6,15,19)-10,20-dimethyl-10,20-diphenyl-5,15-bis
(3,4,5-trimethoxyphenyl)-1H,9H,10H,20H-porphyrin (4d)
Yield (91%); m.p. 271 C; IR (KBr): νmax (cmꢀ 1): 3319 (NH), 1252 (ph-
̊
O-C); 1H NMR (DMSO‑d6): δ (ppm): 1.45 (s, 3H, CH3), 1.87 (s, 3H, CH3),
2.90 (d, 1H, pyrrolic-H), 3.60 (d, 1H, pyrrolic-H), 3.77 (s, 6H, 2OCH3),
3.92 (s, 12H, 4OCH3), 5.12 (d, 1H, pyrrolic-H), 5.53 (d, 1H, pyrrolic-H),
5.62 (t, 1H, pyrrolic-H), 6.81 (d, 1H, pyrrolic-H), 6.24 (d, 1H, pyrrolic-
H), 6.27 (d, 1H, pyrrolic-H), 6.45 (d, 1H, pyrrolic-H), 6.68 (s, 1H, NH),
6.73 (d, 1H, pyrrolic-H), 7.18–7.53 (m, 14H, Ar–H), 12.05 (s, 1H, NH);
13CNMR (DMSO‑d6): δ (ppm): 19.5, 24.7, 46.4, 50.6, 56.3 (4C), 60.9
(2C), 67.5, 76.8, 101.2 (2C), 104.8 (2C), 107.8, 112.2, 117.3, 119.3,
119.8, 122.3, 126.0 (6C), 126.9, 128.2, 128.6 (4C), 130.0, 134.4, 135.5,
138.2 (2C), 142.9, 144.3, 145.0, 146.4, 146.8, 148.6, 151.9, 153.2 (4C),
164.7 (2C); UV–Vis spectrum: (λmax), 276,391 nm. Anal. calcd. For
C52H50N4 O6 (826.99): C, 75.52; H, 6.09; N, 6.77%. Found: C, 75.47; H,
6.19; N, 6.82%.
4.13. Synthesis of 5,15-bis(2,4,5-trimethoxyphenyl)-10,20-bis(3,4,5-tri-
methoxyphenyl)porphyrin (5a)
Yield (85%); m.p. 311 C; IR (KBr): νmax (cmꢀ 1): 3310 (NH), 1254 (ph-
̊
O-C); 1H NMR (DMSO‑d6): δ (ppm): 3.62 (s, 6H, 2OCH3), 3.85 (s, 24H,
8OCH3), 3.98 (s, 6H, 2OCH3), 6.31 (d, 2H, pyrrolic-H), 6.42 (d, 2H,
pyrrolic-H), 6.51 (d, 2H, pyrrolic-H), 6.61–6.88 (m, 8H, Ar–H), 7.90 (d,
2H, pyrrolic-H), 8.71 (s, 1H, NH), 9.52 (s, 1H, NH). 13CNMR (DMSO‑d6):
δ (ppm): 56.3 (8C), 56.4 (2C), 60.7 (2C), 98.6 (2C), 99.4, 101.2 (2C),
103.3, 104.9 (2C), 106.3, 111.1, 112.7, 113.0, 119.7 (2C), 120.6 (2C),
126.7, 131.8 (2C), 134.4, 136.6 (2C), 137.6 (2C), 138.3 (2C), 141.2
(2C), 142.3 (2C), 142.4 (2C), 150.1 (2C), 150.8, 152.7, 153.2 (4C),
155.8 (2C), 161.3 (2C); UV–Vis spectrum: (λmax), 427 nm. Anal. calcd.
For C56H54N4O12 (974.37): C, 68.98; H, 5.58; N, 5.75%. Found: C, 68.97;
H, 5.56; N, 5.73%.
4.10. Synthesis of (4,6,15,19)-10,20-dimethyl-5,15-bis(3-nitrophenyl)-
10,20-diphenyl-1H,9H,10H,20H-porphyrin (4e)
Yield (92%); m.p. 261 C; IR (KBr): νmax (cmꢀ 1): 3335 (NH), 1530
̊
4.14. Synthesis of 5,15-bis(4-methoxyphenyl)-10,20-bis(3,4,5-trime-
thoxyphenyl) porphyrin (5b)
(symm. NO2), 1350 (asymm. NO2); 1H NMR (DMSO‑d6): δ (ppm): 1.47
(s, 3H, CH3), 1.82 (s, 3H, CH3), 2.97 (d, 1H, pyrrolic-H), 3.78 (d, 1H,
pyrrolic-H), 5.13 (d, 1H, pyrrolic-H), 5.29 (t, 1H, pyrrolic-H), 5.33 (d,
1H, pyrrolic-H), 5.55 (t, 1H, pyrrolic-H), 6.23 (d, 1H, pyrrolic-H), 6.25
(d, 1H, pyrrolic-H), 6.45 (d, 1H, pyrrolic-H), 6.55 (s, 1H, NH), 7.35–7.86
(m, 18H, Ar–H), 8.12 (d, 1H, pyrrolic-H), 12.05 (s, 1H, NH); 13CNMR
(DMSO‑d6): δ (ppm): 19.2, 24.7, 46.6, 50.3, 67.5, 76.9, 107.5, 112.2,
117.6, 119.3, 119.6, 120.1 (2C), 122.3, 122.9 (2C), 126.3 (2C), 126.3
(2C), 127.9, 128.2, 128.6 (4C), 129.6 (2C), 130.0, 133.3, 134.3, 134.4,
135.1, 135.5, 141.0, 142.5, 144.2, 145.2, 146.4, 146.8, 148.1 (2C),
148.7, 164.5 (2C); UV–Vis spectrum: (λmax), 279,391 nm. Anal. calcd.
For C46H36N6O4 (736.83): C, 74.98; H, 4.92; N, 11.41%. Found: C,
74.87; H, 4.89; N, 11.51%.
Yield (91%); m.p. 319 C; IR (KBr): νmax (cmꢀ 1): 3312 (NH), 1261 (ph-
̊
O-C); 1H NMR (DMSO‑d6): δ (ppm): 3.62 (s, 6H, 2OCH3), 3.68 (s, 6H,
2OCH3), 3.82 (s, 12H, 4OCH3), 5.98 (d, 2H, pyrrolic-H), 6.38 (d, 2H,
pyrrolic-H), 6.55 (d, 2H, pyrrolic-H), 6.71–7.03 (m, 12H, Ar–H), 7.92 (d,
2H, pyrrolic-H), 10.12 (s, 1H, NH), 12.25 (s, 1H, NH). 13CNMR
(DMSO‑d6): δ (ppm): 55.8 (2C), 56.3 (4C), 60.6 (2C), 101.2 (2C), 103.2
(2C), 105.0 (2C), 114.0 (4C), 120.1 (2C), 120.7 (2C), 126.7, 132.3 (2C),
134.2, 134.5 (2C), 136.1 (4C), 136.8 (2C), 137.5 (2C), 138.3 (2C), 141.1
(2C), 142.3 (2C), 153.2 (4C), 155.6 (2C), 159.7 (2C), 161.2 (2C); UV–Vis
spectrum: (λmax), 429 nm. Anal. calcd. For C52H46N4O8 (854.33): C,
73.05; H, 5.42; N, 6.55%. Found: C, 73.07; H, 5.40; N, 6.54%.
4.15. Synthesis of 5,15-bis(4-chlorophenyl)-10,20-bis(3,4,5-trimethoxy-
phenyl) porphyrin (5c)
4.11. Synthesis of 6,6’-((4,6,15,19)-10,20-dimethyl-10,20-diphenyl-
1H,9H,10H,20H-porphyrin-5,15-diyl)bis(2-methoxyphenol) (4f)
Yield (86%); m.p. 284 C; IR (KBr): νmax (cmꢀ 1): 3350 (NH), 762
̊
(C–Cl); 1H NMR (DMSO‑d6): δ (ppm): 3.55 (s, 6H, 2OCH3), 3.85 (s, 12H,
4OCH3), 6.29 (d, 2H, pyrrolic-H), 6.39 (d, 2H, pyrrolic-H), 6.41 (d, 2H,
pyrrolic-H), 6.54 (s, 2H, Ar–H), 6.59 (s, 2H, Ar–H), 7.22–7.78 (m, 8H,
Ar–H), 7.85 (d, 2H, pyrrolic-H), 9.03 (s, 1H, NH), 9.81 (s, 1H, NH).
13CNMR (DMSO‑d6): δ (ppm): 56.0 (4C), 60.7 (2C), 101.3 (2C), 105.2
(2C), 119.7 (2C), 120.6 (2C), 126.7, 127.6 (2C), 128.2 (4C), 130.2 (2C),
132.3 (2C), 133.4 (2C), 134.2, 136.5 (2C), 137.5 (2C), 138.3 (2C), 140.5
(2C), 141.2 (2C), 142.3 (2C), 153.1 (4C), 155.8 (2C), 161.2 (2C); UV–Vis
spectrum: (λmax), 424 nm. Anal. calcd. For C50H40Cl2N4O6 (862.23): C,
69.52; H, 6.49; N, 6.52%. Found: C, 69.50; H, 6.47; N, 6.53%.
Yield (95%); m.p. 321 C; IR (KBr): νmax (cmꢀ 1): 3400 (OH), 3347
̊
(NH), 1260 (ph-O-C); 1H NMR (DMSO‑d6): δ (ppm): 1.41 (s, 3H, CH3),
1.86 (s, 3H, CH3), 2.96 (d, 1H, pyrrolic-H), 3.62 (d, 1H, pyrrolic-H), 3.93
(s, 6H, 2OCH3), 5.12 (d, 1H, pyrrolic-H), 5.50 (d, 1H, pyrrolic-H), 5.66
(t, 1H, pyrrolic-H), 5.85 (t, 1H, pyrrolic-H), 6.24 (d, 1H, pyrrolic-H),
6.26 (d, 1H, pyrrolic-H), 6.66 (s, 1H, NH), 6.68 (d, 1H, pyrrolic-H),
7.01–7.48 (m, 16H, Ar–H), 7.86 (d, 1H, pyrrolic-H), 12.01 (s, 1H,
NH), 13.29 (s, 2H, 2OH); 13CNMR (DMSO‑d6): δ (ppm): 19.2, 24.7, 46.3,
50.7, 55.8 (2C), 67.5, 76.6, 107.6, 111.8 (2C), 115.7, 115.9, 117.3,
119.3, 119.5, 122.2, 122.3, 125.6, 125.7 (2C), 125.9 (4C), 127.9, 128.5
(4C), 129.9, 135.3, 142.6, 144.3, 145.2, 146.2, 146.4, 146.7, 147.5 (2C),
149.0 (3C), 150.6, 164.5 (2C); UV–Vis spectrum: (λmax), 278,389 nm.
Anal. calcd. For C48H42N4O4 (738.89): C, 78.03; H, 5.73; N, 7.58%.
Found: C, 78.21; H, 5.59; N, 7.41%.
6

A.A. Fadda et al.
Dyes and Pigments 191 (2021) 109008
4.16. Synthesis of 5,15-bis(4-bromophenyl)-10,20-bis(3,4,5-trimethoxy-
phenyl)porphyrin (5d)
of distilled water to achieve complete hemolysis, and the absorbance B
of the supernatant obtained after centrifugation was measured at 540
nm [35].
Yield (86%); m.p. 284 C; IR (KBr): νmax (cmꢀ 1): 3348 (NH), 751
̊
(C–Br); 1H NMR (DMSO‑d6): δ (ppm): 3.53 (s, 6H, 2OCH3), 3.82 (s, 12H,
4OCH3), 6.23 (d, 2H, pyrrolic-H), 6.39 (d, 2H, pyrrolic-H), 6.42 (d, 2H,
pyrrolic-H), 6.53 (s, 4H, Ar–H), 7.24–7.78 (m, 8H, Ar–H), 7.88 (d, 2H,
pyrrolic-H), 9.11 (s, 1H, NH), 10.22 (s, 1H, NH). 13CNMR (DMSO‑d6): δ
(ppm): 56.2 (4C), 60.6 (2C), 101.3 (2C), 102.9 (2C), 105.3 (2C), 119.7
(2C), 120.6 (2C), 122.2 (2C), 126.7, 128.8 (2C), 130.3 (2C), 131.7 (4C),
131.8 (2C), 134.4, 136.6 (2C), 137.3 (2C), 138.5 (2C), 141.4 (4C), 142.3
(2C), 153.2 (4C), 155.8 (2C), 161.3 (2Cd); UV–Vis spectrum: (λmax), 426
nm. Anal. calcd. For C50H40Br2N4O6 (950.13): C, 63.04; H, 4.23; N,
5.88%. Found: C, 63.05; H, 4.21; N, 5.86%.
4.16.4. Antioxidant activity screening assay ABTS method
For each of the investigated dyes, 2 mL of ABTS solution (60 mM)
was added to 3 mL MnO2 solution (25 mg/mL) all prepared in 5 mL
aqueous phosphate buffer solution (pH 7, 0.1 M). The mixture was
shaken, centrifuged, filtered, and the absorbance (A control) of the
resulting green-blue solution (ABTS radical solution) at λmax 734 nm was
adjusted at approx. 0.5. Then, 50 mL of 2 mL solution of the test com-
pound in spectroscopic grade MeOH/phosphate buffer (1:1) was added.
The absorbance (A test) was measured and the reduction in color in-
tensity was expressed as % inhibition percentage. L-Ascorbic acid
(vitamin C) was used as a standard antioxidant (positive control), and a
blank sample was run without ABTS and using MeOH/phosphate buffer
(1:1) instead of the tested compounds [36].
4.16.1. Biochemical assays
(I) Antimicrobial assay:
4.16.5. Bleomycin-dependent DNA damage assay
Standard sterilized filter paper disks (5 mm diameter) impregnated
with a solution of the tested compound in DMF (1 mg/mL) was placed on
an agar plate seeded with the appropriate test organism in triplicates.
The utilized test organisms were: Bacillus Subtilis and Bacillus Thur-
ingiensis as examples of Gram-positive bacteria and Escherichia Coli and
Pseudomonas Aeruginosa as examples of Gram-negative bacteria. We
have been estimated the synthesized compounds for their in vitro anti-
fungal potential against Fusarium Oxysporum and Botrytis Fabae fungal
strains. Chloramphenicol, cephalothin, and cycloheximide were used as
standard antibacterial and antifungal agents, respectively [33]. DMF
alone was used as control at the same above-mentioned concentration.
The plates were incubated at 37 ◦C for 24 h for bacteria and 48 days for
fungi. Compounds that showed significant growth inhibition zones
(>14 mm) using the twofold serial dilution technique, were further
evaluated for their minimal inhibitory concentrations (MICs).
The reaction mixtures contained, in a final volume of 1.0 mL, the
following reagents at the final concentrations stated: DNA (0.2 mg/mL),
bleomycin (0.05 mg/mL), FeCl3 (0.025 mM), MgCl (5 mM),
KH2PO4–KOH buffer pH 7.0 (30 mM), and ascorbic acid (0.24 Mm) or
the dyes tested in MeOH to give a concentration of (0.1 mg/mL). The
reaction mixtures were incubated in a water bath at 378C for 1 h. At the
end of the incubation period, 0.1 mL of 0.1 M EDTA was added to stop
the reaction (the iron–EDTA complex is unreactive in the bleomycin
assay). DNA damage was assessed by adding 1 mL 1% w/v TBA and 1 mL
25% v/v hydrochloric acid (HCl) followed by heating in a water bath
maintained at 808C for 15 min. The chromogen formed was extracted
into butan-1-ol and the absorbance was measured at 532 nm [37–39].
CRediT authorship contribution statement
Ahmed A. Fadda: Supervision, Principle supervisor, suggesting the
point of research, Continuous help, continuous advice, Formal analysis,
check up the analysis, Writing – review & editing. Eman El-Gendy:
Responsible for practical work, Writing – review & editing. Hala M.
Refat: Supervision, Co-supervisor. Eman H. Tawfik: Continuous help.
4.16.2. Minimal inhibitory concentration (MIC) measurement
The minimum inhibitory concentrations (MICs) were determined
using 96-well microtitre plates. The microdilution susceptibility test in
Müller-Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid)
was used for the determination of antibacterial and antifungal activity,
respectively. Stock solutions of the tested compounds, chloramphenicol,
cephalothin, and cycloheximide were prepared in DMF at a concentra-
Declaration of competing interest
tion of 1000
μg/mL followed by twofold dilution at concentrations of
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
(500, 250, 3.125
μ
g/mL). The microorganism suspensions at 106 CFU/
mL (Colony Forming U/mL) concentrations were inoculated to the
corresponding wells. Plates were incubated at 36 ◦C for 24–48 h and the
minimal inhibitory concentrations (MIC) were determined. Control ex-
Appendix A. Supplementary data
periments were also done. The MIC values were expressed in
μg/ml [34].
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2020.109008.
(II) Antioxidant assay:
4.16.3. Antioxidant activity screening assay for erythrocyte hemolysis
Blood was obtained from rats by cardiac puncture and collected in
heparinized tubes. Erythrocytes were separated from plasma and the
buffy coat and washed three times with 10 vol of 0.15 M NaCl. During
the last washing, the erythrocytes were centrifuged at 2500 rpm for 10
min to obtain a constantly packed cell preparation. Erythrocyte hemo-
lysis was mediated by peroxyl radicals in this assay system. A 10%
suspension of erythrocytes in phosphate-buffered saline pH 7.4 (PBS)
was added to the same volume of 200 mM 2,20-azobis (2-amidinopro-
pane) dihydrochloride (AAPH) solution (in PBS) containing samples to
be tested at different concentrations. We used L-Ascorbic acid as a pos-
itive control. The solution was shaken gently while being incubated at
378C for 2 h, diluted with eight volumes of PBS, and centrifuged at
1500g for 10 min. The absorbance A of the supernatant was measured at
540 nm. Similarly, the reaction mixture was treated with eight volumes
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