Chemical constituents with antimicrobial and antioxidant activity from the aerial parts of Callistemon lanceolatus (Sm.) Sweet

Article abstract of DOI:10.1080/14786419.2018.1557174

Callistemon lanceolatus (Sm.) Sweet grows all over the world and used to treat cough and bronchitis. The air-dried powder of the aerial parts was exhaustively extracted with methanol and the concentrated extract was adsorbed on silica gel for preparation

Full text of DOI:10.1080/14786419.2018.1557174

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Chemical constituents with antimicrobial and
antioxidant activity from the aerial parts of
Callistemon lanceolatus (Sm.) Sweet
Syed Nazreen, Mohammad Mahboob Alam, Hinna Hamid, Mohammad Ali &
Mohammad Sarwar Alam
To cite this article: Syed Nazreen, Mohammad Mahboob Alam, Hinna Hamid, Mohammad Ali &
Mohammad Sarwar Alam (2019): Chemical constituents with antimicrobial and antioxidant activity
from the aerial parts of Callistemonꢀlanceolatus (Sm.) Sweet, Natural Product Research, DOI:
10.1080/14786419.2018.1557174
To link to this article: https://doi.org/10.1080/14786419.2018.1557174
View supplementary material
Published online: 08 Jan 2019.
Submit your article to this journal
Article views: 6
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20

NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1557174
SHORT COMMUNICATION
Chemical constituents with antimicrobial and antioxidant
activity from the aerial parts of Callistemon lanceolatus
(Sm.) Sweet
Syed Nazreena,b, Mohammad Mahboob Alama,b, Hinna Hamidb, Mohammad Alic
and Mohammad Sarwar Alamb
aFaculty of Science, Department of Chemistry, Al Baha University, Kingdom of Saudi Arabia;
bFaculty of Science, Department of Chemistry, Jamia Hamdard (Hamdard University), New Delhi,
c
India; Faculty of Pharmacy, Department of Pharmacognosy and Phytochemistry, Jamia Hamdard
(Hamdard University), New Delhi, India
ABSTRACT
ARTICLE HISTORY
Received 2 October 2018
Accepted 5 December 2018
Callistemon lanceolatus (Sm.) Sweet grows all over the world
and used to treat cough and bronchitis. The air-dried powder of
the aerial parts was exhaustively extracted with methanol and
the concentrated extract was adsorbed on silica gel for prepar-
ation of slurry. It was dried and subjected to silica gel column
packed in petroleum ether. The column was eluted with organic
solvents in order of increasing polarity to isolate 1-triacosanol
(1), n-eicosanyl palmitate (2), n-heptadecanyl arachidate (3),
n-tricosanyl palmitate, (4), 4-hydroxyphenethyl carbocerate (5),
4-hydroxyphenethyl gheddate (6), urs-12-en-3a-acetoxy-18b-
H-28-oic acid (7) and stigmast-5-en-3b-ol-3b-D-glucuronopyra-
noside (8). Among them, compound 5 and 6 were new fatty
acid ester isolated from this plant. Compound 7 showed MIC 32
mg/mL against E. coli which was comparable to amoxicillin hav-
ing same MIC 32 mg/mL. Compound 5 and 6 showed significant
antioxidant activity by inhibiting DPPH due to the presence of
phenolic groups.
KEYWORDS
Antimicrobial; Callistemon
lanceolatus; DPPH; isolation;
microdilution
CONTACT Mohammad Sarwar Alam
msalam@jamiahamdard.ac.in; msalam5555@gmail.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1557174.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
S. NAZREEN ET AL.
1. Introduction
Callistemon lanceolatus (Sm.) Sweet, syn. C. citrinus (Curtis) Skeels, lemon bottle brush,
red bottle brush, is originally from Australia and widely planted as an ornamental
plant all over the world including India (Anonymous 1992). The leaves are used as a
tea substitute with a delightfully refreshing flavor. Egyptians utilized the plant volatile
oils as antimicrobial and insecticidal agents and for the treatment of cough and bron-
chitis (Shinde et al. 2012; Das and Singh, 2012). The alcoholic extracts of the leaves
exhibited good antimicrobial effect against Salmonella typhi, Bacillus ereus,
Streptococcus epidermidi and B. anthracis which was comparable with antibiotics
(Seyydnejad et al. 2010). The ethanol extract of C. lanceolatus showed strong elastase
inhibition and DPPH radical scavenging activities (Kim et al. 2009). We have carried
out the antimicrobial and antioxidant activities of the compounds to know the respon-
sible phytoconstituents which are imparting such activity to this plant.
In our earlier study, we have reported new flavones, phenolic esters, a-amyrin,
betulinic acid, oleanolic acid and kaempferol from the chloroform fractions of
Callistemon lanceolatus (Nazreen et al. 2012, 2014). In continuation of our work on the
isolation of chemical constituents, we now report herein the isolation and characteriza-
tion of chemical constituents with their antimicrobial and antioxidant activity.
2. Results and Discussion
Compound 1 was an aliphatic alcohol identified as 1-triacosanol (myricyl alcohol)
(Jaybhay et al. 2010). Compounds 2, 3 and 4 were the known fatty ester characterized
as n-eicosanyl palmitate (eicosanyl-hexacosanoate), n-heptadecanyl arachidate (n-hep-
tadecanyl eicosanoate) and n-tricosanyl palmitate, respectively (Ribechini et al. 2008;
Alam et al. 2011). Compound 7 and 8 were characterized as urs-12-en-3a-acetoxy-18b-
H-28-oic acid and stigmast-5-en-3b-ol-3b-D-glucuronoopyranoside, respectively. (Ali,
2001) (Supplementary data)
Compound 5, designated as 4-hydroxyphenethyl carbocerate, responded positive
phenolic tests and exhibited UV absorption maximum at 281 nm for aromatic com-
pound. The IR spectrum revealed absorption bands 3450 cmꢀ1 (OH), 1725 cmꢀ1 (ester)
and 725 cmꢀ1 (aliphatic chain). Its mass spectrum exhibited a molecular ion peak at
1
m/z 530 (C35H62O3). The H NMR spectrum showed two doublets at d 7.08 (J ¼ 8.4 Hz)
and d 6.76 (J ¼ 8.4 Hz) integrating for two-protons each assigned to aromatic H-2, H-6
and H-3, H-5 protons, respectively, three two-proton triplets at d 4.23 (J ¼ 7.0 Hz), 2.85
(J ¼ 7.0 Hz), 2.27 (J ¼ 7.4 Hz) ascribed correspondingly oxymethylene H2-8, methylene
H2-7 linked to the aromatic ring and methylene H2-20 adjacent to the ester function.
The other methylene protons appeared as multiplets at d 1.57 (2H) and 1.29 (4H) and
as a broad singlet at d 1.23 (42 H). A triplet at d 0.87 (J ¼ 6.3 Hz, 3H, Me-270) was
accounted to terminal C-270 primary methyl protons. The 13C NMR spectrum of 5 dis-
played signals for ester carbon at d 176.23 (C-10), aromatic carbons between d 156.98
and 115.32, oxymethylene carbon at d 63.94 (C-8), other methylene carbons from d
35.09 to 21.33 and methyl carbon at d 13.73 (C-270). From the HSQC correlations
(Figure S2), the aromatic protons at H-2 and H-6 (d 7.08) showed correlation with C-2
and C-6 (d 129.39) and H-3 and H-5 (d 6.76) showed interactions with C-3 and C-5 (d

NATURAL PRODUCT RESEARCH
3
1
1'
20'
16
CH3-(CH2)14--CO-O-CH2-(CH2)18-CH3
CH3-(CH2)28CH2OH
2
1
1
1'
17'
20
1
1'
23'
16
CH3-(CH2)18-CO-O-CH2-(CH2)15-CH3
CH3-(CH2)14-CO-O-CH2-(CH2)21-CH3
4
3
3
2
3
2
1'
1'
8
27'
8
7
7
34'
4
4
1
1
HO
CH2-CH2-O-CO-(CH2)25-CH3 HO
CH2-CH2-O-CO-(CH2)32-CH3
5
6
6
5
5
6
30
29
28
21
29
19
20
17
20
21
18
14
22
27
12
25
12
18
22
28
11
13
13
19
11
16
26
OH
17
16
1
9
25
26
8
1
4
2
14
15
10
9
O
2
6'
3
10
8
15
HOOC
7
O
5
27
O
3
4
6
4'
O
7
5' 2'
5
1'
HO
HO
6
O
3'
H3C
OH
24
23
8
7
Figure 1. Structure of compounds (1–8) isolated from Callistemon lanceolatus.
115.32). Benzylic protons H2-7 (d 2.85) correlated with C-7 (d 35.09); oxymethylene
protons H2-8 (d 4.23) with C-8 (d 63.94); methylene H2-20 (d 2.27) with C-20 (d 35.89);
other methylene protons (d 1.57, 1.29 and 1.23) with C-30 to C-250 (d 32.79–21.33) and
methyl protons H3-270 (d 0.87) with C-270 (d 13.73). From HMBC, 4-OH showed correl-
ation (d 9.27) with C-3 and C-5 (d 115.32); The attachment of long alkyl side chain
1
could be deduced from long-range H–13C correlations observed between H2-7 (d 2.
85) with C-2 and C-6 (d 129.39);H2-8 (d 4.23) with C-1 (d 130.09), C-10 (d 176.23).
(Figure S2). Acid hydrolysis of 5 yielded 4-hydroxyphenethyl alcohol (tyrosol), m.p.
90–92 ꢁC, [M]þþ m/z at 138 (C8H10O2) and carboceric acid (heptacosanoic acid), m.p.
88–89 ꢁC; [M] m/z at 410 (C27H54O2). Analysis of the spectral data and chemical
reactions led to formulate the structure of 5 as 4-hydroxyphenethyl heptacosanoate,
a new fatty acid ester (Figure 1).
Compound 6, named 4-hydroxyphenethyl gheddate, responded positively to phen-
olic tests and showed UV absorption maximum at 277 nm for an aromatic compound,
It also showed IR absorption bands for hydroxyl group (3503 cmꢀ1), ester function
(1735 cmꢀ1), aromatic ring (1647, 1541 cm_1) and a long aliphatic chain (727 cmꢀ1). Its
mass spectrum exhibited a molecular ion peak at m/z 628 consistent with a molecular
formula of a fatty acid ester with aromatic alcohol, C42H76O3. The ion peaks arising at
þ
þ
0
m/z 507 [C8–O fission, OCO(CH2)32CH3] , 491[C1 –O fission, CO(CH2)32CH3] and 137
[M–491]þ indicated that gheddic acid (tetratriacontanoic acid) was esterified with 4-
1
hydroxyphenethyl alcohol (tyrosol). The H NMR spectrum of 6 displayed two two-pro-
ton doublets d 7.08 (J ¼ 8.4 Hz) and 6.76 (J ¼ 8.0 Hz) assigned to aromatic H-2, H-6 and
H-3, H-5 protons, respectively, three two-proton triplets at d 4.23 (J ¼ 7.1 Hz), 2.87
(J ¼ 7.7 Hz) and 2.27 (J ¼ 7.4 Hz) ascribed correspondingly oxymethylene H2-8 and
methylene H2-7 and H2-20 protons, other methylene protons as multiplets at d 1.56
(2H) and 1.28 (4H) and as a broad singlet at d 1.25 (56 H) and a three-proton triplet at

4
S. NAZREEN ET AL.
d 0.87 (J ¼ 6.3 Hz) accounted to terminal primary C-340 methyl protons. The 13C NMR
spectrum of 6 exhibited signals for ester carbon at d 171.80 (C-10), aromatic carbons
between d 159.32 and 115.39, oxymethylene carbon at d 64.95 (C-8), other methylene
carbons in the range of d 34.57–22.70 and methyl carbon at d 14.13 (Me-340). From
the HSQC spectrum (Figure S2), the aromatic protons at H-2 and H-6 (d 7.08) showed
correlations with C-2 and C-6 (d 115.39), the aromatic protons H-3 and H-5 (d 6.76)
interacted with C-5 and C-6 (d 130.05), benzylic protons H2-7 (d 2.87) correlated
with C-7 (d 34.57); oxygenated methylene protons H2-8 (d 4.23) with C-8 (d 64.95);
methylene protons H2-20 (d 2.27) with C-20 (d 34.28); other methylene protons with
C-30 to C-330 (d 31.94 - 22.70) and methyl protons H3-340 (d 0.87) with C-340 (d 14.13).
The attachment of a hydroxyl group at C-4 was based on the long-range 1H–13C
correlations (HMBC) observed between 4-OH (d 10.94) with C-3 and C-5 (d 115.39).
The attachment of long alkyl side chain could be deduced from HMBC correlations
observed between H2-7 (d 2.87) with C-2 and C-6 (d 115.39); H2-8 (d 4.23) with C-1
(d 138.41), C-10 (d 171.80). (Figure S2). Acid hydrolysis of 6 yielded 4-hydroxyphenethyl
alcohol (tyrosol), m. p. 90-92 ꢁC, [M]þþm/z at 138 (C8H10O2) and gheddic acid (tetratria-
contanoic acid), m. p. 93-94ꢁ C; [M] m/z at 508 (C34H68O2). On the basis of spectral
evidences and chemical reactions, the structure of 6 has been elucidated as 4-hydroxy-
phenethyl tetratriacontanoate, a new fatty acid ester (Figure 1).
Compound 7 showed good antibacterial activity against S. aureus, E.coli and
K. pneumoniae with zone of inhibition 28, 28, 26 respectively at concentration 200 mg/
mL comparable to standard drug Amoxycillin (30, 28, 29) respectively at the same
concentration. Compounds 5, 6, and 8 showed moderate antimicrobial activity.
Compound 7 showed MIC 32 mg/mL against E. coli which was comparable to amoxicillin
having same MIC 32 mg/mL (Table S1 and S2).
Compounds 5 and 6 due to presence of phenolic group exhibited good antioxidant
activity by inhibiting DPPH. These compounds strongly scavenged DPPH radical,
with IC50 values of 3.8mM and 3.2 mM, respectively. Vitamin C, used as positive
controls, showed IC50 values of 1.6 mM (Table S3). Compound 7 and 8 was found to
be inactive.
Conclusion
This study led to the isolation of eight compounds (1–8) from Callistemon lanceolatus.
Compound 7 was potent against bacterial strain E. coli. Compounds 5 and 6
showed good antioxidant activity. This work has enhanced understanding about the
phytoconstituents of the undertaken plants. These secondary metabolites can be used
as analytical markers for quality control of the aforementioned herbal drugs. All these
phytoconstituents are reported for the first time from this plant and can be used
for quality control of the plant.
Acknowledgments
The authors are thankful to Al Baha University, Kingdom of Saudi Arabia and Jamia Hamdard,
New Delhi, India for providing the necessary facilities to the Department of Chemistry and

NATURAL PRODUCT RESEARCH
5
Dr T S Thakur, Microbiologist, Regional Laboratory and Central Blood Bank, Albaha, Kingdom
of Saudi Arabia for evaluating antimicrobial activity.
Disclosure statement
No conflict of interest was reported by the authors.
References
Anonymous. 1992. The Wealth of India: A dictionary of indian raw materials and industrial
products, Vol. 3. New Delhi: Ca–Ci, Publication and information Directorate, Council of
Scientific and Industrial Research, 64–65.
Alam P, Ali M, Aeri V. 2011. Isolation of four new phytoconstituents from the roots of Albizzia
lebbeck Benth. Der Pharm Lett. 3(6):74–81.
Ali M. 2001.Techniques in terpenoid identification. Delhi: Birla Publications; 352–60.
Das S, Singh U. 2012. Therapeutic potentials of Callistemon lanceolatus DC. Int J Adv Pharm Biol
Chem. 1(2): 206–210.
Jaybhay S, Chate P, Ade A. 2010. Isolation and Identification of crude triacontanol from rice bran
wax . J Exp Sci. 1(2): 26.
Kim JH, Byun JC, Bandi AKR, Hyun CG and Lee NH. 2009. Compounds with elastase inhibition
and free radical scavenging activities from Callistemon lanceolatus. J Med Plant Res. 3(11):
914–920.
Nazreen S, Alam MS, Hamid H, Dhulap A, Alam MM, Bano S, Haider S, Ali M, Pillai KK. 2014.
New flavone and phenolic esters from Callistemon lanceolatus DC: Their molecular docking
and antidiabetic activities. Arab J Chem. https://doi.org/10.1016/j.arabjc.2014.11.029.
Nazreen S, Kaur G, Alam MM, Shafi S, H Hamid, Ali M, Alam MS. 2012. New flavones
with antidiabetic activity from Callistemon lanceolatus DC. Fitoterapia. 83(8): 1623–1627.
Ribechini E, Modugno F, Colombini MP, Evershed RP. 2008. Gas chromatographic and mass
spectrometric investigations of organic residues from Roman glass unguentaria. J Chromatog
A. 1183: 158–169 .
Seyydnejad SM, Niknejad M, Darabpoor I, Motamedi H. 2010. Antibacterial activity of hydro-
alcoholic extract of Callistemon citrinus and Albizia lebbeck. Am J Appl Sci., 7(1): 13–16.
Shinde PR, Patil PS, Bairagi VA. 2012. Pharmacognostic, phytochemical properties and antibacter-
ial activity of Callistemon citrinus viminalis leaves and stems. Int J Pharm Pharm Sci. 4(4):
406–413.