Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Muntingia calabura: A review of its traditional

uses, chemical properties, and pharmacological
observations

N. D. Mahmood, N. L. M. Nasir, M. S. Rofiee, S. F. M. Tohid, S. M. Ching, L. K.

Teh, M. Z. Salleh & Z. A. Zakaria

To cite this article: N. D. Mahmood, N. L. M. Nasir, M. S. Rofiee, S. F. M. Tohid, S. M. Ching,

L. K. Teh, M. Z. Salleh & Z. A. Zakaria (2014) Muntingia calabura: A review of its traditional
uses, chemical properties, and pharmacological observations, Pharmaceutical Biology, 52:12,
1598-1623, DOI: 10.3109/13880209.2014.908397

To link to this article: https://doi.org/10.3109/13880209.2014.908397

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ISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. Pezzuto
Pharm Biol, 2014; 52(12): 1598–1623
! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.908397

REVIEW ARTICLE

Muntingia calabura: A review of its traditional uses, chemical

properties, and pharmacological observations
N. D. Mahmood1, N. L. M. Nasir1, M. S. Rofiee2, S. F. M. Tohid1, S. M. Ching3, L. K. Teh2, M. Z. Salleh2, and
Z. A. Zakaria1,2,4
1
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia, 2Integrative
Pharmacogenomics Institute, Universiti Teknologi MARA, Selangor, Malaysia, 3Department of Family Medicine, Faculty of Medicine and Health
Sciences, Selangor, Malaysia, 4Halal Product Research Institute, Universiti Putra Malaysia, Selangor, Malaysia

Abstract Keywords
Context: Different parts of Muntingia calabura L. (Elaeocarpaceae), or ‘‘kerukup siam’’ in Malay, Elaeocarpaceae, ethnomedicinal uses,
have been reported to possess medicinal value, supported by a number of scientific studies. pharmacological activities,
Objective: To gather all information related to the ethnomedicinal uses, phytochemical phytoconstituents
compositions, and pharmacological activities of M. calabura and present them as a
comprehensive and systematic review article. History
Materials and methods: Literature has been retrieved from a number of databases (e.g.,
Pubmed, Science Direct, Springer Link, etc.). General web searches were also carried out using Received 19 July 2013
Google and Yahoo search engines by applying some related search terms (e.g., Muntingia Revised 10 February 2014
calabura, phytochemical, pharmacological, extract, and traditional uses). The articles related to Accepted 22 March 2014
agriculture, ecology, and synthetic work and those using languages other than English or Malay Published online 25 July 2014
have been excluded. The bibliographies of papers relating to the review subject were also
searched for further relevant references.
Results and discussion: The literature search conducted using the above-mentioned Internet
search engines only lead to the identification of 36 journals published as early as 1987. From
the articles reviewed, M. calabura possessed various pharmacological activities (e.g., cytotoxic,
antinociceptive, antiulcer, anti-inflammatory), which supported the folklore claims and could be
attributed to its phytoconstituents.
Conclusion: Muntingia calabura possesses remarkable medicinal value, which warrants further
and in-depth studies. Therefore, this review paper is presented to help guide researchers to
plan their future studies related to this plant in the hope of isolating potential leads for future
drug development.

Introduction Muntingia calabura is known throughout the world as

Medicinal plants are sources of important therapeutic aid for
alleviating human ailments. Approximately 80% of the people
in the developing countries all over the world depend on the
traditional medicine for their primary health-care.
Interestingly, approximately 85% of traditional medicine
involves the use of plant extracts. Interest in phytomedicine
started in the last 20 years and with increasing awareness of
the health hazards and toxicities associated with unsystematic
use of synthetic drugs and antibiotics, interest in the use of
plants and plant-based drugs has revived throughout the
world. However, a large number of medicinal plants remain
to be investigated for their possible pharmacological value.
One of the plants that has recently gained a medicinal plant
status is Muntingia calabura L. (Elaeocarpaceae).
Correspondence: Associate Professor. Dr. Zainul Amiruddin Zakaria,
Department of Biomedical Science, Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia. Tel: +603 89472654. Fax: +603 89436178. E-mail:
dr_zaz@yahoo.com
‘‘Jamaican cherry’’ and in Malaysia, particularly among the
Malay, it is known as ‘‘kerukup siam’’. Being the sole species
within the genus Muntingia, it is native to southern Mexico,
tropical South America, Central America, the Greater
Antilles, Trinidad, and St. Vincent. It is also widely cultivated
in warm areas in India and Southeast Asia such as Malaysia,
Indonesia, and the Philippines. Indeed, in Malaysia,
M. calabura is commonly cultivated as roadside trees
(Morton, 1987; Sani et al., 2012; Yusof et al., 2011;
Zakaria et al., 2006a,b, 2007a–f, 2008, 2010, 2011).
Botanical information
This plant is a fast-growing tree of slender proportions,
reaching a height of approximately 7.5–12 m with nearly
horizontal spreading branches (Figure 1a). The leaves of
M. calabura are evergreen approximately 5–12.5 cm long,
alternate lanceolate or oblong, long pointed at the apex,
oblique at the base with dark green color and minutely hairy
on the upper surface, gray- or brown-hairy on the underside
DOI: 10.3109/13880209.2014.908397
Figure 1. The various parts of M. calabura. (a) The tree of M. calabura. (b) The leaves of M. calabura. (c) The flower of M. calabura. (d) The fruits of
M. calabura.
and irregularly toothed (Figure 1b). The flowers are approxi-
mately 1.25–2 cm wide; borne singly or in 2’s or 3’s in the
leaf axils with five green sepals and five white petals and
many prominent yellow stamens (Figure 1c). The fruits are
abundant, in round shape; approximately 1–1.25 cm wide,
with red or yellow, thin, smooth, tender skin and light-brown,
soft, juicy pulp, with very sweet, musky, fig-like flavor, and
filled with exceedingly tiny, yellowish seeds (Figure 1d)
(Morton, 1987).
Traditional uses
The emergence of various types of diseases, both infec-
tious and non-infectious, nowadays have become a major
Review on M. calabura medicinal value 1599
global burden. Various pharmaceutical drugs have been
developed and prescribed to patients to help cure those
diseases. Unfortunately, conventional drugs have also been
associated with various unwanted side effects. For example,
morphine has been known to cause phenomena such as
tolerance and dependence while the appearance of anti-
biotic-resistance bacteria such as methicillin- and vanco-
mycin-resistance bacteria have been well documented
(Katzung, 2012). Due to these problems, patients have
been looking for alternatives to treat their diseases, where
complementary and alternative medicine (CAM), particu-
larly the plant-based medicines, has been one of the
sources of the CAM used. One of the plants that has
1600 N. D. Mahmood et al.
Table 1. The vernacular names of M. calabura.
Vernacular names Country
Kerukup siam Malaysia
Singapore cherry, Jamaican cherry, Panama berry, Britain
cotton candy berry, glade mallow, glademallow
Pau de seda, calabura Brazil
Kersen, talok Indonesia
Bolaina, yamanaza, cacaniqua, capulı́n blanco, Spain
nigua, niguito, memizo or memiso
Alatris, aratilis, manzanitas, sarisa, cereza Philippines
Púan, Capulin rojo Mexico
Gasagase hanninamara India
Trú’ng cá, mat sam; cay trung ca Vietnam
Takhop farang Thailand
krakhob barang Cambodia
Bois ramier, bois de soil, bois France
Páu de seda, calabura Portugal
Bolaina, yamanaza, bolina, lumanasa Peru
Capulina, guasimo bobo, memiso Cuba
Majuaguito, majanjo, chitato, chitoto Colombia
recently gained attention among researchers throughout the
world is M. calabura.
Based on the literature search carried out, this plant
has limited traditional uses throughout the world with medi-
cinal uses recorded in, particularly, Peru, Colombia, Mexico,
Vietnam, and the Philippines. The vernacular names of
M. calabura in various countries are given in Table 1.
This might explain why M. calabura medicinal value is not
well documented in Malaysia and why it is considered as a
neglected plant (Zakaria et al., 2006a, 2007a). Despite the lack
of traditional claims, various parts of the plant have been used
to treat different types of illnesses. In Peruvian folklore
medicine, the flowers and bark are used as an antiseptic and to
reduce swelling in lower extremities, while the leaves, either
boiled or steeped in water, are used to reduce gastric ulcer and
swelling of the prostate gland, and to alleviate headache
and cold (Morton, 1987; Zakaria et al., 2007d). Moreover, the
boiled bark can be used as a wash to reduce swelling in
the lower extremities (Zakaria et al., 2006a). In Colombia, the
infusion of the flowers is used as a tranquillizer and tonic
(see Kaneda et al., 1991; Perez-Arbealaez, 1975). In Mexico,
the plant is used to treat measles, mouth pimples, and
stomachache (Yasunaka et al., 2005). In the Philippines, the
flowers are also used to treat headache and incipient cold or as
tranquillizers, antispasmodics, and antidyspeptics. Other than
that, the roots of M. calabura have been used as an
emmenogogue in Vietnam and as an abortifacient in Malaysia.
Apart from the medicinal uses, the fruits, which are
sometimes eaten fresh, are frequently cooked in tarts or made
into jam, while the leaf infusion is drunk as a tea-like
beverage (Zakaria et al., 2007e).
Phytochemical constituents of M. calabura
From 1991 to the present, various phytochemical constituents
have been isolated from different parts of M. calabura. Kaneda
et al. (1991) were the first to isolate bioactive compounds
from the roots of M. calabura. They reported on the
isolation of 12 flavonoids from the methanol extract of
M. calabura roots (MEMCR), namely, (2S)-50 -hydroxy-7,30 ,40 -
trimethoxyflavan (1), (2S)-7,8,30 ,40 ,50 -pentamethoxyflavan
Pharm Biol, 2014; 52(12): 1598–1623
References
Zakaria et al. (2006a, 2007c, 2008, 2009, 2010, 2011)
Neto Bandeira et al. (2013)
Neto Bandeira et al. (2013)
Wikipedia (2013)
Wikipedia (2013) and Janick and Paull (2008)
Wikipedia (2013) and Janick and Paull (2008)
Yasunaka et al. (2005)
Siddiqua et al. (2010)
Wikipedia (2013) and Janick and Paull (2008)
ICRAF (2004) and Janick and Paull (2008)
ICRAF (2004)
Duke (2009) and Janick and Paull (2008)
Janick and Paull (2008)
Duke (2009) and Janick and Paull (2008)
Duke (2009)
Duke (2009) and Janick and Paull (2008)
(2), (2S)-20 -hydroxy-7,8,30 ,40 ,50 -pentamethoxyflavan (3),
(2S)-50 -hydroxy-7,8,30 ,40 -tetramethoxyflavan (4), (2S)-8-
hydroxy-7,30 ,40 ,50 -tetramethoxyflavan (5), (2S)-8,20 -dihy-
droxy-7,30 ,40 ,50 -tetramethoxyflavan (6), (2S)-8,50 -dihydroxy-
7,30 ,40 -trimethoxyflavan (7), 7,8,30 ,40 ,50 -pentamethoxyflavone
(8), (M),(2S),(200 S)-,(P),(2S),(200 S)-8,800 -50 -trihydroxy-7,70 -
3 ,3 -4 ,4 -5 -heptamethoxy-5,500 -biflavan (9), 50 -hydroxy-
0 000 0 000 000
7,8,30 40 -tetramethoxyflavone (10), (M),(2S),(200 S)-,(P),(2S),
(200 S)-8,800 -50 -5000 -tetrahydroxy-70 ,700 -30 ,3000 -40 ,4000 -hexamethoxy-
50 ,5000 -biflavan (11), and 8,50 -dihydroxy-7,30 ,40 -trimethoxyfla-
vone (12).
Twelve years later, there was another attempt to isolate
the bioactive compounds from the leaves of M. calabura,
collected in Purus, Peru, in October 1997 (Su et al., 2003).
The MEMCL was first partitioned into water, petroleum
ether (PEE), and ethyl acetate (EAE). Only the EAE
partition was further subjected to the isolation procedures,
which led to the identification of 25 compounds consisting
of one new (2R,3R)-7-methoxy-3,5,8-trihydroxyflavanone
(13) and 24 known compounds [(2S)-7-hydroxyflavanone
(14), (2S)-5,7-dihydroxyflavanone (pinocembrin, 15),
(2R,3R)-3,5,7-trihydroxyflavanone (pinobanksin, 16), (2S)-
5-hydroxy-7-methoxyflavanone (pinostrobin, 17), 7-hydro-
xyflavone (18), 5,7-dihydroxyflavone (chrysin, 19),
3-methoxy-5,7,40 -trihydroxyflavone (isokaemferide, 20),
3,30 -dimethoxy-5,7,40 -trihydroxyflavone (21), 3,8-
dimethoxy-5,7,40 -trihydroxyflavone (22), 3,5-dihydroxy-
7,40 -dimethoxyflavone (ermanin, 23), 3,5-dihydroxy-7,
8-dimethoxyflavone (gnaphaliin, 24), 5-hydroxy-3,7,8-tri-
methoxyflavone (25), 5,40 -dihydroxy-3,7,8-dimethoxyflavone
(26), 5-hydroxy-3,7,8,40 -tetramethoxyflavone (27), 20 ,40 -
dihydroxychalcone (28), 4,20 ,40 -trihydroxychalcone (isoli-
quiritigenin, 29), 7-hydroxyisoflavone (30), 7,30 ,40 -tri-
methoxyisoflavone (cabreuvin, 31), (2S)-50 -hydroxy-
7,8,30 ,40 -tetramethoxyflavan (4), 20 ,40 -dihydroxydihydrochal-
cone (32), 3,4,5-trihydroxybenzoic acid (33), lupenone (34),
and 2a,3b-dihydroxy-olean-12-en-28-oic acid (35)].
In other studies published by Chen et al. (2004), isolation
of flavonoids was performed on the methanol extract of
M. calabura stem bark (MEMCSB), collected from Kaohsiung
City, Taiwan in June 2001. MEMCSB was partitioned between
DOI: 10.3109/13880209.2014.908397
H2O–CHCl3 prior to the isolation processes, where the
CHCl3-soluble fraction was collected for the isolation
purposes. Isolation procedures carried out on the CHCl3-
soluble fraction led to the identification of 15 compounds
of which two are new compounds 8-hydroxy-7,30 ,40 ,50 -
tetramethoxyflavone (36) and 8,40 -dihydroxy-7,30 ,50 -tri-
methoxyflavone (37) and 13 are known compounds
6,7-dimethoxy-5-hydroxyflavone (38), 5,7-dimethoxyflavone
(39), 3,5-dihydroxy-6,7-dimethoxyflavone (40), (2S)-50 -
hydroxy-7,8,30 ,40 -tetramethoxyflavan (4), b-sitostenone (41),
6b-hydroxystigmast-4-en-3-one (42), b-sitosterol (43), syrin-
gic acid (44), vanillic acid (45), 3-hydroxy-1-(3,5-dimethoxy-
4-hydroxyphenyl)propan-1-one (46), tetracosyl ferulate (47)
and, a mixture of 1-tetracosanol (48), and 1-hexacosanol (49).
This was followed a year later by another report from the
same group on the isolation of chalcones and flavonoids from
the leaves of M. calabura (Chen et al., 2007), collected from
Kaohsiung City, Taiwan, in June 2001. Prior to the isolation
processes, the MEMCL was partitioned using H2O–CHCl3 to
obtain the water-soluble and CHCl3-soluble partitions with
the former also further partitioned using H2O and n-BuOH
to afford an n-BuOH-soluble and H2O-soluble fractions.
The CHCl3-soluble partition and, n-BuOH-soluble and H2O-
soluble fractions were then subjected to the isolation proced-
ures. From this study, 20 compounds were isolated of
which four were new compounds (20 ,40 -dihydroxy-30 -methox-
ydihydrochalcone (50), ()-30 -methoxy-20 ,40 ,b-trihydroxydi-
hydrochalcone (51), (2S)-()-50 -hydroxy-7,30 ,
0
4 -trimethoxyflavanone (52), and 8-hydroxy-10-methoxy-
5H-isochromeno[4,3-b]chromen-7-one (muntingone, 53)
while the remaining 16 were known compounds [7-hydroxy-
flavanone (14), 20 ,40 -dihydroxychalcone (28), 20 ,40 -dihydrox-
ydihydrochalcone (32), 6,7-dimethoxy-5-hydroxyflavone
(38), 3,5-dihydroxy-6,7-dimethoxyflavone (40), 5-hydroxy-
7-methoxyflavone (54), 3,7-dimethoxy-5-hydroxyflavone
(55), 5-hydroxy-3,6,7-trimethoxyflavone (56), 3,5-dihy-
droxy-7-methoxyflavone (57), 8-methoxy-3,5,7-trihydroxy-
flavone (58), 5,7-dihydroxy-3,8-dimethoxyflavone (59),
galangin (60), chrysin (61), 7-hydroxy-8-methoxyflavanone
(62), 40 -hydroxy-7-methoxyflavanone (63), and 20 ,40 -dihy-
droxy-30 -methoxychalcone (64)].
Then 2 years later, upon vigorous phytochemistry studies
on the same sample of M. calabura leaves that were collected
in Kaohsiung City, Taiwan, in June 2001, Chen et al. (2007)
again reported on the isolation of 22 compounds from the
CHCl3-soluble partitions and n-BuOH-soluble fraction pre-
pared from the MEMCL as described earlier (Chen et al.,
2005). Of the isolated compounds, three were new com-
pounds [2,3-dihydroxy-4,30 ,40 ,50 -tetramethoxydihydrochal-
cone (65), 4,20 ,40 -trihydroxy-30 -methoxydihydrochalcone
(66), and (2R,3R)-()-3,5-dihydroxy-6,7-dimethoxyflavanone
(67)], and the others were known compounds [b-sitostenone
(41), mixture of b-sitosterol (43) and stigmasterol (68), 7-
methoxyflavone (69), 5,7-dihydroxy-3-methoxyflavone (70),
5,7-dihydroxy-6-methoxyflavone (71), 5,40 -dihydroxy-3,7-
dimethoxyflavone (72), (2S)-7,8,30 ,40 ,50 -pentamethoxyflavan
(73), (2S)-50 -hydroxy-7,8,30 ,40 -tetramethoxyflavan (74),
methyl 4-hydrobenzoate (75), isovanillic acid (76), p-nitro-
phenol (77), methyl gallate (78), trans-methyl p-coumarate
Review on M. calabura medicinal value 1601
(79), b-amyrenone (80), a-tocopherylquione (81), d-tocoph-
erol (82), a-tocospiro A (83), and a-tocospiro B (84)].
A recent attempt has been made to isolate the bioactive
compounds with potential antimicrobial and cytotoxic proper-
ties from the leaves of M. calabura collected in Shah Alam,
Selangor, Malaysia, in January 2008 (Sufian et al., 2013). The
MEMCL was suspended in H2O and then partitioned using
PEE and EAE to afford the PEE, EAE, and H2O (WEE)
extracts. Based on the antimicrobial and cytotoxic effective-
ness, the EAE was chosen for further fractionation proced-
ures, which, in turn, yielded seven major fractions. Of these
fractions, fraction 5 was the most effective and, therefore, was
subjected to further isolation of bioactive compounds. This
led to the isolation of four known compounds, namely, 20 ,40 -
dihydroxychalcone (28), 5,7-dihydroxy-3,8-dimethoxyflavone
(59), 5-hydroxy-3,8-dimethoxyflavone (85), and 3,5,7-trihy-
droxy-8-methoxyflavone (86).
In another recently published article, Yusof et al. (2011)
studied the antinociceptive activity of MEMCL, which was
later suspended in H2O before being partitioned into PEE,
EAE, and WEE, as earlier described by Sufian et al. (2013).
Following the antinociceptive investigation of those parti-
tions, the PEE showed the most effective results, which
was further fractionated to yield seven separate fractions,
labeled as Fractions A–G. Subsequent antinociceptive
studies demonstrated that Fraction D was the most effective
fraction and, upon separation processes led to the identifica-
tion of one new compound [8-hydroxy-6-methoxyflavone
(calaburone), (87)] and three known compounds, namely
5-hydroxy-3,7,8-trimethoxyflavone (25), 3,7-dimethoxy-5-
hydroflavone (55), and 20 ,40 -dihydroxy-30 -methoxychalcone
(64). Table 2 shows the chemical structures of several new
bioactive compounds isolated for the first time from
M. calabura.
Pharmacological studies
For the past 22 years, attempts to establish the pharmaco-
logical value of M. calabura through rigorous scientific
investigations has been taken by researchers all over the
world. Despite the differences in term of cultural, geograph-
ical, location, and climate, different parts of M. calabura have
been medicinally used to treat various ailments and many
have been scientifically proven. Interestingly, various new
medicinal potential of M. calabura have been reported based
on the scientific investigations. All the findings and observa-
tions are described in detailed below and also summarized in
Table 3.
Acute toxicity
Despite the first report on the pharmacological activity of
M. calabura published in 1991, the first attempt to determine
the plant acute toxicity was published only in 2011. The acute
oral toxicity was determined on M. calabura leaves collected
from the Station Ghanpur, Warangal, Andhra Pradesh, India
(Sridhar et al., 2011). The methanol extract of the leaves,
MEMCL, in doses ranging from 300, 500, and 2000 mg/kg,
was administered orally to rats. Signs of toxicity were
observed for the first 2–3 h after extract administration,
1602 N. D. Mahmood et al. Pharm Biol, 2014; 52(12): 1598–1623

Table 2. List of new bioactive compounds isolated from M. calabura according to the extracts used.

Name of compound Structure Types of extract Reference

0 0 0
(2S)-5 -Hydroxy-7,3 ,4 -trimethox- MEMCR Kaneda et al. (1991)
O CH3
yflavan (1)
CH3

O O O
H3C

OH

(2S)-7,8,30 ,40 ,50 -pentamethoxyfla-

van (2)

O CH3
H3C O

O
O CH3

CH3 O
CH3
0 0 0 0
(2S)-2 -hydroxy-7,8,3 ,4 ,5 -penta-
H3C
methoxyflavan (3)
O CH3

CH3 HO O
O

O O CH3
H3C O

(2S)-50 -Hydroxy-7,8,30 ,40 -tetra-

O H3C
methoxyflavan (4)
O

H3C O

CH3 O O CH3

CH3

(2S)-8-Hydroxy-7,30 ,40 ,50 -tetra-

O CH3
methoxyflavan (5)
OH CH3

O O O
H3C

O CH3

(2S)-8,20 -Dihydroxy-7,30 ,40 ,50 -tet-

H3C
ramethoxyflavan (6)
O

HO O
OH CH3

O O CH3
H3C O

(continued )
DOI: 10.3109/13880209.2014.908397 Review on M. calabura medicinal value 1603
Table 2. Continued

Name of compound Structure Types of extract Reference

0 0 0
(2S)-8,5 -Dihydroxy-7,3 ,4 -tri-
OH
methoxyflavan (7)
OH
OH

O O CH3
H3C O

7,8,30 ,40 ,50 -Pentamethoxyflavone

O
(8)

CH3

O CH3
O O

H3C O
O CH3

O
CH3

(M),(2S),(200 S)-,(P),(2S),(200 S)-

O CH3
8,800 -50 -Trihydroxy-7,700 -30 ,3000 -
40 ,4000 -5000 -heptamethoxy-5,500 - OH CH3
biflavan (9)
O O O
H3C

OH

O CH3

CH3

O O

CH3 OH
O CH3

50 -Hydroxy-7,8,30 40 -tetramethoxy-
O
flavone (10)
H3C OH

H3C O

O O CH3

CH3

(continued )
1604 N. D. Mahmood et al. Pharm Biol, 2014; 52(12): 1598–1623

Table 2. Continued

Name of compound Structure Types of extract Reference

00 00
(M),(2S),(2 S)-,(P),(2S),(2 S)-
O CH3
8,800 -50 -5000 -Tetrahydroxy-70 ,700 -
30 ,3000 -40 ,4000 -hexamethoxy- OH CH3
50 ,5000 -biflavan (11)
O O O
H3C

OH

OH

CH3

O O

CH3 OH
O CH3

8,50 -Dihydroxy-7,30 ,40 -trimethoxy-

H3C
flavone (12)
O
OH

O O O
H3C
CH3

OH

(2R,3R)-7-Methoxy-3,5,8- Ethyl acetate-soluble Su et al. (2003)

trihydroxyflavanone(13) OH partition of MEMCL

O O
H3C

OH

OH O
0 0 0
8-Hydroxy-7,3 ,4 ,5 -tetramethoxy- CHCl3-soluble partition Chen et al. (2004)
CH3
flavone (36) of MEMCSB
O

O CH3
OH

O O
H3C O

CH3
CH3

8,40 -Dihydroxy-7,30 ,50 -trimethoxy-

CH3
flavone (37)
O

OH
OH

O O
H3C O

CH3
CH3

(continued )
DOI: 10.3109/13880209.2014.908397 Review on M. calabura medicinal value 1605
Table 2. Continued

Name of compound Structure Types of extract Reference

0 0 0
2 ,4 -Dihydroxy-3 -methoxydihy- CHCl3-soluble partition Chen et al. (2005)
drochalcone (50) and, n-BuOH-soluble
and H2O-soluble frac-
HO tions of MEMCL

CH3 OH O

0 0 0
()-3 -Methoxy-2 ,4 ,b-trihydrox-
ydihydrochalcone (51)

HO HO

CH3 OH O
0 0 0
(2S)-()-5 -Hydroxy-7,3 ,4 -tri-
H3C
methoxyflavanone (52)
O CH3

O O CH3
H3C O

8-hydroxy-10-methoxy-5H-iso-
chromeno[4,3-b]chromen-7-one
(muntingone, 53)
O O
H3C

OH O
0 0 0
2,3-Dihydroxy-4,3 ,4 ,5 -tetra- CHCl3-soluble parti- Chen et al. (2007)
CH3
methoxydihydrochalcone (65) tions and n-BuOH-sol-
uble fraction of
H3C O
MEMCL
O

H3C O
OH

OH
O

CH3 O

4,20 ,40 -Trihydroxy-30 -methoxydi-

OH
hydrochalcone (66)

HO

CH3 O

(continued )
1606 N. D. Mahmood et al. Pharm Biol, 2014; 52(12): 1598–1623

Table 2. Continued

Name of compound Structure Types of extract Reference

(2R,3R)-()-3,5-Dihydroxy-6,7-
dimethoxyflavanone (67) CH3

H3C
O OH

OH O

8-Hydroxy-6-methoxyflavone PEP of MEMCL Yusof et al. (2011)

(calaburone, 87)
OH

CH3 O

Table 3. Information on pharmacological activities, parts of M. calabura and types of extracts used, location, and period of collection of plant samples.

Activity Part Type of extract Location of collection Period of collection References

Acute toxicity Leaves MEMCL, Station Ghanpur, Warangal, NG Sridhar et al. (2011)
Andhra Pradesh, India
Leaves EEMCL, Selangor, Malaysia NG Ibrahim et al. (2012)
Fruits Not clear; NG NG Karthyaini and Suresh (2012)
possibly EEMCFr
Leaves MEMCL Shah Alam, Selangor, May and August 2010 Balan et al. (2013)
Malaysia
Cytotoxic Roots MEMCR Sarabuti Province, Thailand NG Kaneda et al. (1991)
Stem barks MEMCSB Kaohsiung City, Taiwan June 2001 Chen et al. (2004)
Leaves MEMCL Kaohsiung City, Taiwan June 2001 Chen et al. (2005)
Leaves MEMCL Shah Alam, Selangor, January 2008 Sufian et al. (2013)
Malaysia
Antiproliferative Leaves MEMCL Shah Alam, Selangor, August and September 2006 Zakaria et al. (2011)
Malaysia
Leaves CEMCL Shah Alam, Selangor, August and September 2006 Zakaria et al. (2011)
Malaysia
Leaves AEMCL Shah Alam, Selangor, August and September 2006 Zakaria et al. (2011)
Malaysia
Insecticidal Flowers EEMCFl Universidade Rural Federal July 2008 Bandeira et al. (2013)
de Pernambuco (UFRPE),
Recife, Brazil
Fruits EEMCFr Universidade Rural Federal July 2008 Bandeira et al. (2013)
de Pernambuco (UFRPE),
Recife, Brazil
Flowers HEMCFL Universidade Rural Federal July 2008 Bandeira et al. (2013)
de Pernambuco (UFRPE),
Recife, Brazil
Fruits HEMCFr Universidade Rural Federal July 2008 Bandeira et al. (2013)
de Pernambuco (UFRPE),
Recife, Brazil
Hypotensive Leaves MEMCL fractionated Kaohsiung City, Taiwan June 2001 Shih et al. (2006)
sequentially using a mixture
of dH2O and n-butanol
Leaves Butanol-soluble fraction Kaohsiung City, Taiwan NG Shih (2009)
(BSF)
Leaves MEMCL partitioned using Kaohsiung City, Taiwan NG Shih (2009)
dH2O
Leaves MEMCL partitioned using Kaohsiung City, Taiwan NG Shih (2009)
chloroform

(continued )
DOI: 10.3109/13880209.2014.908397 Review on M. calabura medicinal value 1607
Table 3. Continued

Activity Part Type of extract Location of collection Period of collection References

Antinociceptive Leaves AEMCL Shah Alam, Selangor, January and February 2004 Zakaria et al. (2006a)
Malaysia
Leaves AEMCL Shah Alam, Selangor, January and February 2004 Zakaria et al. (2007e)
Malaysia
Leaves AEMCL Shah Alam, Selangor, July and August 2005 Zakaria et al. (2007d,f)
Malaysia
Leaves CEMCL Shah Alam, Selangor, July and August 2005 Zakaria et al. (2007a)
Malaysia
Leaves MEMCL Shah Alam, Selangor, January 2008 Yusof et al. (2011)
Malaysia
Leaves PEP partition Shah Alam, Selangor, January 2008 Yusof et al. (2011)
Malaysia
Leaves EAP partition Shah Alam, Selangor, January 2008 Yusof et al. (2011)
Malaysia
Leaves AQP partition Shah Alam, Selangor, January 2008 Yusof et al. (2011)
Malaysia
Leaves MEMCL Shah Alam, Selangor, NG Sani et al. (2012)
Malaysia
Cardioprotective Leaves AEMCL NG NG Nivethetha et al. (2009)
Antipyretic Leaves CEMCL Shah Alam, Selangor, July and August 2005 Zakaria et al. (2007a)
Malaysia
Leaves AEMCL Shah Alam, Selangor, July and August 2005 Zakaria et al. (2007b)
Malaysia
Antiplatelet aggregation Leaves Methanol Kaohsiung City, Taiwan 2001 Chen et al. (2007)
Leaves CHCl3 Kaohsiung City, Taiwan 2001 Chen et al. (2007)
Leaves BuOH Kaohsiung City, Taiwan 2001 Chen et al. (2007)
Antioxidant Leaves AEMCL Shah Alam, Selangor, June and September 2005 Zakaria et al. (2007b)
Malaysia
Fruits HEMCFr Erode District, Tamil Nadu, May and June 2008 Preethi et al. (2010)
India
Fruits CEMCFR Erode District, Tamil Nadu, May and June 2008 Preethi et al. (2010)
India
Fruits EAEMCFR Erode District, Tamil Nadu, May and June 2008 Preethi et al. (2010)
India
Fruits BEMCFR Erode District, Tamil Nadu, May and June 2008 Preethi et al. (2010)
India
Fruits MEMCFR Erode District, Tamil Nadu, May and June 2008 Preethi et al. (2010)
India
Leaves MEMCL Bangalore, Karnakata March 2009 Siddiqua et al. (2010)
Leaves AEMCL Shah Alam, Selangor, August and September 2006 Zakaria et al. (2011)
Malaysia
Leaves MEMCL Shah Alam, Selangor, August and September 2006 Zakaria et al. (2011)
Malaysia
Leaves CEMCL Shah Alam, Selangor, August and September 2006 Zakaria et al. (2011)
Malaysia
Fruits NG NG NG Karthyaini and Suresh (2012)
Anti-inflammation Leaves AEMCL Shah Alam, Selangor, July and August 2005 Zakaria et al. (2007b)
Malaysia
Fruits MEMCFr Erode district Tamilnadu, NG Preethi et al. (2012)
India
Fruits MEMCFr NG NG Karthyaini and Suresh (2012)
Fruits AEMCFr NG NG Karthyaini and Suresh (2012)
Anti-diabetic Leaves MEMCL Roman Catholic Church, September Sridhar et al. (2011)
Station Ghanpur, Warangal,
Andhra Pradesh, India
Antiulcer Leaves EEMCL Ethno Resources Sdn. Bhd, NG Ibrahim et al. (2012)
Selangor, Malaysia
Leaves MEMC Shah Alam, Selangor, May–August 2010 Balan et al. (2013)
Malaysia
Antibacterial Leaves AEMCL Shah Alam, Selangor, January and February 2005 Zakaria et al. (2006b)
Malaysia
Leaves MEMCL Shah Alam, Selangor, January and February 2005 Zakaria et al. (2006b)
Malaysia
Leaves CEMCL Shah Alam, Selangor, January and February 2005 Zakaria et al. (2006b)
Malaysia
Leaves MEMCL Cuetzal’an del Progreso in Between 2000 and 2003 Yasunaka et al. (2005)
the State of Puebla
Fruits MEMCFr Cuetzal’an del Progreso in Between 2000 and 2003 Yasunaka et al. (2005)
the State of Puebla
Leaves AEMCL Shah Alam, Selangor, June 2006 Zakaria et al. (2007c)
Malaysia
Leaves CEMCL Shah Alam, Selangor, June 2006 Zakaria et al. (2007c)
Malaysia

(continued )
1608 N. D. Mahmood et al. Pharm Biol, 2014; 52(12): 1598–1623

Table 3. Continued

Activity Part Type of extract Location of collection Period of collection References

Leaves MEMCL Shah Alam, Selangor, June 2006 Zakaria et al. (2007c)
Malaysia
Leaves AEMCL Shah Alam, Selangor, January 2008 Zakaria et al. (2010)
Malaysia
Leaves CEMCL Shah Alam, Selangor, January 2008 Zakaria et al. (2010)
Malaysia
Leaves MEMCL Shah Alam, Selangor, January 2008 Zakaria et al. (2010)
Malaysia
Leaves PEEMCL Shah Alam, Selangor, January 2008 Zakaria et al. (2010)
Malaysia
Leaves EAEMCL Shah Alam, Selangor, January 2008 Zakaria et al. (2010)
Malaysia
Leaves WEEMCL Shah Alam, Selangor, January 2008 Zakaria et al. (2010)
Malaysia
Leaves MEMCL Shah Alam, Selangor, NG Sufian et al. (2013)
Malaysia
Leaves PEEMCL Shah Alam, Selangor, NG Sufian et al. (2013)
Malaysia
Leaves EAEMCL Shah Alam, Selangor, NG Sufian et al. (2013)
Malaysia
Leaves WEEMCL Shah Alam, Selangor, NG Sufian et al. (2013)
Malaysia
Leaves AEMCL Bengaluru, Karnataka, India NG Sibi et al. (2012)
Barks AEMCB Bengaluru, Karnataka, India NG Sibi et al. (2012)
Fruits AEMCFr Bengaluru, Karnataka, India NG Sibi et al. (2012)
Leaves MEMCL Bengaluru, Karnataka, India NG Sibi et al. (2012)
Barks MEMCB Bengaluru, Karnataka, India NG Sibi et al. (2012)
Fruits MEMCFr Bengaluru, Karnataka, India NG Sibi et al. (2012)

followed by observation on the percentage of mortality
beginning from 24 h up to a period of 14 d. The results
obtained showed no sign of toxicity and no mortality was
recorded up to the dose of 2000 mg/kg of extract.
Another attempt to study the acute toxicity of M. calabura
leaves, collected in Selangor, Malaysia, was carried out by
Ibrahim et al. (2012). The ethanol extract of the leaves,
EEMCL, in the doses of 2000 and 5000 mg/kg, was admin-
istered orally. Again, no mortality was recorded up to 14 d
following the extract administration and no visible clinical
signs of general weakness in the animals were observed.
Moreover, this observation was supported by further histo-
pathological, hematological, and serum biochemical studies
which revealed the inviolability of the extracts to retain the
rat’s normal conditions. These findings also indicate that the
EEMCL will not induce acute toxicity and is safe for
consumption even at the highest dose (5000 mg/kg).
In the same year, Karthyaini and Suresh (2012) studied the
acute toxicity effect of M. calabura fruits using the limit test
dose of 2000 mg/kg (OECD guidelines 420). However, the
type of solvents used for extraction for the toxicity study was
not described in any part of the report (neither in the
Methodology nor Results sections), despite their claim that
there were no signs of toxicity or mortality recorded at
2000 mg/kg. In addition, it was also reported that the ethanol
extract of M. calabura fruits (EEMCFr) did not show signs of
toxicity at 1000 mg/kg.
In a recent attempt to determine the acute toxicity of M.
calabura leaves, collected from Shah Alam, Selangor,
Malaysia, between May and August 2010, the MEMCL was
prepared for a single dose (2000 mg/kg) acute oral toxicity
test (Balan et al., 2013). Interestingly, 2000 mg/kg MEMCL
also did not cause any signs of toxicity and mortality in the
treated animals up to 14 d.
Cytotoxic activity
The first attempt to study the cytotoxic activity of M.
calabura was performed using the roots of the plant collected
in Sarabuti Province, Thailand (Kaneda et al., 1991). The
methanol extract of the roots, MEMCR, was first subjected to
the isolation of bioactive compounds and then tested against
BC1 (human breast cancer), HT-1080 (human fibrosarcoma),
Lu1 (human lung cancer), Me12 (human melanoma), Co12
(human colon cancer), KB (human nasopharyngeal carcin-
oma), KB-V (vincristine-resistant KB), and P-388 (murine
lymphocytic leukemia) cell lines. Twelve compounds were
isolated from MEMCR, namely, seven flavans (compounds 1–
7), three flavones (compounds 8, 10, and 12), and two
biflavans (compounds 9 and 11). Of all the isolated
compounds, only compound 8 was not tested against all cell
lines. Compounds 1–7 and 9–12 exerted cytotoxicity activity
against P-388 cells with the ED50 values ranging between 2.0
and 16.7 mg/mL. As for KB and KB-V cells, the cytotoxic
effect was shown by all compounds except for compounds 10
and 12, while compounds 6, 10, and 12 with the recorded
ED50 ranging between 2.2–15.5 and 2.1–13.3 mg/mL, respect-
ively. As for BC-1, HT-1080, and Lu-1, only compounds 3, 9,
and 11 , respectively, caused cyotoxic effect with the recorded
ED50 ranging among 10.9–16.0, 3.3–5.5, and 13.5–15.6 mg/
mL, respectively. In addition, all compounds, except for 10
and 12, exerted a cytotoxic effect against ME-12 cells with
the recorded ED50 ranging between 8.7 and 14.6 mg/mL.
Lastly, all compounds, except for 1 and 4–7, exerted cytotoxic
DOI: 10.3109/13880209.2014.908397
effect against the CO-12 with recorded ED50 ranging between
5.9 and 15.8 mg/mL.
A few years later, Chen et al. (2004) continued the
cytotoxicity studies of M. calabura, where 15 bioactive
compounds isolated from the MEMCSB were tested against
P-388, A549, and HT-29 cells using the MTT colorimetric
method. Of all the isolated compounds, compound 36, 37, 40,
41, 45, 46, and 47 exerted remarkable cytotoxic activities
against P-388 cells with recorded ED50 values of 3.56,3.71,
9.39, 6.28, 10.72, 15.62, and 3.27 mg/mL, respectively, in
comparison with mithramycin (ED50 ¼ 0.06 mg/mL). Further
cytotoxicity studies against A549 and HT-29 revealed that only
compound 4 exerted remarkable activity with the recorded
ED50 of 16.81 and 26.60 mg/mL in comparison with mithra-
mycin (ED50 ¼ 0.07 and 0.08 mg/mL), respectively.
This was followed a year later by another cytotoxic
investigation on 20 bioactive compounds isolated from
MEMCL against P-388 and HT-29 cells (Chen et al., 2005).
Of these isolated compounds, compounds 32, 38, 53, and 60
were not cytotoxic against both types of cancer cells while
compounds 40, 50, and 51 were not cytotoxic against the HT-
29 cells indicated by their ED50 values that were 450 mg/mL.
On one hand, the ED50 values recorded against P-388 and the
respective cytotoxic compounds were the following: (i)
415 mg/mL for compound 50; (ii) 15 mg/mL4X410 mg/mL
consist of compounds 14, 51, 54, and 58; (iii)
10 mg/mL4X45 mg/mL consist of compounds 40, 55, 56,
57, 59, 61, and 62; and (iv) 5 mg/mL4X40.01 mg/mL consist
of compounds 28, 52, 63, and 64. On the other hand, the ED50
values recorded against HT-29 and the respective cytotoxic
compounds were the following: (i) 415 mg/mL consist of
compounds 52, 56, 57, and 58; (ii) 15 mg/mL4X410 mg/mL
consist of compounds 14, 54, 55, 59, and 61; (iii)
10 mg/mL4X45 mg/mL consist of compounds 62 and 63;
and (iv) 5 mg/mL4X40.01 mg/mL consist of compounds 28
and 64. Comparison was made against mithramycin, which
recorded an ED50 of 0.06 and 0.08 against theP-388 and
HT-29 cells, respectively.
In the most recent cytotoxicity studies, Sufian et al. (2013)
used the bioassay-guided approaches to isolate at least four
compounds from the MEMCL. The MEMCL together with its
partitions (e.g., PEE, EAE, and water (WEE)) were tested
against a panel of cancer cell lines, namely, MCF-7 (human
breast adenocarcinoma), HL-60 (human acute lymphoblastic
leukemia), and HCT-116 (colonic carcinoma tumor types), as
well as a normal cell line WRL-68 (human embryonic liver
non-tumor type). On one hand, the IC50 values against MCF-
7, HL-60, HCT-116, and WRL-68 recorded for MEMCL were
30.9, 34.7, 61.3, and 4100 mg/mL, respectively. On the other
hand, the IC50 values recorded for PEE and EAE against
the four cells were as follows: (i) 29.5, 42.1, 47.2, and
73.6 mg/mL; and (ii) 17.3, 38.6, 58.4, and 78.3 mg/mL,
respectively. Unfortunately, the WEE was non-cytotoxic
against the four cells with the IC50 recorded 4100 mg/mL.
Based on the IC50 values obtained, the EAE was further
fractionated to yield seven fractions (labeled as F1–F7) that
were again tested for cytotoxicity against MCF-7, HL-60, and
WRL-68 cells. Based on the results obtained, F1 was effective
only against HL-60 (IC50 ¼ 32.1 mg/mL); F2 was effective
against all cells with the recorded IC50 ranging between 35.6
Review on M. calabura medicinal value 1609
and 40.8; F3 was effective against only HL-60 and WRL-68
with the recorded IC50 of 84.5 and 34.2 mg/mL, respectively;
F4 was effective against all cells with the recorded IC50
ranging between 30.8 and 35.6 mg/mL; F5 was effective
against all cells with the recorded IC50 ranging between 4.0
and 34.9 mg/mL; F6 was effective against all cells with
recorded IC50 ranging between 6.0 and 40.8 mg/mL, and; F7
was effective against all cells with the recorded IC50 ranging
between 28.1.0 and 40.1 mg/mL. Based on the IC50 of the
fractions, F5 was found to be the most effective fraction, and
therefore was subjected to the isolation of bioactive com-
pounds, which, in turn, led to the isolation of compounds 28,
59, 85 and 86. Of these compounds, only compounds 28 and
85 produced IC50 values below 20 mg/mL against HL-60 (3.43
and 3.34 mg/mL) and MCF-7 (11.78 and 18.88 mg/mL) in
comparison with the reference drug, doxorubicin (0.02 and
0.05 mg/mL), respectively.
Antiproliferative activity
The only attempt to study the antiproliferative activity of M.
calabura was made by Zakaria et al. (2011) while the authors
studied the antioxidant activity of the leaves. The leaves,
prepared as AEMCL, CEMCL, and MEMCL, at the concen-
trations between 12.5 and 100.0 mg/mL, were tested using
3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. The in vitro antiproliferative activity of M.
calabura extracts was evaluated against several cancer cell
lines, namely, MCF-7 (estrogen-dependent human breast
adenocarcinoma), HeLa (human cervical adenocarcinoma),
HT-29 (human colon cancer), HL-60 (acute promyelocytic
leukemia), K-562 (chronic myelogenous leukemia), and
MDA-MB-231 (human breast carcinoma) with 3T3 (normal
mouse fibroblast) being the normal non-cancerous cell. The
results showed that the three extracts, as well as DMSO which
was used to dissolve the extracts, did not produce any
antiproliferative or cytotoxic effect against 3T3 or MDA-MB-
231 cell lines. In contrast, AEMCL, MEMCL, and CEMCL
showed antiproliferative activity against the following:
(i) MCF-7 with the recorded IC50 of 18, 98, and 22 mg/mL;
(ii) HeLa with the recorded IC50 of 52, 23, and 22 mg/mL,
and; (iii) K-562 with the recorded IC50 of 18, 42, and
39 mg/mL, respectively. Moreover, only the AEMCL and
MEMCL exerted antiproliferative activity against the HT-29
cell with IC50 values of 16 and 46 mg/mL while for the HL-60
cell, only the CEMCL and MEMCL demonstrated antiproli-
ferative activity with IC50 values of 29 and 7 mg/mL,
respectively.
Quinone reductase activity
Su et al. (2003) also studied the quinine reductase (QR)
induction potential of compounds isolated from the leaves of
M. calabura using the mouse culture Hepa IcIc7 cell. In this
study, the enzyme activity which is the concentration required
to double the specific activity of QR (expressed as CD), half-
maximal inhibitory concentration of cell viability (IC50), and
chemoprevention index (CI) were measured. Except for
lupenone, all isolated compounds were tested against the
assays and only compounds 13, 17, 18, 22, 23, 24, and 25
exerted significant QR induction activity with the recorded
1610 N. D. Mahmood et al.
CD of 50.15, 4.77, 5.22, 0.70, 1.40, 1.70, and 1.42 mg/mL,
which was equivalent to 50.56, 15.8, 17.4, 2.9, 5.5, 5.7, and
4.6 mM, respectively, when compared with the reference drug,
sulforaphane (0.08 mg/mL; 0.43 mM). In terms of the IC50, the
values recorded for compounds 13, 17, 18, 22, 23, 24, and 25
were 420, 420, 15.8, 45, 45, 420, and 18.6 mg/mL that were
equivalent to 474, 466.2, 52.7, 420.8, 419.5, 67.1, and
59.6 mM, respectively, in comparison with the reference drug,
sulforaphane (1.95 mg/mL; 11.0 mM). From the CD and IC50
values obtained, the CI for each compounds was determined
to be 4132, 44.2, 3.0, 47.2, 43.5, 411.8, and 13.0. The
results also revealed that compound 17 had the highest CI
value (4132) in comparison with the reference drug,
sulforaphane (25.0).
Antiplatelet aggregation activity
Studies were performed to determine the antiplatelet aggre-
gation of 22 compounds isolated from the leaves of M.
calabura (Chen et al., 2007). The platelets were obtained
from rabbit’s blood and platelet aggregation was measured by
an in vitro turbidimetric method using a Chrono–Log Lumi
aggregometer. Washed rabbit platelets were induced by 0.1 U/
mL thrombin, 100 mM arachidonic acid, 10 mg/mL collagen,
or 2 ng/mL platelet-activating factor (PAF). All compounds
were tested at 100 mg/mL, except for compounds 65, 70, 71,
72, 73, and 74, which were tested at 50 and 100 mg/mL, and
compound 78, which was serially diluted and tested at 100,
50, 20, 10, 5, 2, and 1 mg/mL. Aspirin was used as a reference
drug and tested at the concentrations of 100, 50, and 20 mg/
mL. In the thrombin-induced assay, all compounds produced
the percentage platelet aggregation inhibition ranging
between 1.4 and 43.2% in comparison with 100 mg/mL
aspirin, which caused only 1.5% inhibition. In the arachidonic
acid-induced assay, several compounds (e.g., compounds 65,
70, 71, 72, 73, 74, and 78) exhibited remarkable anti-platelet
aggregation activity indicated by the high percentage of
platelet aggregation inhibition (80–100%) at the concentration
of 100 mg/mL. Of these, compound 78 exerted more than 80%
inhibitory effect even at the concentration of 10 mg/mL
indicating its remarkable effectiveness in comparison with
aspirin, which lost its activity to only 5.6% at the concentra-
tion of 20 mg/mL. The same compounds (e.g., compound 65,
70, 71, 72, 73, 74, and 78) were also effective against the
collagen-induced platelet aggregation with the percentage of
inhibition recorded above 80% at the concentration of 100 mg/
mL. Interestingly, aspirin was not effective in this model with
the recorded percentage of inhibition of 5.1 mg/mL. Finally, in
the PAF-induced assay, only compounds 65 and 73 exerted
the percentage of anti-platelet aggregation of 485% at the
concentration of 100 mg/mL in comparison with aspirin that
caused only 2.5% inhibition.
Antibacterial activity
The first attempt to study the antibacterial activity of
M. calabura was carried out by Yasunaka et al. (2005)
using the leaves and fruits collected in the State of Puebla and
State of Veracruz, Mexico. The MEMCL and methanol extract
of M. calabura fruits (MEMCFr) diluted in 100 mg/mL of
DMSO concentration were subjected to two-fold serial
Pharm Biol, 2014; 52(12): 1598–1623
dilutions and then tested against Escherichia coli (C600)
and Staphylococcus aureus (209 P) using the micro-dilution
assay. Both MEMCL and MEMCFr exhibited antibacterial
activity against E. coli and S. aureus with the recorded MIC
of 512 and 1024 mg/mL, and 128 and 256 mg/mL, respectively.
This was followed by another antibacterial activity report
by Zakaria et al. (2006b), who studied the antibacterial
properties of MEMCL, aqueous (AEMCL), and chloroform
(CEMCL) extract of M. calabura leaves, collected from Shah
Alam Selangor, Malaysia, between January and February
2005. These extracts, prepared at various concentrations
(10 000, 40 000, 70 000 and 100 000 ppm), were tested against
Corneybacterium diphtheria, S. aureus, Bacillus cereus,
Proteus vulgaris, Staphylococcus epidermidis, Kosuria rhizo-
phila, Shigella flexneri, E. coli, Aeromonashydrophila, and
Salmonella typhi using the in vitro disc diffusion method. The
results showed that CEMCL was less effective as compared
with the AEMCL and MEMCL. At all concentrations tested,
AEMCL inhibited the growth of S. aureus and K. rhizophila
while MEMCL exerted antibacterial activity against S.
flexneri, B, cereus, S. aureus, P. vulgaris, A. hydrophila,
and K. rhizophila. At the concentration of 40 000 ppm and
above, the AEMCL exerted antibacterial activity against C.
diptheriae, P. vulgaris, S. epidermidis, and A. hydrophila
while the MEMCL inhibited the growth of C. diptheriae and
L. monocytogenes, and the CEMCL showed antibacterial
activity only against S. aureus. Chloramphenicol, used as a
reference antibiotic at the concentration of 30 mg/mL, was
effective against all bacteria.
Another study was performed to study the antistaphylo-
coccal effect of the AEMCL, CEMCL, and MEMCL (Zakaria
et al., 2007c). The leaves, collected in June 2006 and prepared
into the respective extracts, were tested against various strains
of S. aureus, namely, S. aureus 29213a, S. aureus 33591,
S. aureus 700699, vancomycin-intermediate S. aureus
(VISA), and vancomycin-resistant S. aureus (VRSA) using
an in vitro single concentration liquid microdilution method.
The results showed that all extracts of M. calabura exerted
antibacterial activity against S. aureus 29213a, S. aureus
33591, and S. aureus 700699. Further attempts were made to
determine the minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) values of each
extract. The recorded MIC for AEMCL, CEMCL and MEMCL
was 5.00, 1.25, and 1.25–2.50 mg/mL, respectively, while the
recorded MBC was 2.50 mg/mL for MEMCL and CEMCL.
Based on the earlier findings reported above, further
studies were carried out to determine the antimicrobial
activity of various extracts, partitions, and fractions of
M. calabura leaves (Zakaria et al., 2010). The leaves were
collected in Shah Alam, Selangor, Malaysia, in January 2008
and prepared as AEMCL, CEMCL, and MEMCL. These
extracts were tested against S. aureus ATCC 25923
[methicilin sensitive S. aureus (MSSA)], S. aureus ATCC
33591 [methicilin-resistant S. aureus (MRSA)], E. coli ATCC
10536, Pseudomonas aeruginosa ATCC 27853, Candida
albicans ATCC 10231, and Microsporum canis ATCC 36299
using the liquid–liquid microdilution assay. From the results
obtained, only MEMCL successfully inhibited the growth of
MSSA and MRSA with the MIC value of 1250 mg/mL and the
MBC value of 2500 mg/mL. In contrast, AEMCL and CEMCL
DOI: 10.3109/13880209.2014.908397
did not successfully inhibited C. albicans, M. canis,
P. aeruginosa, and E. coli. Further studies involved partition-
ing of the MEMCL into PEE, EAE and water-(WEE) extracts
followed by the antibacterial study using the MSSA and
MRSA. EAE exerted antistaphylococcal activity against
MSSA and MRSA with the recorded MIC and MBC values
of 156 and 313 mg/mL, respectively. This is followed by the
WEE, which exerted an antistaphylococcal activity with the
recorded MIC value of 625 mg/mL and MBC value of
1250 mg/mL. Further attempts were made to fractionate the
EAE partition yielded 15 fractions, labeled as A1–A15,
which was further tested against MSSA and MRSA. Only
fractions A9–A15 inhibited the growth of (i) MSSA with MIC
and MBC values ranging from 78 to 156 mg/mL; and
(ii) MRSA with the MIC and MBC values ranging from
313 to 625 mg/mL. The most effective antibacterial fraction
was A10, which exhibited MIC and MBC values of 78 mg/mL.
Two years later, another attempt was made to investigate the
antimicrobial activity of M. calabura (Sibi et al., 2012). Using
various parts of the plant, namely the leaf, bark, and fruits,
collected from Bengaluru, Karnataka, India, the aqueous and
methanol extracts of the respective M. calabura parts were
prepared. The extracts (50 mL) were tested against several
bacterial isolates of clinical importance (e.g., B. cereus, K.
pneumonia, Micrococcus luteus, Proteus vulgaris, P. aerugi-
nosa, and Serratia marcescens) and fungal phytopathogens
(e.g., Aspergillus oryzae, Fusarium sp., and Penicillium sp.)
using the agar well diffusion method. Despite lack of
information on the concentration of the extracts used, the
extracts were reported to be effective antimicrobial agents.
The AEMCL was effective only against M. luteus and
P. aeruginosa; the aqueous extract of M. calabura bark
(AEMCB) was effective against B. cereus, M. luteus, and
P. aeruginosa; and, AEMCFr was effective against only M.
luteus. In contrast, MEMCL exerted the most effective
antimicrobial activity indicated by its ability to inhibit the
growth of B. cereus, M. luteus, P. aeruginosa, A. oryzae,
Fusarium sp., and Penicillium sp.; the methanol extract of
M. calabura bark (MEMCB) was effective against B. cereus,
M. luteus, Fusarium sp., and Penicllium sp., and; the MEMCFr
was effective only against B. cereus, M. luteus, P. aeruginosa,
and S. marcescens. All aqueous extracts of M. calabura only
exhibited antibacterial activity, in contrast to the methanol
extracts which inhibited the growth of several bacterial and
fungal, indicating its ability to exert antimicrobial activity.
In recent studies, Sufian et al. (2013) attempted to isolate
antibacterial compounds from M. calabura leaves.
The sample was collected from Shah Alam, Selangor,
Malaysia, and prepared as MEMCL. The extract, prepared
in the concentrations ranging between 78 and 5000 mg/mL via
serial two-fold dilutions, was tested against P. aeruginosa
ATCC27853, E. coli ATCC 10536, MSSA, MRSA, B. cereus
ATCC 11778, and B. subtilis ATCC 6633 using the micro-
dilution broth method. The MEMCL exerted notable antibac-
terial activity only against MSSA and MRSA with recorded
MIC values of 1250 and 2500 mg/mL, respectively. The
extract was further partitioned into PEE, EAE, and WEE and
subjected to the antibacterial study against MSSA and MRSA.
Interestingly, partitioning of the crude extract improved the
EAE and WEE antibacterial activity indicated by reduction in
Review on M. calabura medicinal value 1611
the values of recorded MIC (156 mg/mL for both bacteria) and
MBC (1250 mg/mL for both bacteria). In addition, the PEE
was not effective against any of the tested bacteria. The most
effective partition, which is the EAE, was further fractionated
leading to the isolation of seven fractions, labeled as F1–F7.
These fractions were also subjected to the antibacterial study
against MSSA and MRSA, and it was observed that fractions
F3, F4, and F5 exerted notable antibacterial activity against
both bacteria with the recorded MIC value of 400, 400,
and 100 mg/mL, and 400, 400, and 200 mg/mL, respectively.
As for the MBC value, only fraction F5 produced MBC value
against MSSA and MRSA at 200 and 400 mg/mL, respect-
ively. The most effective fraction, which was fraction F5, was
further purified leading to the isolation and identification of
four known flavonoids, which were compounds 28, 59, 85,
and 86. Of these compounds, only 59, 28, and 85 inhibited the
growth of MSSA and MRSA with the recorded MIC of 200,
50, and 200 mg/mL, and 400, 100, and 400 mg/mL, respect-
ively. Further attempt to determine the MBC value of the
three flavonoids revealed that the MBC value recorded
against: (i) MSSA was 400, 100, and 200 mg/mL; and (ii)
MRSA were 4800, 200, and 4800 mg/mL, respectively.
Unfortunately, compound 86 was not tested due to low
yield. Comparison was made against standard antibiotic,
chloramphenicol, which showed MIC and MBC against
MSSA at 6.25 mg/mL and against MRSA with the recorded
MIC and MBC values of 6.25 mg/mL.
Antioxidant activity
The first attempt to determine the antioxidant potential of
M. calabura was made by Zakaria et al. (2007b). In their
study, the leaves of M. calabura, collected from Shah Alam,
Selangor, Malaysia between June and September 2005, were
prepared as AEMCL and subjected to the DPPH free radical-
and superoxide anion radical-scavenging assays. From the
test, AEMCL showed approximately 94.80 ± 1.14 and
83.70 ± 2.05%, respectively, of antioxidant capacity when
measured using both assays.
Three years later, Preethi et al. (2010) investigated the
antioxidant activity of fully ripened fruits M. calabura.
The fruits, collected between May and June, 2008 from Erode
District, Tamil Nadu, India, were prepared as hexane
(HEMCFr), chloroform (CEMCFr), ethyl acetate (EAEMCFr),
butanol (BEMCFr), and methanol (MEMCFr) extracts and
subjected to several antioxidant assays such as total phenolic
content (TPC), DPPH radical scavenging activity, reductive
ability, superoxide anion-, hydroxyl ion (‘‘OH) radical- and
nitric oxide-scavenging activity, ferric ion chelating activity,
and lipid peroxidation reduction assays. From the report,
the TPC, expressed in terms of gallic acid equivalent
in mg/100 g of fresh material (mg GAE/100 g FW), was
highest in MEMCFr (1486 ± 0.028 mg GAE/100 g FW)
followed by EAEMCFr (1140 ± 0.02 mg GAE/100 g) FW,
BEMCFr (940 ± 0.03 mg GAE/100 g FW) and CEMCFr
(447 ± 0.025 mg GAE/100 g FW). HEMCFr extract had the
lowest content of phenolics (358 ± 0.020 mg/100g). In the
DPPH radical scavenging assay, all extracts, at the concen-
trations of 500, 400, 300, 200, and 100 mg/mL, exerted
significant DPPH radical quenching property as indicated
1612 N. D. Mahmood et al.
by the recorded IC50 in the following sequence:
MEMCFr (90.00 ± 0.04 mg/mL), HEMCFr (98.00 ± 0.62 mg/
mL), EAEMCFr (100.24 ± 0.24 mg/mL), CEMCFr
(245.42 ± 0.22 mg/mL), and BEMCFr (350.12 ± 0.88 mg/mL);
in comparison with butyl hydroxyl toluene (BHT), a synthetic
reference drug (IC50 ¼ 26.00 ± 0.65 mg/mL). In the reductive
ability study, EAEMCFr displayed the highest reductive
capacity (OD ¼ 0.73 ± 0.04 at 1000 mg/mL) followed by
BEMCFr, MEMCFr, HEMCFr, and CEMCFr; in comparison
with BHT (1.63 ± 0.02 mg/mL at 1000 mg/mL). Further inves-
tigation using superoxide anion scavenging assay revealed
that MEMCFr exerted the highest effect (IC50 ¼ 79.20 ±
0.04 mg/mL) followed by EAEMCFr (240.5 ± 0.2 mg/mL),
BEMCFr (250.50 ± 0.48 mg/mL), HEMCFr (310.20 ± 0.04 mg/
mL), and CEMCFr (378.20 ± 0.08 mg/mL); in comparison
with BHT (IC50 ¼ 83.00 ± 2.35 mg/mL). In hydroxyl radical
scavenging assay, MEMCFr showed the most effective activity
with the recorded IC50 of 49.98 ± 0.20 mg/mL followed by
BEMCFr (52.00 ± 0.40 mg/mL), HEMCFr (79.46 ± 0.08 mg/
mL), EAEMCFr (198.20 ± 0.02 mg/mL), and CEMCFr
(280.40 ± 0.80 mg/mL); in comparison with BHT
(IC50 ¼ 41.50 ± 2.23 mg/mL). In nitric oxide radical scaven-
ging assay, the results indicated that all extracts were dose
dependently inhibiting the formation of nitrite with MEMCFr
exhibiting the highest activity (IC50 ¼ 187.00 ±0.60 mg/mL)
followed by BEMCFr (189.00 ± 0.26 mg/mL), HEMCFr
(207.00 ± 0.02 mg/mL), CEMCFr (250.00 ± 0.08 mg/mL), and
EAEMCFr (497.20 ± 0.08 mg/mL) in comparison with BHT
(IC50 ¼ 33.50 ± 2.12 mg/mL). The ability to chelate ferric ion
was also studied and it was found that the MEMCFr exhibited
the highest chelating activity with an IC50 value of
80.26 ± 0.08 mg/mL. This was followed by EAEMCF
(81.40 ±0.04 mg/mL), CEMCFr (91.20 ± 0.64 mg/mL),
BEMCFr (290.20 ± 0.24 mg/mL), and HEMCFr
(480.60 ± 0.02 mg/mL); comparison was not made against
BHT. In the final antioxidant studies, the inhibition of lipid
peroxidation (LPO) assay was carried out and MEMCFr
(IC50 ¼ 110.4 ± 0.64 mg/mL) was found to be the most
effective extract in inhibiting the generation of LPO. This
was followed by EAEMCFr (190.20 ± 0.62 mg/mL), HEMCFr
(240.20 ± 0.04 mg/mL), CEMCFr (490.23 ± 0.24 mg/mL), and
BEMCFr (540.10 ±0.02 mg/mL); comparison was also not
made against BHT.
A second attempt was made in 2010 to determine the
antioxidant potential of the leaves of M. calabura collected
from Bangalore, Karnakata, during March, 2009 (Siddiqua
et al., 2010). The sample was prepared as MEMCL, in the
concentration of 5, 10, 15, 20, and 25 mg/mL, and tested only
using the DPPH radical scavenging assay. From this study, the
authors reported that MEMCL exerted a radical scavenging
activity with a recorded IC50 value of 22 mg/mL in compari-
son with ascorbic acid, the reference drug, which produced an
IC50 value of 12 mg/mL. In addition, the TPC value of
MEMCL was also determined but was not clearly expressed as
no unit for TPC value was given. The TPC value of MEMCL,
assessed using the Folin–Ciocalteau method with gallic acid
and tannic acid as calibration standards, was found to be 0.903
and 2.900, respectively.
This is followed by another antioxidant study by Zakaria
et al. (2011) using the same leaf sample collected between
Pharm Biol, 2014; 52(12): 1598–1623
August and September 2006 from Shah Alam, Selangor,
Malaysia. The leaves were prepared as AEMCL, CEMCL, and
MEMCL, in the concentrations of 20, 100, and 500 mg/mL,
and tested using the DPPH radical- and superoxide anion
radical-scavenging assay. In the former assay, MEMCL
exerted a radical scavenging activity ranging between 92.1
and 99.9% followed by the AEMCL (30.7–94.9%) and
CEMCL (27.2–99.9%) while in the latter assay, MEMCL,
AEMCL, and CEMCL demonstrated the radical scavenging
activity ranging between 85.7 and 89.0%, 79.8 and 77.9, and
60.0 and 87.2%, respectively. Additional studies to determine
the TPC value of those extracts were also carried out and it
was found that all extracts recorded their respective TPC
value of 41000 mg GAE/100 g FW, which is considered high.
At 6.25 mg/mL, the MEMCL, AEMCL, and CEMCL demon-
strated the TPC values of 2978.10 ± 4.34, 2970.40 ± 6.58, and
1279.90 ± 6.12 mg GAE/100 g FW, respectively.
The latest report on antioxidant activity related to
M. calabura was made by Karthyaini and Suresh (2012),
who used fruit extracts in their study. Unfortunately, although
the study was aimed to report the use of two types of extracts,
MEMMFr and AEMCFr in their pharmacognostic, anti-inflam-
matory and antioxidant studies, only the data of the anti-
inflammatory activity of the two extracts were adequately
reported, while the study on acute toxicity was not mentioned
in the report. Meanwhile, antioxidant data presented was only
for one extract, which was also not specifically described. The
unspecified extract at the concentrations of 100, 200, 300, 400,
and 500 mg/mL, was tested using the DPPH radical scavenging
assay. From the doses used (100–500 mg/mL), the percentage
of radical scavenging recorded ranged between 55 and 94%
with the IC50 value at 90 mg/mL.
Insecticidal activity
Only one study related to investigation of insecticidal activity
of M. calabura was recorded by Bandeira et al. (2013), who
used the flowers and fruits collected from the Universidade
Rural Federal de Pernambuco (UFRPE), Recife, Brazil. The
authors prepared two types of extracts from M. calabura
flowers and fruits, namely ethanol extracts [flowers (EEMCFl)
and fruits (EEMCFr)], and hexane extracts [flowers (HEMCFl)
and fruits (HEMCFr)], at concentrations ranging from 0.25 to
30.0 mg/mL, and tested them against Plutella xylostella larvae
and pupae using leaf disc immersion assay. All extracts were
reported to be toxic to the larvae and pupae of P. xylostella.
Moreover, the EEMCFl and EEMCFr were the most toxic
against first instar P. xylostella larvae with the recorded LC50
of 0.61 mg/mL and 1.63 mg/mL, respectively. This is followed
by the HEMCFr (LC50 ¼ 5.5 mg/mL) and HEMCFl
(LC50 ¼ 18.9 mg/mL). When comparing their relative toxi-
cities, it is worth highlighting that EEMCFl was 31.0-fold
more toxic than HEMCFl, and 4.2 - and 8.9-fold more toxic
than EEMCFr and HEMCFr, respectively. Overall, these
extracts were more effective than cordycepin, the reference
drug, which produced 100% mortality only at 500 mg/mL in
72 h. In addition, the EEMCFr and HEMCFr exerted greater
mortality against pupae which were 39% and 77% following
prior exposure of the larvae to 9 and 20 mg/mL of the
respective extracts. The pupal mortalities were 11% and 18%
DOI: 10.3109/13880209.2014.908397
following prior exposure of the larvae to 3.5 and 30 mg/mL of
EEMCFl and HEMCFl, respectively. In addition, EEMCFl,
EEMCFr, HEMCFl, and HEMCFr also prolonged larval
duration by approximately 2 d in some cases as compared
with the control (7.2 d).
Antinociceptive activity
The first attempt to investigate the antinociceptive activity of
M. calabura was made in 2006 by Zakaria et al. (2006a). Using
the leaves collected from Shah Alam, Selangor, Malaysia,
between January and February, 2004, AEMCL was prepared in
concentrations of 10, 50, and 100% strength that is equivalent
to 27, 135, and 270 mg/kg, was subjected to the acetic acid-
induced abdominal constriction test followed by another
studies to determine the role of L-arginine/nitric oxide/
cyclic-guanosine monophosphate (L-arginine/NO/cGMP)
pathway in the observed antinociceptive activity of AEMCL.
From the results obtained, AEMCL exerted a significant and
concentration-dependent antinociceptive activity when
assessed using the abdominal constriction test. Pre-treatment
with L-arginine significantly blocked the antinociceptive
activity of the extract at the highest concentration while pre-
treatment with NG-nitro-L-arginine methyl esters (L-NAME)
significantly enhances the antinociceptive effects at low,
concentration but inhibit its effect at higher concentration of
AEMCL. Methylene blue (MB) significantly enhanced
AEMCL antinociceptive activity at all concentrations used.
Co-treatment of L-arginine with L-NAME or MB together
significantly reversed the antinociceptive activity of AEMCL
at low concentration without affecting other concentrations of
the AEMCL. These findings suggested the involvement of L-
arginine/NO/cGMP pathway in modulating the antinociceptive
activity of AEMCL. Acetylsalicylic acid (ASA), in the dose of
10 mg/kg, was used as the reference drug.
A year later, another report on the antinociceptive activity
of M. calabura leaves was released (Zakaria et al., 2007a).
This time, the leaves were collected between July and August,
2005 from Shah Alam, Selangor, Malaysia, and prepared as
CEMCL, in the concentrations of 10, 50, and 100% strength.
CEMCL was tested for its antinociceptive activity using the
abdominal constriction test, the hot plate test, and the
formalin test. In the abdominal constriction test, the extract
exhibited a concentration-dependent activity with CEMCL at
the highest concentration producing 495% analgesia while
CEMCL at 50% concentration produced an activity that was
equieffective to that of 100 mg/kg ASA (the reference drug).
The extract also exerted an antinociceptive effect, but in a
concentration-independent manner, when assessed using the
hot plate test with the onset of activity depending on the
concentration of CEMCL. However, the activity exerted by
CEMCL, at all concentrations used, was overshadowed by the
activity exhibited by 5 mg/kg morphine. The extract also
demonstrated antinociceptive activity when assessed using the
formalin test, which was seen in both early and late phases of
the test. However, the concentration-dependent activity by
CEMCL was observed only in the early phase of the formalin
test. The reference drugs used in the formalin test were 5 mg/
kg morphine for the early and late phase, and 100 mg/kg ASA,
for the late phase.
Review on M. calabura medicinal value 1613
In a subsequent study published in response to the finding
reported in 2006, Zakaria et al. (2007e) continued to build the
antiociceptive profile of AEMCL, collected in Shah Alam,
Selangor, Malaysia, between January and February 2004.
AEMCL, in the concentrations of 1, 5, 10, 50, and 100%
(which is equivalent to the doses of 2.7, 13.5, 27, 135, and
270 mg/kg, respectively), was tested for its antinociceptive
activity using the abdominal constriction test and hot plate
test. In addition, the role of temperature and opioid receptors
on the antinociceptive activity of the extract was also
investigated. From the results obtained, AEMCL exerted
significant antinociceptive activity in both tests with a
concentration-dependent (analgesia range between 8 and
83%) effect seen only in the abdominal constriction test.
The 10% AEMCL produced percentage of analgesia that was
equivalent to that of 0.8 mg/kg morphine (47.9% versus
46.2%) while the 50% AEMCL produced analgesia that was as
equieffective as 100 mg/kg ASA (63.4% versus 71.2%) when
measured using the abdominal constriction test. The esti-
mated IC50 value for AEMCL was 12.5% (equivalent to
33.75 mg/kg extract). Although a concentration-independent
effect was observed in the hot plate test, AEMCL exerted a
significant antinociceptive activity at all concentrations
tested. The onset of antinociception range between 60 and
120 min after the extract administration and lasted until the
end (180 min) of the experiment in comparison to 5 mg/kg
morphine, which lost its activity after 180 min of the drug
administration. The 50% AEMCL, when heated at different
sets of temperature (40, 60, 80, and 100 C), did not show
sign of loss of activity as indicated by the recorded percentage
of analgesia ranging between 55 and 62% in comparison with
AEMCL prepared at room temperature (63%). Moreover, pre-
treatment with 2 or 10 mg/kg naloxone, a non-selective opioid
receptor antagonist, significantly blocked the activity of
AEMCL indicating the role of opioid receptors in the
modulation of its action.
In another study by Zakaria et al. (2007f), the leaves of M.
calabura, collected from Shah Alam, Selangor, Malaysia,
between July and August, 2005, were prepared as AEMCL, in
the concentrations of 10 (27 mg/kg), 50 (135 mg/kg), and
100% (270 mg/kg), and tested using the formalin test. The
extract exerted a concentration-independent antinociceptive
activity in both the early and late phases of the formalin test.
In comparison, 100 mg/kg ASA exerted antinociceptive action
only in the late phase, which is an inflammatory-mediated
pain stage, while 5 mg/kg morphine inhibited the late as well
as the early phase, which is related to non-inflammatory-
mediated/neurogenic pain).
Following their success in establishing the antinociceptive
profiles of CEMCL and AEMCL, and the involvement of L-
arginine/NO/cGMP pathway and opioid receptors in mod-
ulating the antinociceptive activity of AEMCL, Zakaria et al.
(2007d) took another step forward to determine the involve-
ment of non-opioid receptor systems in the central anti-
nociceptive activity of M. calabura extracts, particularly the
AEMCL and CEMCL. The M. calabura leaves were collected
between July and August, 2005, from Shah Alam, Selangor,
Malaysia, and prepared as AEMCL and CEMCL in the
concentrations of 10, 50, and 100%, which were equivalent to
the dosages of 27, 135, and 270 for AEMCL and 50, 250, and
1614 N. D. Mahmood et al.
500 mg/kg for CEMCL, respectively. In this study, the
AEMCL exerted its antinociceptive activity 120 min after its
administration while the CEMCL exerted its antinocicpetive
activity 60 min after its administration. The antinociceptive
activity of both extracts lasted the whole duration of the
experiment. Interestingly, the CEMCL exhibited antinocicep-
tive activity that was comparable with 5 mg/kg morphine.
From this study, the 50% AEMCL and CEMCL were chosen
for further studies to determine the involvement of non-
opioid receptor systems in mediating the antinociceptive
activity of the extract. The 50% concentration extracts were
pre-challenged with various types of non-opioid receptor
antagonists, namely the antagonists of muscarinic (5 mg/kg
atropine), nicotinic (5 mg/kg mecamylamine), a1-adrenergic
(10 mg/kg phenoxybenzamine), a2-adrenergic (10 mg/kg
yohimbine) and b-adrenergic (10 mg/kg pindolol), dopamin-
ergic (1 mg/kg haloperidol), and g-aminobutyric acid (GABA;
10 mg/kg bicuculline) receptors. From the results obtained,
atropine, phenoxybenzamine, yohimbine, pindolol, haloperi-
dol, and bicuculline significantly reversed the antinociceptive
activity of AEMCL either partially or completely. In contrast,
phenoxybenzamine, yohimbine, pindolol, and bicuculline also
significantly decreased the antinociceptive activity of
CEMCL. Only mecamylamine, a nicotinic receptor antagon-
ist, failed to block the antinociceptive activity of AEMCL
and CEMCL.
In another study, Zakaria et al. (2008) investigated the
effects of various receptor antagonists, pH, and enzymes on
the antinociceptive effect of AEMCL using the abdominal
constriction test. The leaves of M. calabura were collected
between January and February, 2004 from Shah Alam,
Selangor, Malaysia, and prepared as AEMCL, in the concen-
trations of 5, 50, and 100%. In the first study, the extract was
subjected directly to the abdominal constriction test to
develop its antinociceptive profile. In the second study, the
50% concentration AEMCL, which was chosen based on the
first study, was pre-challenged with various types of opioid
and non-opioid receptors’ antagonists, namely 10 mg/kg
naloxonazine, 10 mg/kg naltrindole, 10 mg/kg pindolol,
10 mg/kg phenoxybenzamine, 10 mg/kg bicuculine, 5 mg/kg
atropine, and 5 mg/kg mecamylamine. In the third study, the
50% concentration AEMCL, with recorded pH of 5.1, was
subjected to a series of different pH (3, 5, 7, 9, 11, or 13) for
2 h and then neutralized back to pH 5.1. Those modified
AEMCLs were then subjected to the abdominal constriction
test. In the fourth study, the AEMCL was pretreated either
with 10% concentration a-amylase, 10% concentration lipase
or 1% concentration protease for 2 h in a water bath at 40  C.
The pre-treated extracts were then subjected to the abdominal
constriction test. In the first study, the AEMCL exhibited
significant antinociceptive activity in a concentration-depend-
ent manner when assessed using the abdominal constriction
test. The 5 and 50% concentration AEMCL produced an
activity that has a similar potency as the reference drugs,
morphine (0.8 mg/kg), or ASA (100 mg/kg), respectively. In
the second study, the peripheral antinociceptive activity of
AEMCL was reduced following pre-treatment with only
naloxonazine (opioid receptor antagonist), pindolol (b-adren-
ergic receptor antagonist), and atropine (muscarinic receptor
antagonist). In the third study, the antinociceptive activity of
Pharm Biol, 2014; 52(12): 1598–1623
AEMCL increased significantly after modification of pH to
alkaline condition, which is at pH 9–11. Moreover, the
activity was still preserved at the extreme acidic condition
(pH 2) and extreme alkaline condition (pH 13). In the fourth
study, its antinociceptive activity was not distorted after
pretreatment with amylase, protease, lipase, or their combin-
ation when compared with the untreated AEMCL.
A further attempt was made by Yusof et al. (2011) to study
the antinociceptive activity of semipurified fractions derived
from PEE extract using the formalin test. Seven fractions,
labeled as A–G, were isolated from the PEE extract of M.
calabura. From the results obtained, fraction D exerted the
most significant antinociceptive activity when compared with
other fractions and at the dose of 300 mg/kg produced 66.2%
and 81.4% antinociception in the early phase and late phase of
the formalin test, respectively. However, Fraction D exhibited
no significant difference when compared with the reference
drug, 100 mg/kg ASA.
In the latest study, Sani et al. (2012) reported on the
antinociceptive activity and the possible mechanisms of
antinociception of M. calabura leaves collected from Shah
Alam, Selangor, Malaysia. The leaves were prepared as
MEMCL in the concentrations of 100, 250, and 500 mg/kg
and tested using the abdominal constriction test, hot plate test,
and formalin test to build the antinociceptive profile of the
extract. Results showed that the MEMCL exerted a significant
and dose-dependent antinociceptive activity in the abdominal
constriction test with the 250 mg/kg MEMCL exerted an
activity that was as equieffective as 100 mg/kg ASA. In the
hot plate test, only 500 mg/kg MEMCL exerted significant
ability to prolong the latency of response to discomfort
throughout the whole experiment. Generally, 5 mg/kg mor-
phine was more effective then the extract. In the formalin test,
MEMCL showed significant antinociceptive activity in both
phases of the formalin test with dose-dependent activity seen
only in the early phase. Only the 250 and 500 mg/kg of
MEMCL showed antinociceptive effect in the early phase
whereas all doses of MEMCL exerted antinociceptive activity
in the late phase. The ability of MEMCL to attenuate
nociception in the early and late phases of the formalin test
was comparable with 5 mg/kg morphine.
In the second part of the study, the mechanisms of
antinociception (e.g., the role of vanilloid receptors, gluta-
matergic systems, opioid receptors, and L-arginine/NO/cGMP
pathway) were determined for MEMCL. To determine the
involvement of vanilloid receptors, MEMCL was subjected to
the capsaicin-induced paw licking test. From the results
obtained, all doses of MEMCL showed a dose-dependent
attenuation of capsaicin-induced nociception with the per-
centage of analgesia ranging between 20 and 62%. To
determine the involvement of glutamatergic system,
MEMCL was subjected to the glutamate-induced paw licking
test. The results showed that the extract exerted a dose-
dependent antinociceptive activity against the glutamate-
induced nociception with the percentage of analgesia ranging
between 35 and 72%. Both studies indicated the involvement
of vanilloid receptors and glutamatergic system in the
modulation of the antinocicpetive activity of MEMCL.
To determine the role of opioid receptors, the MEMCL was
pre-challenged with 5 mg/kg naloxone, a non-selective opioid
DOI: 10.3109/13880209.2014.908397
receptor antagonist followed by the abdominal constriction
test, hot plate test, and formalin test. Result showed that the
antinociceptive activity was significantly attenuated by
naloxone in all the tests used indicating the involvement
of opioid receptors in the modulation of antinociceptive
activity of MEMCL. To determine the involvement of
L-arginine/NO/cGMP pathway, the extract was pre-challenged
separately with 20 mg/kg L-arginine, 20 mg/kg L-NAME,
20 mg/kg MB, or their respective combination followed
by the abdominal constriction test. The results showed that
L-arginine alone did not affect the acetic acid-induced
nociception but significantly reversed the antinociceptive
activity of MEMCL. Meanwhile, L-NAME alone exerted
significant antinociceptive activity and maintained the
MEMCL-induced antinociception. L-Arginine was found to
reverse the L-NAME-induced antinociceptive activity but
when given together, L-arginine and L-NAME failed to affect
the antinociceptive activity. In the second part of this study,
MB alone exhibited significant antinociceptive activity,
but when pre-challenged with MEMCL, failed to affect the
antinociceptive activity. In addition, L-arginine failed to
attenuate the antinociceptive activity of MB while the
combination of L-arginine and MB also failed to inhibit the
antinociception of MEMCL.
Another recently published finding related to the anti-
nociceptive potential of M. calabura leaves also described the
successful isolation and identification of pain-relieving bio-
active compounds. The leaves were collected from Shah
Alam, Selangor, Malaysia, in January, 2008, and prepared as
MEMCL, in the doses of 100, 500, and 1000 mg/kg, and
subjected to the formalin test. MEMCL was later partitioned
into PEE, EAE, and WEE and prepared in the same doses
range of 100–1000 mg/kg. From the results obtained, MEMCL
exerted significant (p50.05) antinociceptive activity in both
the early and late phases of the formalin test. The extract
(100–1000 mg/kg) reduced the amount of time the rat spent
licking the pain-induced paw (indicator of nociception) in
both phases by 38.3–20.0 and 67.5–25.7 s in comparison with
the time recorded by the negative control (10% DMSO-
treated) group, which was 70.7 and 138.2 s, respectively.
In addition, PEE and EAE, but not WEE, in the dose range of
100–1000 mg/kg, also caused significant (p50.05) reduction
of the nociceptive latency at the early phase in the range of
63.5–20.8 and 69.0–25.8 s, respectively, while in the
late phase, all partitions reduced the latency in the range of
85.0–16.0, 100.2–35.2, and 129.5–101.0, respectively.
From the results obtained, PEE was considered as the most
effective partition and, thus, subjected to the fractionation
processes to yield seven fractions, labeled A–G. Following the
formalin test, only fractions C, D, and E caused significant
(p50.05) reduction of nociceptive latency in the early phase
to 57.2, 27.7, and 42.2 s while in the late phase, Fractions B,
C, D, E, and F reduced the latency of nociception to 137.8,
86.2, 27.2, 77.8, and 86.7 s, respectively. For comparison
purposes, the latency of nociception for both phases of the
formalin test observed in the negative control group was 83.2
and 149.0 s, respectively. Since fraction D showed the most
significant antinociception in both phases of the formalin test,
it was subjected to the isolation and identification processes
leading to the isolation of 25, 55, 64, and 87. Except for
Review on M. calabura medicinal value 1615
compound 64 that was prepared only in the dose of 50 mg/kg,
the rest of the compounds were prepared in the doses of 50
and 100 mg/kg. Upon testing using the formalin test, all
compounds were effective against both phases of formalin-
induced nociception wherein all doses tested were effective
against the late phase of formalin-induced nociception.
However, in the early phase of the test, particularly, only
compounds 64 and 87 exerted an antinociceptive activity even
at the lowest dose used (50 mg/kg). Only a single dose of
compound 64 was studied as the yields obtained from
extraction were rather too small for multiple dose study.
From the results obtained, compound 64, at the single dose of
50 mg/kg, was considered the most effective antinociceptive
agent as it caused an approximately 34% and 44%
antinociception in comparison with the other compounds at
the same dose used.
Anti-inflammatory activity
The earlier report on anti-inflammatory potential of
M. calabura was published by Zakaria et al. (2007a). The
leaves were prepared as CEMCL, in the concentrations of 10,
50, and 100%, and tested using the carrageenan-induced paw
edema test. ASA (100 mg/kg) was used as a reference drug.
All concentrations of CEMCL exerted an inconsistent anti-
inflammatory activity that was less effective than the ASA
Another report on the anti-inflammatory activity of
M. calabura leaves was also published in 2007 (Zakaria
et al., 2007f) while attempting to determine the antinocicep-
tive activity of AEMCL. In this study AEMCL was prepared
in the concentrations of 10, 50, and 100% (equivalent to
the doses of 27, 135, and 270 mg/kg, respectively) and
subjected to the carrageenan-induced paw edema assay. The
results obtained demonstrated that the extract exhibited
concentration-independent anti-inflammatory activity. The
anti-inflammatory activity of the 10 and 50% AEMCL were
completely lost after 7 h of its administration while the anti-
inflammatory activity of 100% AEMCL was lost after only 6 h
of its administration. It is worth mentioning that the anti-
inflammatory activity of AEMCL, at the concentrations of 10
and 50%, was significantly greater than the reference drug,
100 mg/kg ASA, at the interval of 3 and 4 h after their
administration.
In the recent attempt to study the pharmacological
properties of the fruits of M. calabura, the MEMCFr and
AEMCFr were prepared in doses of 200 and 400 mg/kg and
tested using the carrageenan-induced paw edema test (Preethi
et al., 2012a). The results obtained demonstrated that both
extracts exerted dose-dependent inhibition of carrageenan-
induced localized edema at 4 h after the administration of
extracts. The significant anti-inflammatory activity was
recorded at 24.5 and 44.2% for both doses of MEMCFr and
at 20.4 and 46.2% for both doses of AEMCFr. Indomethacin,
in the dose of 10 mg/kg, was used as the reference drug and
caused 84.3% inhibition of carrageenan-induced edema
formation in comparison with the extracts.
Preethi et al. (2012b), in another recently published study,
also investigated the anti-inflammatory activity of M. calabura
fruits, collected from Erode District, Tamil Nadu, India using
the carrageenan-induced paw edema model. The fruits were
1616 N. D. Mahmood et al.
prepared as MEMCFr, at doses of 100, 200, and 300 mg/kg, and
subjected to the carrageenan-induced paw edema test. The
results showed that the extract exerted a significant and dose-
dependent anti-inflammatory activity indicated by the reduc-
tion in edema formation irrespective of the dose used. At the
doses tested, a dose-dependent inhibition of carrageenan-
induced localized edema was observed at 4 h. However, the
activity seen with MEMCFr, at all doses (percentage of anti-
inflammation ranging between 24 and 46%), was lower than
that of the reference drug, 10 mg/kg indomethacin (percentage
of anti-inflammation was 80.48%). Despite this second report
by Preethi et al. (2012b), some of the data have already been
presented in Preethi et al. (2012a). The only differences
observed were the dose of extract used and the percentage of
anti-inflammation recorded.
Antipyretic activity
The first attempt to determine the antipyretic potential of M.
calabura was made by Zakaria et al. (2007a) using the leaves
that was prepared as CEMCL. The extract, at the concentra-
tions of 10, 50, and 100%, was tested using Brewer’s yeast
(BY)-induced pyrexia test. The extract exhibited a concen-
tration-independent antipyretic activity. Comparison made
against the 100 mg/kg ASA, as the reference drug, showed
that the CEMCL antipyretic activity was less effective than
the drug.
Zakaria et al. (2007f) also reported on the antipyretic
activity of another extract of M. calabura, namely AEMCL,
while studying the antinociceptive and anti-inflammatory
activities. The AEMCL exerted a concentration-independent
antipyretic effect with the onset of effects of 27 and 135 mg/
kg AEMCL was recorded after 240 min of their administra-
tion. Overall, the antipyretic activity of AEMCL was less
effective than the reference drug, 100 mg/kg ASA.
Antiulcer activity
The investigation of antiulcer potential of M. calabura was
initiated only in 2012 with one study published. This
preliminary study was carried out by Ibrahim et al. (2012)
involving the use of M. calabura leaves obtained from a
company, Ethno Resources Sdn. Bhd., Selangor, Malaysia.
The leaves were prepared as EEMCL, in the dose of 250 and
500 mg/kg, and assayed only against the ethanol-induced
gastric ulcer model. The extract demonstrated significant and
dose-dependent antiulcer activity indicated by the reduction
in the areas of gastric ulcer injuries (112.5 ± 2.11 and
95.08 ± 2.18 mm2) in comparison with the negative control
group (735.25 ± 2.12 mm2) and 20 mg/kg omeprazole-treated
group (the reference drug; 90.33 ± 2.02 mm2). Further study
on the ethanol-treated stomach samples revealed that the
EEMCL reduces the acidity of gastric content while increases
the mucus production of gastric mucosa when compared with
the negative control. Moreover, the subsequent microscopic
observations supported the macroscopic findings.
Another study on the antiulcer potential of M. calabura
leaves was recently published in 2013 (Balan et al., 2013). In
this study, the leaves were prepared as MEMCL and subjected
to ethanol- and indomethacin-induced gastric ulcers wherein
in the former assay the doses of 25, 50, 100, 250, and 500 mg/
Pharm Biol, 2014; 52(12): 1598–1623
kg were used while in the later assay, the doses of 100, 250,
ad 500 mg/kg were used. The discrepancy in the range of
doses used was attributed to preliminary findings using the
ethanol-induced gastric ulcer model wherein the extract
exerted a dose-independent antiulcer activity. Therefore, an
additional study using lower doses (25 and 50 mg/kg) was
performed. Moreover, the role of NO and sulfhydryl groups in
mediating the antiulcer activity of MEMCL was also
investigated using the ethanol-induced gastric ulcer. From
the results obtained, MEMCL, at all doses tested, exhibited a
significant and dose-dependent reduction of ethanol-induced
gastric ulcer formation with the percentage of antiulcer
ranging between 63 and 95% in comparison with the reference
drug, 100 mg/kg ranitidine, that produced 70% protection. In
addition, all doses of MEMCL exerted significant and dose-
dependent inhibition of indomethacin-induced gastric ulcer
formation with the percentage of protection ranging between
47 and 69%. In comparison, 100 mg/kg ranitidine exhibited
78% antiulcer activity. Histopathological evaluation revealed
the extract potential to reverse the toxic effect of ethanol and
indomethacin and returned the stomach to almost normal
mucosal architecture that is comparable with protection
exerted by ranitidine. Moreover, pre-treatment with 70 mg/
kg L-NAME significantly worsened the gastric ulcers in
MEMCL- and 100 mg/kg carbenoxolone-treated groups and
this unwanted effect of L-NAME was reversed by 200 mg/kg
L -arginine. These findings indicate the participation of NO in
the antiulcer potential exerted by MEMCL. Pre-treatment with
10 mg/kg NEM, in contrast, significantly reversed the
antiulcer activity of MEMCL and increased the gastric ulcer
formation in comparison with saline pretreated group that is
also receiving MEMCL. These findings indicate the partici-
pation of endogenous sulfhydryl compounds in the gastro-
protective activity demonstrated by MEMCL.
Antidiabetic activity
The first report on antidiabetic activity of the leaves of
M. calabura was published in 2011 (Sridhar et al., 2011). The
leaves of M. calabura, collected from Station Ghanpur,
Warangal, Andhra Pradesh, India, were prepared as MEMCL,
in doses of 300 and 500 mg/kg, and subjected to the
antidiabetic studies. Firstly, the serum glucose level was
observed at 2, 4, 6, and 8 h after the administration of the
extract. The results showed that both doses of MEMCL
produced significant hypoglycemic effects after 6 and 4–8 h,
respectively, in the normal fasted rats. The 500 mg/kg of
MEMCL caused significant reduction in the blood glucose
level from 83.19 mg/dL at 0 h to 62.62 mg/dL (24.81%) at the
end of the 6 h. In comparison, the reference drug, 5 mg/kg
glipizide, caused significant reduction in the blood glucose
level after 2 h of administration that lasted for another 6 h. In
the second study, the effect of 500 mg/kg MEMCL on the oral
glucose tolerance test (OGTT) was also investigated. The
results showed that pre-treatment with 500 mg/kg MEMCL
caused significant reduction in the rise of blood glucose at 1 h
interval (116.46 ± 6.94 mg/dL) when compared with the
control group pre-treated with 5% gum acacia, which
showed a rapid increase of blood glucose (144.73 ±
7.86 mg/dL). For the standard group (glipizide 5 mg/kg), the
DOI: 10.3109/13880209.2014.908397
glucose levels reached the fasting values at the end of 1 h
interval (72.09 ± 2.98 mg/dL). In the third study, the 500 mg/
kg MEMCL was subjected to the alloxan-induced diabetic
assay. Following the experiments, 500 mg/kg of MEMCL
significantly reduced the alloxan-induced hyperglycemia with
maximum effect observed at 6 h (27%) in comparison with the
reference drug, 5 mg/kg glipizide, which produced 37%
reduction in blood glucose level.
Antihypertensive activity
The first report on the antihypertensive activity of
M. calabura was published in 2006 (Shih et al., 2006). The
leaves of M. calabura were collected in June, 2001, from
Kaohsiung City, Taiwan and prepared as a methanol extract
(MEMCL). The crude MEMCL was then partitioned using
dH2O and chloroform in the ratio of 1:1, and the aqueous
fraction obtained was further fractionated sequentially using a
mixture of dH2O and n-butanol (1:1). The water-soluble
fraction (WSF) was collected, prepared in the dose range of
10, 25, 50, 75, and 100 mg/kg, and systemically injected into
the femoral vein of the animals. In the first study, the mean
systemic arterial pressure (MSAP), heart rate (HR), baseline
blood pH, gas (partial pressure of CO2 and O2), and
electrolytes (Na+, K+, hematocrit) were measured for 3 h
from blood samples withdrawn from the arterial blood
following pre-treatment with isotonic normal saline, 5 mg/kg
acetylcholine (the reference drug), or WSF (10, 25, 50, 75, or
100 mg/kg). In the second study, the plasma nitrate level was
measured using blood samples withdrawn from the femoral
artery using the chemiluminescense assay. In the third study,
the biochemical analysis involving protein extraction and
Western blot analysis were carried out. The apical heart or
segment of thoracic aorta was rapidly removed from
sacrificed rats and later subjected to Western blot analysis
of iNOS, eNOS, nNOS, or b-actin protein. Another study was
performed to delineate the causative relationship between NO
and M. calabura-induced cardiovascular responses wherein
the temporal change in MSAP or HR elicited by 50 mg/kg
WSF was measured for 180 min in rats subjected to pre-
treatment with L-NAME, L-NIO, SMT, 7-NI, or ODQ
administered 20 min prior to administration of WSF.
The findings revealed that intravenous administrations of
WSF significantly and dose dependently caused an immediate
decrease in MSAP (initial phase) that returned to the
pre-injection baseline within 10 min post-injection without
affecting the HR. The decrease in MSAP was followed by a
delayed hypotensive effect (delayed phase) that started at
90 min and lasted for approximately 180 min post-injection.
Acetylcholine (5 mg/kg) also caused a significant decrease in
MSAP that reached its peak within the first 30 s and lasted for
less than 5 min post-administration. The authors also reported
that treatment with a 50 mg/kg WSF caused no significant
change to the baseline systemic arterial blood gases, electro-
lytes, Hct, and pH when measured at 10, 30, 60, 120, and
180 min post-injection as seen with saline and WSF, at the
doses of 25, 75, and 100 mg/kg. Moreover, intravenous pre-
treatment with 0.65 mg/kg/min, as well as 0.13 and 0.35 mg/
kg/min L-NAME, a non-selective NOS inhibitor, significantly
attenuated both the initial and delayed phases of hypotension
Review on M. calabura medicinal value 1617
induced by the WSF. An inhibitor of eNOS, L-NIO, given
intravenously at the dose of 1.0 mg/kg/min, significantly
suppressed only the initial phase of WSF-induced hypoten-
sion while, to the contrary, 0.5 mg/kg/min SMT, a selective
inhibitor of iNOS, given through the same route strongly
inhibited only the delayed phase of the same response. Of all
doses of L-NAME, L-NIO, 7-NI or SMT used, only L-NMAE,
at the highest dose (0.65 mg/kg/min), given alone evoked a
significant and transient increase in MSAP by 12% and a
decrease in HR by 11%. In addition, 0.2 mg/kg/min ODQ, an
sGC inhibitor that had no significant effect on baseline MSAP
or HR when given alone, markedly suppressed both the initial
and delayed phases of WSF-decreased MSAP. WSF also
induced a significant increase in iNOS, but not eNO or nNOS,
protein expression in the heart or aorta detected at 90 or
180 min post-administration.
Shih (2009) had again reported on the antihypertensive
effect of butanol-soluble fraction (BSF) of M. calabura
leaves. The leaves of M. calabura, collected from Kaohsiung
City, Taiwan, were prepared as MEMCL and then partitioned
using dH2O and chloroform in the ratio of 1:1. The aqueous
fraction was collected and further fractionated sequentially
using a mixture of dH2O and n-butanol (1:1). This time, the
BSF was collected and prepared in the dose range of 10, 25,
50, 75, and 100 mg/kg. Together with the isotonic normal
saline or 5 mg/kg acetylcholine (the reference drug), the BSF
were systemically injected into femoral vein of the animals. In
the first study, the temporal changes in MSAP and HR after
the administration of test solutions were determined for 2 h. In
the second study, which attempted to delineate the involve-
ment of the NO/sCG/cGMP/PKG signaling pathway, the
temporal changes in MSAP and HR induced by intravenous
administrations of BSF, at 25 mg/kg, were evaluated for
120 min in rats exposed earlier to pre-treatment with
L -NAME (0.33, 0.5, and 1.3 mg/kg/min), L -NIO (1 mg/kg/
min), SMT (0.5 mg/kg/min), 7-NI (6 mg/kg/min), ODQ
(0.2 mg/kg/min), or KT5823 (7 mg/kg/min). From the results
obtained, intravenous administrations of BSF, in the dose
range of 10–100 mg/kg, caused a dose-dependent hypotensive
and bradycardiac responses in normotensive Witar–Kyoto
(WKR) and spontaneously hypertensive (SHR) rats. The
biphasic responses evoked by BSF in WKR were character-
ized by immediate but transient decreases in MSAP and HR,
which returned to pre-injection baseline within 2–3 min post-
injection. In contrast, the SHR with established hypertension
demonstrated initial decreases in MSAP and HR, which was
followed by a delayed phase of vasodepressor and bradycar-
diac responses that began at 40 min and prolonged for at least
120 min post-injection. The fraction also exerted significantly
greater hypotensive responses in SHR than in WKR.
Moreover, the BSF-induced depressor response was greater
in the initial than the delayed phase while the bradycardia was
significantly greater in the delayed phase than the initial phase
in the SHR.
In comparison, 5 mg/kg acetylcholinem induced a signifi-
cant MSAP reduction in WKR and SHR with a peak detected
within the first 30 min and prolonged for less than 5 min post-
treatment. In addition, pre-treatment with L-NAME signifi-
cantly attenuated the hypotensive and bradycardia effects of
BSF in the normotensive WKR whereas, in the SHR, L-
1618 N. D. Mahmood et al.
NAME dose dependently antagonized the depressive effect of
BSF on the cardiovascular initial and delayed phases. Of the
three isoforms of NOS, only L-NIO (1 mg/kg; an eNOS
inhibitor) given intravenously caused significant suppression
of the BSF-induced depression of the initial phase in WKR or
SHR, and the delayed phase of SHR. Furthermore, the iNOS
inhibitor, SMT (0.5 mg/kg/min) which was given intraven-
ously, greatly inhibited the delayed phase in SHR, but not the
initial phase of the BSF-induced cardiovascular response in
WKR or SHR. In addition, L-NAME, but not L-NIO, SMT or
7-NI, when given alone caused transient increase in MSAP
and a decrease in HR, which returned to baseline prior to the
BSF administration. From the third study, intravenous pre-
treatment with 0.2 mg/kg/min ODQ, a sGC inhibitor, or 7 mg/
kg/min KT5823, a PKG inhibitor, caused significant attenu-
ation of the decrease MSAP and HR at the initial phase in
WKR, and both the initial and delayed phases in SHR. When
given alone, ODQ and KT5823 did not cause significant
change on the baseline MSAP or HR.
It is, therefore, concluded that the BSF demonstrated a
transient followed by delayed antihypertensive and bradycar-
diac effects through the activation of NO-dependent cGC/
cGMP/PKG signaling pathways. Furthermore, the eNOS-
derived NO was shown to induce initial cardiovascular
depressive response whereas the iNOS-derived NO was
responsible for modulating the delayed response elicited by
BSF.
Cardioprotective activity
Only one report was published on the cardioprotective
potential of M. calabura leaves by Nivethetha et al. (2009).
Using the AEMCL, of which the location and period of leaves
collection were not given, the authors studied the extract
ability to attenuate isoproterenol-induced myocardial infarc-
tion in rats. Several parameters (e.g., aspartate transaminase
(AST), alanine transaminase (ALT), lactate dehydrogenase
(LDH), and creatinine phosphokinase (CK)) were estimated
in both the serum and heart tissues, and the serum uric acid
level was also estimated. From the results obtained, AEMCL
caused significant reduction in the activity of marker enzymes
(AST, ALT, CK, and LDH) and the level of uric acid
when compared with the isoproterenol-induced myocardial
infarction group. In all parameters estimated, only 200 and
300 mg/kg AEMCL exerted significant effects.
Discussion
The assets of a country, particularly Malaysia, dwell to a large
extent in its plant heritage. Malaysia is one of the countries
with large biodiversity and prosperous flora, and convention-
ally estimated to include approximately 15 000 species of
higher order plants, many of which are endemic. Plants play a
key part in the cure of ailments and still remain the leading
treatment choice for a large majority of people including
people of Malaysia. In many countries throughout the world,
herbal products are either consumed in traditional medical
setting or taken as food supplements. A number of medicinal
plants have been shown to offer an alternative to synthetic
drugs in preventing and treating some chronic and mild
diseases. The facts that synthetic and chemical therapeutic
Pharm Biol, 2014; 52(12): 1598–1623
approaches possess severe adverse effects have triggered the
search for natural products with less or, possibly, no side
effect (Preethi et al., 2012).
Current attention towards the pharmacological potential of
medicinal plants have been escalating globally as indicated by
the increase in publications on the pharmacological potential
of various traditionally claimed or newly discovered medi-
cinal plants. In an attempt to find a pure and effective lead
from plants, pharmacologists have also collaborated with
phytochemists to isolate, identify, and determine potential
bioactive compounds with specific ability to treat any
particular disease through a process known as bioassay-
guided fractionation. Several drugs that are currently avail-
able in the international market were the result of exhaustive
scientific and systematic explorations of the traditional claims
of the plants and ethnopharmacology. Regardless of the global
rise in scientific investigation on medicinal plants, only small
numbers of plant-derived bioactive compounds have reached
the market, locally or internationally, due to their evidence-
based therapeutic potential (Mitchell & Ahmad, 2006). The
reasons for a low number of plant-based drugs reaching the
market could be associated to the lack of endeavors taken to
determine or validate the evidence related to the safety of
the respective plant, which, in turn, is mistakenly assumed
to be safe due to their plant-based and naturally occurring
facts (Yob et al., 2011).
In traditional medicine worldwide, treatment of sympto-
matologies related to the respective ailment (i.e., gastric
ulcers) with medicinal plant is quite common. When using
crude drugs to treat a respective ailment, several important
questions should be raised such as the necessary amount of
plant to provide adequate healing response, traditional way of
preparation (e.g., infusion, decoction, maceration, etc.),
concentration (plant/solvent ration), and frequency and dur-
ation of treatment. Unfortunately, these questions are left
unattended during the ethnopharmacological studies and this
fact is surprising as there should be a realistic approach in
acquiring the right doses to confirm the reputed effectiveness
of the crude drugs. Normally, plants used in traditional
medicine are prepared either as infusions or as decoctions, but
in some regions, the plants are macerates, either in water or in
alcoholic beverages. Based on the traditional claims recorded,
various parts of M. calabura (e.g., flowers, bark, leaves and
roots) possess medicinal values. In lieu of this, various types
of extracts (e.g., AEMCL, AEMCFr, AEMCB, AEMCF,
MEMCR, MEMCL, MEMCSB, MEMCFr, MEMCB, EEMCL,
EEMCFr, EEMCFl, EAEMCFr, CEMCL, CEMCFr, BEMCFr,
HEMCFr, HEMCFl, and HEMCF) were prepared for scientific
studies with the hope of finding the most effective extract for
future drug development. In line with this, several studies
have been carried out to isolate various bioactive compounds
from different parts of M. calabura. A total of 88 pure
compounds have been isolated and identified from different
extracts of different parts of M. calabura, of which 26 of them
(e.g., compounds 1–13, 36, 37, 50–53, 65–67, and 85–88)
were new compounds isolated directly from M. calabura. The
remaining compounds were first isolated from other plants
but later found to be present in M. calabura.
Many factors influence the quality of herbs and these
include species variation, environmental conditions, and the
DOI: 10.3109/13880209.2014.908397
time of harvesting, storage, and processing. For these reasons,
the quality control of herbal extracts is an essential part in any
research involving safety, efficacy, and therapeutic reprodu-
cibility. Quality control is a difficult task because medicinal
plant extracts are complex mixtures of different compounds,
which can vary due to various factors including location and
period of collection of samples. Despite the importance of
some of the above-mentioned factors for scientific reprodu-
cibility and validity, there are several manuscripts that failed
to provide information on the factors such as the place and
period of samples collection (Table 3).
It is worth mentioning that according to the World Health
Organization (1999), a medicinal plant is any plant which, in
one or more of its parts, contains substances that can be used
for therapeutic purposes, or which are precursors for semi-
synthesis of chemo-pharmaceutical (Doughari, 2012). Such a
plant will have its parts including leaves, flowers, stems,
barks, roots, rhizomes, fruits, grains or seeds, employed in the
control or treatment of a disease condition and, therefore,
contains chemical components that are medically active.
By referring to Table 3 in regard of M. calabura, all parts of
the plant, namely the leaves, fruits, flowers, stem bark, bark,
and roots have been used traditionally to treat various
ailments as described earlier. However, the scientific
approaches used by the researchers in their attempts to
prove scientifically the traditional claims of M. calabura’s
medicinal values were focusing mainly on the use of its leaves
and fruits, followed in the decreasing order by the flowers,
bark, stem bark and roots. This might be due to the nature of
the plant wherein the leaves and fruits are the parts that are
easy to collect in abundance throughout the year. The roots or
bark are not well studied probably because of their collections
which could lead to damage or death of the tree. Moreover,
studies which use animal models require a large amount of
samples in order to get sufficient amount of bioactive
compounds.
With regard to the relationship between the observed
pharmacological activities and the different parts of M.
calabura tested, several conclusions can be proposed. Only
the leaves and fruits of M. calabura were confirmed to be safe
for consumption and have antioxidant effects. This is in
accordance with the claims that the leaves are consumed
directly as a tea-like beverage in Peru while the fruits are
freshly eaten or prepared as tart or jam in Mexico. Moreover,
the acute toxicity study was performed using the animal
model and required a huge amount of extract, which can be
easily prepared using the leaves or fruits in comparison with
the other parts of the plant. These arguments are supported by
our observations that in all the in vitro assays (e.g., cytotoxic,
insecticidal, antioxidant and antibacterial), which required
smaller amount of samples, parts like the stem bark, flowers,
and roots, were also tested for the respective pharmacological
activity. The use of the leaves to study the antinociceptive,
anti-inflammatory, antipyretic, antiulcer, and antiproliferative
activities of M. calabura are concomitant with the traditional
claims of the leaves potential to treat headache and cold,
gastric ulcer, and swelling of prostate gland. In contrast, the
antibacterial activity of M. calabura was determined against
almost all parts of the plant, which is possibly attributed to the
general knowledge that plants developed their own defense
Review on M. calabura medicinal value 1619
mechanisms to prevent infection at any of its parts via
accumulation of antimicrobial secondary metabolites
(González-Lamothe et al., 2009). Furthermore, the insecti-
cidal activity was investigated using the samples of flowers
and fruits as these two parts are where the insects are mostly
attracted to. However, the insecticidal effect of other parts of
M. calabura should also be studied. The antidiabetic potential
of M. calabura was studied using only the leaves probably due
to the earlier claims on its use as a tea-like beverage and
previous scientific reports (e.g., Kaneda et al., 1991; Su et al.,
2003) of high flavonoids content (Babu et al., 2013).
The other activities (e.g., hypotensive, cardioprotective,
and antiplatelet aggregation) were determined only from the
leaves sample even though no traditional claim related to
the cardiovascular treatment had been reported. This might be
contributed by the researchers attempt to look at additional
pharmacological potential of M. calabura regardless of
whether those activities were justified by the traditional
claims.
In the quest for potent, effective and relatively safe plant
medicines, there is a need to study and validate the safety and
efficacy of the plant. Despite their promising potential and
increase in use among patients, many medicinal plants or their
products are untested and their use is not properly monitored.
Consequently, knowledge of their potential toxicity and side
effects is limited. As for M. calabura, only data for acute, but
not chronic or sub-chronic toxicity are available with the first
toxicity study performed only in 2011. Between 2011 and
2013, four acute toxicity studies were reported on various
extracts of different parts of M. calabura by Sridhar et al.
(2011), Ibrahim et al. (2012), Karthyaini and Suresh (2012),
and Balan et al. (2013), who used 300–2000 mg/kg MEMCL,
2000 and 5000 mg/kg EEMCL, 1000 mg/kg EEMCFr or
2000 mg/kg MEMCL, respectively. Overall, M. calabura
leaves and fruits are safe for oral consumption up to a dose
of 2000 mg/kg.
Toxicity studies were also performed at the cell level either
against the normal or cancerous cells. Usually, antiprolifera-
tive and cytotoxic studies using normal non-cancerous cells
aimed at evaluating the safety of the extracts/compounds used
at the cell level while those involving the use of cancerous
cells are usually aimed at determining the anticancer potential
of extracts/compounds. Only one antiproliferative study was
reported by Zakaria et al. (2011) wherein 12.5–100.0 mg/mL
of AEMCL, CEMCL, or MEMCL were tested against normal
3T3 cells (normal non-cancerous mouse fibroblast) and found
to cause no cytotoxic effect to the 3T3 cells indicating that the
extracts were safe towards normal cells. Furthermore, of the
four cytotoxic papers cited in this review paper (Kaneda et al.,
1991; Chen et al., 2004, 2005; Sufian et al., 2013), which
involved studies using various cancerous cells, only Sufian
et al. (2013) carried out additional cytotoxic experiments
using the normal WRL-68 cells (human embryonic liver non-
tumor type cell lines). From the results obtained, Sufian et al.
(2013) reported that the MEMCL was safe and non-toxic
towards the WRL-68 cells even at the highest concentration
(100 mg/mL) tested. Despite the successes of Kaneda et al.
(1991) and Chen et al. (2004, 2005), in particular, in
identification of bioactive compounds with cytotoxic activity
against several cancer cells, their failure to evaluate those
1620 N. D. Mahmood et al.
isolated compounds for safety towards any normal cells were
not justified. Therefore, it is suggested that any in vitro
antiproliferative or cytotoxic studies using cancerous cells
should be accompanied by additional studies using normal
non-cancerous cells. The major impediment in the utilization
of traditional medicinal plant preparations is poor under-
standing on the efficacy and safety of the medicinal plants as
those plants are regarded as safe. This is further worsened by
the negligence among the researchers towards the importance
of evaluating toxicity of the medicinal plants, as well as their
adverse drug reactions. Thus, to encourage the use of
medicinal plants, it is important to establish the safety of
these preparations through toxicological assessments.
Other than the issues of quality and safety, the usefulness
of medicinal plant preparations is also affected by factors such
as dosage and route of administration. According to Schmeda-
Hirschmann and Yesilada (2005), the recommended doses
range to be used when studying the in vivo gastroprotective
potential of plant extracts prepared from single herbs or
herbal mixtures is between 100 and 300 mg/kg. As for the
pure compounds, the proposed doses range was between 50
and 300 mg/kg. Of all the pharmacological activities reported
above, only the antinociceptive, anti-inflammatory, antipyr-
etic, antiulcer, antidiabetic, antihypertensive, and cardiopro-
tective effects were performed using in vivo assays. Taking
these doses range as basis for all in vivo investigations cited in
this review, reports on antinociceptive using AEMCL,
CEMCL, and MEMCL at the doses ranging between 27 and
270, 50 and 500, and 100 and 500 mg/kg, respectively, are
considered acceptable (Sani et al., 2012; Zakaria et al., 2006a,
2007d–f). Therefore, these findings support the traditional
uses of M. calabura in the treatment of headache and
stomachache. In addition, the anti-inflammatory, antipyretic,
and antiulcer studies by Zakaria et al. (2007a,f), Preethi et al.
(2012a, b), Ibrahim et al. (2012), and Balan et al. (2013),
which used AEMCL, AEMCFr, MEMCFr, EEMCL, and
MEMCL at the doses range of 27–270, 200, and 400, 100–
300, 250, and 500, and 25–500 mg/kg, respectively, were also
acceptable and supported the traditional claims of using the
plant to reduce cold, gastric ulcer, and swelling of the prostate
gland. Other than the above studies, the antidiabetic (Sridhar
et al., 2011), antihypertensive (Shih, 2006, Shih et al., 2009),
and cardioprotective (Nivethetha et al., 2009) reports, which
used MEMCL (300 and 500 mg/kg), WSF (10–100 mg/kg),
and BSF (10–100 mg/kg) of MEMCL, or AEMCL (200–
300 mg/kg), were carried out according to the doses range
suggested by Schmeda-Hirschmann and Yesilada (2005).
Another opinion that can be taken into consideration when
discussing about the appropriate dose to be given to the
laboratory animals was outlined by Food and Drug
Administration (FDA) (2010). Using the maximum tolerated
dose (MTD) as a guideline, which suggests 1000 mg/kg/d as
the limit for acute, sub-chronic and chronic toxicity studies in
rodents and non-rodents, the doses regime selected for the
in vivo studies, which range between 27 and 500 mg/kg was
considered acceptable.
Therefore, the extracts of M. calabura as described above
can be proposed to possess in vivo antinociceptive, anti-
inflammatory, antipyretic, antiulcer, antidiabetic, antihyper-
tensive, and cardioprotective activities. However, it is still
Pharm Biol, 2014; 52(12): 1598–1623
inappropriate and difficult to associate the selected doses
regime described above to the actual amount of plant extract
used in traditional medicine since different methods of
medicinal plant preparations were used by different tribes or
ethnic groups. For example, the Peruvian used the leaves
boiled in water for the treatment of headache and gastric
ulcer while the flowers and bark were used to reduce swelling
in the lower extremities. As comparison, the people of
Philippines used the flowers to treat headache and incipient
cold while the people of Colombia used the flowers as a
tranquillizer and tonic. With regard to the route of adminis-
tration used, the in vivo studies described above used various
ways to administer the extracts such as oral (acute toxicity,
antiulcer, antidiabetic, and antinociceptive) (Ibrahim et al.,
2012; Sridhar et al., 2011; Yusof et al., 2011), subcutaneous
(antinociceptive, anti-inflammatory, and antipyretic) (Zakaria
et al., 2006a, 2007a,b, 2008), intraperitoneal (antinociceptive
and anti-inflammatory) (Preethi et al., 2012; Sani et al.,
2012), intravenous (hypotensive and cardioprotective)
(Nivethetha et al., 2009; Shih et al., 2006, 2009). Taking
into account that most medicinal plants are consumed orally,
only investigations on the acute toxicity, antiulcer, antidia-
betic, and antinociceptive activities were found to emulate the
traditional ways of consuming medicinal plants.
The rest of the pharmacological activities were studied
using in vitro techniques and, thus, the reports made should be
interpreted with caution to avoid making false conclusion on
the effectiveness of certain medicinal plants. For any in vitro
technique, any compounds/extracts assayed should demon-
strate significant EC50 or IC50 values of less than or equal to
30 mg/mL (30 mg/mL) for them to be regarded as active
(Meyer et al., 1982). Based on the literature review, several
bioactive compounds have been identified to have potential
cytotoxic activity against various types of cancerous cells
based on the fact that their recorded ED50 values were
30 mg/mL. For the cytotoxicity studies, (i) only compounds
1–7, 9–12, 14, 28, 36, 37, 40, 41, 45–47, 50-52, 54–59, and
61–64 were cytotoxic towards P-388; (ii) only compounds 1–9
and 11 were cytotoxic towards KB and ME-12 while for KBV,
only compounds 1–5, 7–9, and 11 were cytotoxic; (iii) only
compounds 3, 9, and 11 exerted cytotoxic effect against BC-1,
HT-1080, and Lu1, respectively, and; (iv) only compounds 2,
3, and 8–12 were cytotoxic towards CO-12. Moreover,
compounds 4, 14, 28, 52, 54–59, and 61–64 were cytotoxic
towards HT-29 with compound 4 also cytotoxic against A549
cells (Chen et al., 2004, 2005; Kaneda et al., 1991; Su et al.,
2003). Of all reports on cytotoxic investigations cited above,
only Sufian et al. (2013) examined the cytotoxic activity in a
proper manner, a process known as bioassay-guided fraction-
ation, whereby the investigation was carried out beginning
with the extract (MEMCL) followed by its partitions (PEE,
EAE, and WEE) and the fractions against MCF-7, HL-60,
HCT-116, and WRL-68 cell lines. The MEMCL was effective
only against the HL-60 cells (IC50 ¼ 30.9 mg/mL) while the
PEE and EAE, but not WEE, was also effective only against
the HL-60 cells with the recorded IC50 of 29.5 and 17.3 mg/
mL, respectively. Fractionation of EAE leads to the isolation
of seven fractions, labeled as F1–F7, of which only F5, F6,
and F7 were effective against the HL-60 cells with the
recorded IC50 of 4.0, 6.0, and 28.1 mg/mL, respectively. In
DOI: 10.3109/13880209.2014.908397
addition, only the F4 was effective against MCF-7 with the
recorded IC50 of 30.8. However, only the F5 was selected for
isolation and identification of bioactive compounds, which
led to the identification of compounds 28, 59, 85, and 86.
Cytotoxicity study demonstrated that only compounds 28 and
85 exerted potential cytotoxic activity against HL-60 (IC50 is
3.4 and 3.3 mg/mL) and, in addition, MCF-7 (IC50 is 11.8 and
18.9 mg/mL).
In the in vitro antiproliferative study, (i) AEMCL was
effective only against MCF-7, K-562, and HT-29 with IC50
values ranging between 16 and 18 mg/mL; (ii) CEMCL was
effective only against MCF-7, HeLa, and HL-60 with IC50
values ranging between 22 and 29 mg/mL, and; (iii) MEMCL
was effective only against HeLa, and HL-60 with the recorded
IC50 values of 23.0 and 7.0 mg/mL. As for the antiplatelet
investigation, only compound 78 can be confirmed to possess
that activity, as at 20 mg/mL, the antiplatelet activity recorded
was above 80%. For the other compounds, it is difficult
to suggest on their effectiveness as they were tested at
the concentrations that were above 30 mg/mL (e.g., 50 and
100 mg/mL). Moreover, only two concentrations were used,
which was not sufficient for calculation of IC50. Despite
several antibacterial reports on various extracts and parts of
M. calabura, this activity should be ignored or removed from
the list of scientific findings of the plant. The reason was
based on the findings in all reports that the antibacterial
activity was observed at a very high dose and that the MIC
and MBC values recorded were above 30 mg/mL.
In addition to the antioxidant assays, the TPC value has
been generally accepted to reflect the antioxidant capacity of
the extracts/compounds. Any extracts/compounds with the
TPC value of 1000 mg GAE/100 g FW could be considered
as having a high TPC value. Of the various extracts of M.
calabura fruits, only the MEMCFr and EAEMCFr have been
reported to contain high TPC value. Although the TPC value
of MEMCL had been determined by Siddiqua et al. (2010), it
was not clearly expressed in the unit of mg GAE/100 g FW.
The TPC value was assessed using the Folin–Ciocalteau
method with gallic acid and tannic acid as the calibration
standard and the values recorded was 0.903 and 2.900,
respectively. The authors also failed to describe the range of
value for any extracts/compounds to be considered as having a
high TPC value. In another study by Balan et al. (2013), the
TPC values of 6.25 mg/mL MEMCL, AEMCL, and CEMCL
were recorded to be above 1000 mg GAE/100 g FW indicating
their high antioxidant capacity. In another study by Zakaria
et al. (2007b), the AEMCL was found to exert high
antioxidant activity when measured using the DPPH and
superoxide radical scavenging with the recorded percentage
of inhibition of 94.8% and 83.7%, respectively. However, no
concentration of extract was given in this study, which made
the validation of AEMCL antioxidant activity less convincing.
Furthermore, Preethi et al. (2010) reported on the antioxidant
potential of various extracts of M. calabura fruits (e.g.,
MEMCFr, HEMCFr, EAEMCFr, CEMCFr, and BEMCFr)
should also be interpreted with cautious despite the results
obtained using various models of antioxidant assays. In the
DPPH-, superoxide anion-, hydroxyl-, nitric oxide-radical
scavenging assay, all extracts exerted antioxidant effect with
the recorded IC50 values ranging between 90.0 and 351.0,
Review on M. calabura medicinal value 1621
79.0 and 379.0, 49.0 and 281.0, and 187.0 and 498.0 mg/mL in
comparison with BHT, which recorded the respective IC50 of
26.0, 83.0, 41.0, and 33.0 mg/mL. Moreover, in the ferric ion
chelating assay, all extracts recorded an activity with IC50
values ranging between 80.0 and 481.0 mg/mL while in the
LPO inhibitory assay all extracts exerted an activity with
recorded IC50 values ranging between 110.0 and 541.0 mg/
mL. No comparison was made against the reference drug
effect in the ferric ion chelating and LPO inhibitory assays.
In all the assays used, the IC50 value recorded for each of
the extract was430 mg/mL for the extracts to be considered as
effective antioxidants. However, it is important to highlight
that, except for the DPPH radical scavenging assay, the IC50
value of the reference drug, BHT, for the rest of the assays,
was also430 mg/mL. Further studies by Siddiqua et al. (2010)
on the MEMCL antioxidant activity using the DPPH radical
scavenging assay seem to confirm earlier report on the highest
antioxidant potential of AEMCL (Zakaria et al., 2007b). The
IC50 value recorded for MEMCL (22.0 mg/mL) and ascorbic
acid (the reference drug; 10 mg/mL) was 30 mg/mL and,
thus, fulfilled the FDA requirement. Taking into account that
the ascorbic acid produced an IC50 value that is approximately
two-fold lower than that of the BHT (12 mg/mL versus 26 mg/
mL) when assessed using the DPPH radical scavenging assay,
it is suggested that Preethi et al. (2010) should have used more
than one reference drug (e.g., ascorbic acid) for the compari-
son purposes. Unfortunately, adding the number of reference
drugs will not help to contradict the fact that the fruits and its
extracts did not meet the FDA requirements to be considered
effective antioxidants due to the high IC50 recorded. The
report by Zakaria et al. (2011) demonstrated that the
20–500 mg/mL MEMCL exerted the highest percentage of
radical scavenging activity followed by the AEMCL and
CEMCL when assessed using the DPPH- and superoxide
anion-radical scavenging assays. Despite using three concen-
trations in their studies, no attempt to extrapolate the IC50
value was done. However, these findings have also further
supported the previous reports by Zakaria et al. (2007d) and
Siddiqua et al. (2010). Lastly, Karthyaini and Suresh (2012)
also reported on the antioxidant potential of M. calabura
fruits while studying the fruits’ anti-inflammatory activity.
Although two types of extracts, MEMMFr and AEMCFr, were
used in the anti-inflammatory studies, they were not described
anywhere in the antioxidant study. To make things worse, the
antioxidant data presented was only for one extract, which is
not described specifically. The authors used very high doses
ranging between 100 and 500 mg/mL and the IC50 recorded
was approximately 90 mg/mL. These findings further support
our comment that the fruits of M. calabura, despite having
antioxidant activity, were not potent antioxidant agent.
In the investigation of potential of QR induction, several
bioactive compounds were isolated from the leaves of M.
calabura and tested using the cultures mouse Hepa IcIc7
cells. Of all compounds tested for CD, IC50, and CI, only
compounds 13, 17, 18, 22, 23, 24, and 25 exerted significant
QR induction activity. However, based on the IC50 evaluation,
only compounds 13, 17, and 24 could be considered as safe
due to the higher IC50 values (420 mg/mL). The final in vitro
activity reported on M. calabura up to this moment is the
insecticidal activity by Bandeira et al. (2013). Using EEMCFl,
1622 N. D. Mahmood et al.
EEMCFr, HEMCFl, and HEMCFr, the insecticidal activity
was determined against P. xylostella larvae and pupae
using leaf disc immersion assay at the concentrations range
(0.25–30.0 mg/mL) accepted within the FDA procedures. All
extracts exerted toxicity effects against the first instar
P. xylostella larvae with the IC50 recorded within the range
of 0.61–18.9 mg/mL. In comparison, cordycepin, the reference
drug, caused 100% mortality at 500 mg/mL. Moreover, upon
assessment of mortality against pupae, the HEMCFr exerted
the highest mortality against P. xylostella pupae followed
by the EEMCFr, HEMCFl, and EEMCFl and the IC50
range recorded was between 3.5 and 30 mg/mL. Based
on the recorded IC50, the extracts of fruits and flowers of
M. calabura could be proceded for further investigations as a
potential insecticidal agent. Overall, some of the in vitro
pharmacological activities cited above should be ignored due
to several factors such as the use of unrealistic doses range
and failure to properly compare the respective activity against
a proper reference drug. Moreover, the failure to extrapolate/
measure the IC50 for any in vitro studies, which could be due
to negligence or insufficient doses range used, should also be
taken into consideration as the IC50 value will directly
indicates the extracts/compounds potential to be considered as
the lead for future drug development based on the FDA
requirement.
Despite all these negative factors and the consideration that
should be taken in analyzing and accepting the respective
pharmacological claim, factor such as route of administration
should also be taken into consideration. Considering the fact
that the medicinal plants are normally taken orally, some of
the potential pharmacological activities reported in this
review paper (e.g., antinociceptive, anti-inflammatory, anti-
pyretic, antihypertensive and cardioprotective) were not
determined via oral administration. Although some of these
positive findings (e.g., antihypertensive and cardioprotective)
did not reflect traditional uses of the M. calabura, their
significant contribution to the drug discovery field should not
be denied. Moreover, these activities would not have been
observed if the extracts were not administered intravenously.
For example, the ability of WSF and BSF derived from
MEMCL to exhibit antihypertensive or AEMCL to exert
cardioprotective activities indirectly indicates the extracts
ability to produce the said activities in their crude form if
administered directly into the blood stream (intravenously).
This review paper aimed to summarize the progress related
to medicinal research of M. calabura for the past 22 years
since the first scientific publication in 1991. Various scientific
and non-scientific literature related to M. calabura were
analyzed to collect all information related to the traditional
uses, phytochemistry and, in vitro and in vivo pharmaco-
logical activities of M. calabura. Although various scientific
papers were published to report on the pharmacological
properties of M. calabura (30 journals), detail and careful
analysis should be carried out on the results obtained before
the reported pharmacological effects could be accepted. This
is to make sure that the procedures taken to carry out the
experiments were acceptable and within the requirement of
the regulatory bodies such as the FDA.
Concurrent with the lack of ethnomedicinal uses of
M. calabura as described in the Ethnobotanical section, the
Pharm Biol, 2014; 52(12): 1598–1623
therapeutics efficacy of this plant has not been fully studied
thoroughly. Moreover, the pharmacological potential of those
isolated bioactive compounds and their contribution towards
the claimed medicinal uses are not fully studied. Therefore,
the search for bioactive compounds from M. calabura
with specific pharmacological activity remains unsettled.
It is suggested that research should currently focus on the
isolation and identification of new bioactive compounds, and
collection of known bioactive compounds for their pharma-
cological potential could be investigated thoroughly if they
are to be developed as candidates for new drug development
in the future. In conclusion, it is hoped that this review will
serve as an encouragement for others to further explore the
pharmacological potential of M. calabura with the goal of
developing it as a new therapeutic agent.
Acknowledgements
The authors thank the Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, Malaysia, for providing
the facilities to carry out this study.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article. This
study was supported by the Science Fund Research Grant
(Reference no. 06-01-04-SF1127) awarded by the Ministry of
Science Technology and Innovation (MOSTI), Malaysia and
the Research University Grant Scheme (Reference no. 04-02-
12-2019RU) from the Universiti Putra Malaysia, Malaysia.
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