Muntingia calabura: a review of its traditional uses, chemical properties, and pharmacological observations.

http://informahealthcare.com/phb 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 Pharmaceutical Biology Downloaded from informahealthcare.com by University of Waterloo on 01/07/15 For personal use only.

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, have been reported to possess medicinal value, supported by a number of scientific studies. Objective: To gather all information related to the ethnomedicinal uses, phytochemical compositions, and pharmacological activities of M. calabura and present them as a comprehensive and systematic review article. 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 Google and Yahoo search engines by applying some related search terms (e.g., Muntingia calabura, phytochemical, pharmacological, extract, and traditional uses). The articles related to agriculture, ecology, and synthetic work and those using languages other than English or Malay 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.

Elaeocarpaceae, ethnomedicinal uses, pharmacological activities, phytoconstituents

Introduction 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: [email protected]

History Received 19 July 2013 Revised 10 February 2014 Accepted 22 March 2014 Published online 25 July 2014

Muntingia calabura is known throughout the world as ‘‘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

Review on M. calabura medicinal value

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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 approximately 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 infectious and non-infectious, nowadays have become a major

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 antibiotic-resistance bacteria such as methicillin- and vancomycin-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), particularly the plant-based medicines, has been one of the sources of the CAM used. One of the plants that has

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Table 1. The vernacular names of M. calabura.


Vernacular names Kerukup siam Singapore cherry, Jamaican cherry, Panama berry, cotton candy berry, glade mallow, glademallow Pau de seda, calabura Kersen, talok Bolaina, yamanaza, cacaniqua, capulıń blanco, nigua, niguito, memizo or memiso Alatris, aratilis, manzanitas, sarisa, cereza Puán, Capulin rojo Gasagase hanninamara Tru´’ng ca´, mat sam; cay trung ca Takhop farang krakhob barang Bois ramier, bois de soil, bois Paú de seda, calabura Bolaina, yamanaza, bolina, lumanasa Capulina, guasimo bobo, memiso Majuaguito, majanjo, chitato, chitoto

Country

References

Malaysia Britain

Zakaria et al. (2006a, 2007c, 2008, 2009, 2010, 2011) Neto Bandeira et al. (2013)

Brazil Indonesia Spain

Neto Bandeira et al. (2013) Wikipedia (2013) Wikipedia (2013) and Janick and Paull (2008)

Philippines Mexico India Vietnam Thailand Cambodia France Portugal Peru Cuba Colombia

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)

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 medicinal 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

(2), (2S)-20 -hydroxy-7,8,30 ,40 ,50 -pentamethoxyflavan (3), (2S)-50 -hydroxy-7,8,30 ,40 -tetramethoxyflavan (4), (2S)-8hydroxy-7,30 ,40 ,50 -tetramethoxyflavan (5), (2S)-8,20 -dihydroxy-7,30 ,40 ,50 -tetramethoxyflavan (6), (2S)-8,50 -dihydroxy7,30 ,40 -trimethoxyflavan (7), 7,8,30 ,40 ,50 -pentamethoxyflavone (8), (M),(2S),(200 S)-,(P),(2S),(200 S)-8,800 -50 -trihydroxy-7,70 0 000 0 000 000 3 ,3 -4 ,4 -5 -heptamethoxy-5,500 -biflavan (9), 50 -hydroxy7,8,30 40 -tetramethoxyflavone (10), (M),(2S),(200 S)-,(P),(2S), (200 S)-8,800 -50 -5000 -tetrahydroxy-70 ,700 -30 ,3000 -40 ,4000 -hexamethoxy50 ,5000 -biflavan (11), and 8,50 -dihydroxy-7,30 ,40 -trimethoxyflavone (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-hydroxyflavone (18), 5,7-dihydroxyflavone (chrysin, 19), 3-methoxy-5,7,40 -trihydroxyflavone (isokaemferide, 20), 3,30 -dimethoxy-5,7,40 -trihydroxyflavone (21), 3,8dimethoxy-5,7,40 -trihydroxyflavone (22), 3,5-dihydroxy7,40 -dimethoxyflavone (ermanin, 23), 3,5-dihydroxy-7, 8-dimethoxyflavone (gnaphaliin, 24), 5-hydroxy-3,7,8-trimethoxyflavone (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 (isoliquiritigenin, 29), 7-hydroxyisoflavone (30), 7,30 ,40 -trimethoxyisoflavone (cabreuvin, 31), (2S)-50 -hydroxy7,8,30 ,40 -tetramethoxyflavan (4), 20 ,40 -dihydroxydihydrochalcone (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 CHCl3soluble 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 -trimethoxyflavone (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), syringic acid (44), vanillic acid (45), 3-hydroxy-1-(3,5-dimethoxy4-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 H2Osoluble fractions were then subjected to the isolation procedures. From this study, 20 compounds were isolated of which four were new compounds (20 ,40 -dihydroxy-30 -methoxydihydrochalcone (50), ()-30 -methoxy-20 ,40 ,b-trihydroxydihydrochalcone (51), (2S)-()-50 -hydroxy-7,30 , 0 4 -trimethoxyflavanone (52), and 8-hydroxy-10-methoxy5H-isochromeno[4,3-b]chromen-7-one (muntingone, 53) while the remaining 16 were known compounds [7-hydroxyflavanone (14), 20 ,40 -dihydroxychalcone (28), 20 ,40 -dihydroxydihydrochalcone (32), 6,7-dimethoxy-5-hydroxyflavone (38), 3,5-dihydroxy-6,7-dimethoxyflavone (40), 5-hydroxy7-methoxyflavone (54), 3,7-dimethoxy-5-hydroxyflavone (55), 5-hydroxy-3,6,7-trimethoxyflavone (56), 3,5-dihydroxy-7-methoxyflavone (57), 8-methoxy-3,5,7-trihydroxyflavone (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 -dihydroxy-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 prepared from the MEMCL as described earlier (Chen et al., 2005). Of the isolated compounds, three were new compounds [2,3-dihydroxy-4,30 ,40 ,50 -tetramethoxydihydrochalcone (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), 7methoxyflavone (69), 5,7-dihydroxy-3-methoxyflavone (70), 5,7-dihydroxy-6-methoxyflavone (71), 5,40 -dihydroxy-3,7dimethoxyflavone (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-nitrophenol (77), methyl gallate (78), trans-methyl p-coumarate


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(79), b-amyrenone (80), a-tocopherylquione (81), d-tocopherol (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 properties 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 effectiveness, the EAE was chosen for further fractionation procedures, 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-trihydroxy-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 partitions, 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 identification 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-5hydroflavone (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 pharmacological value of M. calabura through rigorous scientific investigations has been taken by researchers all over the world. Despite the differences in term of cultural, geographical, 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 observations 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,

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Table 2. List of new bioactive compounds isolated from M. calabura according to the extracts used. Name of compound 0

Structure 0

0

(2S)-5 -Hydroxy-7,3 ,4 -trimethoxyflavan (1)

O

CH3

Types of extract

Reference

MEMCR

Kaneda et al. (1991)

CH3 O

O

O

H3C OH

(2S)-7,8,30 ,40 ,50 -pentamethoxyflavan (2) O

CH3


H3C

O O O

CH3 O

CH3

CH3 0

0

0

0

(2S)-2 -hydroxy-7,8,3 ,4 ,5 -pentamethoxyflavan (3)

H3C O

CH3

HO

CH3

O

O O

CH3

O O

H3C

(2S)-50 -Hydroxy-7,8,30 ,40 -tetramethoxyflavan (4)

O H3C O O O

H3C O

O

CH3

O

CH3

CH3

(2S)-8-Hydroxy-7,30 ,40 ,50 -tetramethoxyflavan (5)

O

CH3

OH O

CH3 O

O

H3C O

(2S)-8,20 -Dihydroxy-7,30 ,40 ,50 -tetramethoxyflavan (6)

CH3

H3C

O HO

O

OH O H3C

CH3 CH3

O O

(continued )


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Table 2. Continued

Name of compound 0

Structure 0

Types of extract

Reference

0

(2S)-8,5 -Dihydroxy-7,3 ,4 -trimethoxyflavan (7)

OH OH OH O

O

CH3 O

H3C


7,8,30 ,40 ,50 -Pentamethoxyflavone (8)

O

CH3 CH3

O O

O O

H3C O

CH3 O CH3

(M),(2S),(200 S)-,(P),(2S),(200 S)8,800 -50 -Trihydroxy-7,700 -30 ,3000 40 ,4000 -5000 -heptamethoxy-5,500 biflavan (9)

O

CH3

OH O

CH3 O

O

H3C OH

O

CH3 CH3

O

O OH

CH3

O

50 -Hydroxy-7,8,30 40 -tetramethoxyflavone (10)

CH3

O H3C

OH O O

H3C

O O

O

CH3

CH3

(continued )

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Table 2. Continued

Name of compound 00

Structure

Types of extract

Reference

Ethyl acetate-soluble partition of MEMCL

Su et al. (2003)

CHCl3-soluble partition of MEMCSB

Chen et al. (2004)

00

(M),(2S),(2 S)-,(P),(2S),(2 S)8,800 -50 -5000 -Tetrahydroxy-70 ,700 30 ,3000 -40 ,4000 -hexamethoxy50 ,5000 -biflavan (11)

CH3

O OH

CH3

O

O

O

H3C OH

OH CH3


O

O OH

CH3

O

8,50 -Dihydroxy-7,30 ,40 -trimethoxyflavone (12)

CH3

H3C O OH O

O

O

H3C

CH3 OH O

(2R,3R)-7-Methoxy-3,5,8trihydroxyflavanone(13)

OH O

O H3C

OH O

OH 0

0

0

8-Hydroxy-7,3 ,4 ,5 -tetramethoxyflavone (36)

CH3 O CH3

O OH O

O O

H3C

CH3 CH3 O

8,40 -Dihydroxy-7,30 ,50 -trimethoxyflavone (37)

CH3 O OH OH O

O O

H3C

CH3 CH3 O

(continued )


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Table 2. Continued

Name of compound 0

0

Structure

0

2 ,4 -Dihydroxy-3 -methoxydihydrochalcone (50) HO

Types of extract

Reference

CHCl3-soluble partition and, n-BuOH-soluble and H2O-soluble fractions of MEMCL

Chen et al. (2005)

CHCl3-soluble partitions and n-BuOH-soluble fraction of MEMCL

Chen et al. (2007)

O CH3 0

0

OH

O

0

()-3 -Methoxy-2 ,4 ,b-trihydroxydihydrochalcone (51)


HO

HO

O CH3 0

0

OH

O

0

(2S)-()-5 -Hydroxy-7,3 ,4 -trimethoxyflavanone (52)

H3C O

CH3 O

O

O

CH3 O

H3C

O

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

O

H3C

O OH 0

0

O

0

2,3-Dihydroxy-4,3 ,4 ,5 -tetramethoxydihydrochalcone (65)

CH3 H3C

O O

O

H3C

OH OH O O

CH3

4,20 ,40 -Trihydroxy-30 -methoxydihydrochalcone (66)

OH

HO

O CH3

O

(continued )

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Table 2. Continued

Name of compound

Structure

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

Types of extract

Reference

PEP of MEMCL

Yusof et al. (2011)

CH3 O

H3C O

OH OH

8-Hydroxy-6-methoxyflavone (calaburone, 87)

O

OH


O

O 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 Acute toxicity

Cytotoxic

Antiproliferative

Insecticidal

Hypotensive

Part

Type of extract

Location of collection

Period of collection

References

Leaves

MEMCL,

NG

Sridhar et al. (2011)

Leaves Fruits

NG NG

Ibrahim et al. (2012) Karthyaini and Suresh (2012)

Leaves

EEMCL, Not clear; possibly EEMCFr MEMCL

Station Ghanpur, Warangal, Andhra Pradesh, India Selangor, Malaysia NG

Roots Stem barks Leaves Leaves

MEMCR MEMCSB MEMCL MEMCL

Leaves

MEMCL

Leaves

CEMCL

Leaves

AEMCL

Flowers

EEMCFl

Fruits

EEMCFr

Flowers

HEMCFL

Fruits

HEMCFr

Leaves

MEMCL fractionated sequentially using a mixture of dH2O and n-butanol Butanol-soluble fraction (BSF) MEMCL partitioned using dH2O MEMCL partitioned using chloroform

Leaves Leaves Leaves

Shah Alam, Selangor, May and August 2010 Malaysia Sarabuti Province, Thailand NG Kaohsiung City, Taiwan June 2001 Kaohsiung City, Taiwan June 2001 Shah Alam, Selangor, January 2008 Malaysia Shah Alam, Selangor, August and September 2006 Malaysia Shah Alam, Selangor, August and September 2006 Malaysia Shah Alam, Selangor, August and September 2006 Malaysia Universidade Rural Federal July 2008 de Pernambuco (UFRPE), Recife, Brazil Universidade Rural Federal July 2008 de Pernambuco (UFRPE), Recife, Brazil Universidade Rural Federal July 2008 de Pernambuco (UFRPE), Recife, Brazil Universidade Rural Federal July 2008 de Pernambuco (UFRPE), Recife, Brazil Kaohsiung City, Taiwan June 2001

Balan et al. (2013) Kaneda et al. (1991) Chen et al. (2004) Chen et al. (2005) Sufian et al. (2013) Zakaria et al. (2011) Zakaria et al. (2011) Zakaria et al. (2011) Bandeira et al. (2013) Bandeira et al. (2013) Bandeira et al. (2013) Bandeira et al. (2013) Shih et al. (2006)

Kaohsiung City, Taiwan

NG

Shih (2009)


NG

Shih (2009)


NG

Shih (2009)

(continued )


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Table 3. Continued Activity

Part

Type of extract



References

Leaves

AEMCL

January and February 2004

Zakaria et al. (2006a)

Leaves

AEMCL


Zakaria et al. (2007e)

Leaves

AEMCL

July and August 2005

Zakaria et al. (2007d,f)

Leaves

CEMCL


Zakaria et al. (2007a)

Leaves

MEMCL

January 2008

Yusof et al. (2011)

Leaves

PEP partition

January 2008

Yusof et al. (2011)

Leaves

EAP partition

January 2008

Yusof et al. (2011)

Leaves

AQP partition

January 2008

Yusof et al. (2011)

Leaves

MEMCL

NG

Sani et al. (2012)

Leaves Leaves

AEMCL CEMCL

NG July and August 2005

Nivethetha et al. (2009) Zakaria et al. (2007a)

Leaves

AEMCL


Zakaria et al. (2007b)

Leaves Leaves Leaves Leaves

Methanol CHCl3 BuOH AEMCL

2001 2001 2001 June and September 2005

Chen et al. (2007) Chen et al. (2007) Chen et al. (2007) Zakaria et al. (2007b)

Fruits

HEMCFr

May and June 2008

Preethi et al. (2010)

Fruits

CEMCFR

May and June 2008


Fruits

EAEMCFR

May and June 2008


Fruits

BEMCFR

May and June 2008


Fruits

MEMCFR

May and June 2008


Leaves Leaves

MEMCL AEMCL

March 2009 August and September 2006

Siddiqua et al. (2010) Zakaria et al. (2011)

Leaves

MEMCL

August and September 2006

Zakaria et al. (2011)

Leaves

CEMCL

August and September 2006


Fruits Leaves

NG AEMCL

NG July and August 2005

Karthyaini and Suresh (2012) Zakaria et al. (2007b)

Fruits

MEMCFr

NG


Anti-diabetic

Fruits Fruits Leaves

MEMCFr AEMCFr MEMCL

NG NG September

Karthyaini and Suresh (2012) Karthyaini and Suresh (2012) Sridhar et al. (2011)

Antiulcer

Leaves

EEMCL

NG

Ibrahim et al. (2012)

Leaves

MEMC

May–August 2010

Balan et al. (2013)

Leaves

AEMCL



Leaves

MEMCL



Leaves

CEMCL



Leaves

MEMCL

Between 2000 and 2003

Yasunaka et al. (2005)

Fruits

MEMCFr

Between 2000 and 2003

Yasunaka et al. (2005)

Leaves

AEMCL

June 2006

Zakaria et al. (2007c)

Leaves

CEMCL

Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia NG Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Kaohsiung City, Taiwan Kaohsiung City, Taiwan Kaohsiung City, Taiwan Shah Alam, Selangor, Malaysia Erode District, Tamil Nadu, India Erode District, Tamil Nadu, India Erode District, Tamil Nadu, India Erode District, Tamil Nadu, India Erode District, Tamil Nadu, India Bangalore, Karnakata Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia NG Shah Alam, Selangor, Malaysia Erode district Tamilnadu, India NG NG Roman Catholic Church, Station Ghanpur, Warangal, Andhra Pradesh, India Ethno Resources Sdn. Bhd, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Cuetzal’an del Progreso in the State of Puebla Cuetzal’an del Progreso in the State of Puebla Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia

June 2006



Antinociceptive

Cardioprotective Antipyretic

Antiplatelet aggregation Antioxidant

Anti-inflammation

Antibacterial

(continued )

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Pharm Biol, 2014; 52(12): 1598–1623

Table 3. Continued


Activity

Part

Type of extract



References

Leaves

MEMCL

June 2006


Leaves

AEMCL

January 2008


Leaves

CEMCL

January 2008


Leaves

MEMCL

January 2008


Leaves

PEEMCL

January 2008


Leaves

EAEMCL

January 2008


Leaves

WEEMCL

January 2008


Leaves

MEMCL

NG

Sufian et al. (2013)

Leaves

PEEMCL

NG


Leaves

EAEMCL

NG


Leaves

WEEMCL

NG


Leaves Barks Fruits Leaves Barks Fruits

AEMCL AEMCB AEMCFr MEMCL MEMCB MEMCFr

Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Shah Alam, Selangor, Malaysia Bengaluru, Karnataka, India Bengaluru, Karnataka, India Bengaluru, Karnataka, India Bengaluru, Karnataka, India Bengaluru, Karnataka, India Bengaluru, Karnataka, India

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 administered 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 histopathological, 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

NG NG NG NG NG NG

Sibi Sibi Sibi Sibi Sibi Sibi

et et et et et et

al. al. al. al. al. al.

(2012) (2012) (2012) (2012) (2012) (2012)

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 carcinoma), 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, respectively. 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 mithramycin (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 HT29 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 MCF7, 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


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 compounds, 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 concentrations 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-MB231 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 antiproliferative 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), halfmaximal 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



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 aggregation 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 concentration 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 rhizophila, 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 antistaphylococcal 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 partitioning 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. aeruginosa, 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 microdilution broth method. The MEMCL exerted notable antibacterial 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


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, respectively. 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, respectively. 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 radicaland 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 concentrations of 500, 400, 300, 200, and 100 mg/mL, exerted significant DPPH radical quenching property as indicated


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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 investigation 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 scavenging 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 comparison 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

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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 demonstrated 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-inflammatory and antioxidant studies, only the data of the antiinflammatory 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 toxicities, 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 acidinduced 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 pretreatment 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 Larginine/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.


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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 estimated 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, pretreatment 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-inflammatorymediated/neurogenic pain). Following their success in establishing the antinociceptive profiles of CEMCL and AEMCL, and the involvement of Larginine/NO/cGMP pathway and opioid receptors in modulating the antinociceptive activity of AEMCL, Zakaria et al. (2007d) took another step forward to determine the involvement of non-opioid receptor systems in the central antinociceptive 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


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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 antinociceptive 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 nonopioid 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), dopaminergic (1 mg/kg haloperidol), and g-aminobutyric acid (GABA; 10 mg/kg bicuculline) receptors. From the results obtained, atropine, phenoxybenzamine, yohimbine, pindolol, haloperidol, 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 antagonist, 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 concentrations 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-dependent 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-adrenergic receptor antagonist), and atropine (muscarinic receptor antagonist). In the third study, the antinociceptive activity of

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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 combination 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 morphine 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, glutamatergic 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 percentage 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 dosedependent antinociceptive activity against the glutamateinduced 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 antinociceptive potential of M. calabura leaves also described the successful isolation and identification of pain-relieving bioactive 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% DMSOtreated) 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


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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 formalininduced 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 antiinflammatory 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 antinociceptive 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 antiinflammatory activity of 100% AEMCL was lost after only 6 h of its administration. It is worth mentioning that the antiinflammatory 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 carrageenaninduced 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

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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 dosedependent anti-inflammatory activity indicated by the reduction in edema formation irrespective of the dose used. At the doses tested, a dose-dependent inhibition of carrageenaninduced localized edema was observed at 4 h. However, the activity seen with MEMCFr, at all doses (percentage of antiinflammation 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.

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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 concentrations of 10, 50, and 100%, was tested using Brewer’s yeast (BY)-induced pyrexia test. The extract exhibited a concentration-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 administration. Overall, the antipyretic activity of AEMCL was less effective than the reference drug, 100 mg/kg ASA.

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 dosedependent 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 participation of endogenous sulfhydryl compounds in the gastroprotective activity demonstrated by MEMCL.

Antiulcer activity

Antidiabetic 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/

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

Antipyretic activity

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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 pretreatment 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, electrolytes, 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 pretreatment 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


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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 hypotension 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 involvement 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 characterized by immediate but transient decreases in MSAP and HR, which returned to pre-injection baseline within 2–3 min postinjection. In contrast, the SHR with established hypertension demonstrated initial decreases in MSAP and HR, which was followed by a delayed phase of vasodepressor and bradycardiac 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 significant MSAP reduction in WKR and SHR with a peak detected within the first 30 min and prolonged for less than 5 min posttreatment. In addition, pre-treatment with L-NAME significantly attenuated the hypotensive and bradycardia effects of BSF in the normotensive WKR whereas, in the SHR, L-


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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 intravenously, 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 pretreatment with 0.2 mg/kg/min ODQ, a sGC inhibitor, or 7 mg/ kg/min KT5823, a PKG inhibitor, caused significant attenuation 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 bradycardiac effects through the activation of NO-dependent cGC/ cGMP/PKG signaling pathways. Furthermore, the eNOSderived 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 infarction 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 conventionally 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

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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 medicinal 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 bioassayguided fractionation. Several drugs that are currently available 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 evidencebased 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 symptomatologies 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 duration 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


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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 reproducibility. 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 reproducibility 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 semisynthesis 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


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mechanisms to prevent infection at any of its parts via accumulation of antimicrobial secondary metabolites (Gonza´lez-Lamothe et al., 2009). Furthermore, the insecticidal 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, antiproliferative 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 nontumor 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


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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 understanding 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 SchmedaHirschmann 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, antipyretic, antiulcer, antidiabetic, antihypertensive, and cardioprotective 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, antiinflammatory, antipyretic, antiulcer, antidiabetic, antihypertensive, and cardioprotective activities. However, it is still

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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 administration 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, antidiabetic, 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 demonstrate 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 fractionation, 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


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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,


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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 comparison 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 concentrations 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,


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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, antipyretic, 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 pharmacological 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 pharmacological 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. 04-0212-2019RU) from the Universiti Putra Malaysia, Malaysia.

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