Melastoma malabathricum (L.) Smith Ethnomedicinal Uses, Chemical Constituents, and Pharmacological Properties: A Review

Melastoma malabathricum L. (Melastomataceae) is one of the 22 species found in the Southeast Asian region, including Malaysia. Considered as native to tropical and temperate Asia and the Pacific Islands, this commonly found small shrub has gained herbal status in the Malay folklore belief as well as the Indian, Chinese, and Indonesian folk medicines. Ethnopharmacologically, the leaves, shoots, barks, seeds, and roots of M. malabathricum have been used to treat diarrhoea, dysentery, hemorrhoids, cuts and wounds, toothache, and stomachache. Scientific findings also revealed the wide pharmacological actions of various parts of M. malabthricum, such as antinociceptive, anti-inflammatory, wound healing, antidiarrheal, cytotoxic, and antioxidant activities. Various types of phytochemical constituents have also been isolated and identifed from different parts of M. malabathricum. Thus, the aim of the present review is to present comprehensive information on ethnomedicinal uses, phytochemical constituents, and pharmacological activities of M. malabathricum.


Introduction
Melastomataceae plants originate in the tropic and subtropic regions, with a total of more than 4000 species in the world. In the Southeast Asian region alone, the genus Melastoma comprises 22 species, 2 subspecies, and 3 varieties [1]. Malaysia, particularly, with a tropical climate, is home to at least 12 species, many of which are used by natives in folk medicine. One of the plants within the Melastomataceae family that have gained herbs status in the Malay folklore belief is Melastoma malabathricum Linn., which has been known to comprise two subspecies, namely, M. malabathricum L. ssp. malabathricum and M. malabathricum Linn ssp. normale [2].
In general, M. malabathricum is a small shrub commonly found in previously cleared land, waste places, and road-side throughout the Southeast Asian countries, including Malaysia [3]. It is native to tropical and temperate Asia and the Pacific Islands [4]. The plant is one of the most common weeds that grow wildly and abundantly throughout the tropics, especially in the moist areas, and can be found in the Indian Ocean Islands, throughout South and South-East Asia, China, Taiwan, Australia, and the South Pacific Ocean [5]. Throughout Malaysia, particularly, the plant is very common in the lowland and mountain forests, chiefly in open places. M. malabathricum has different vernacular names depending on the location (e.g., Malaysia, Indonesia, China, and India) where the plant was found and the communities or tribes (e.g., Malay, Chinese, and Indian) that used them traditionally for medicinal purposes [4,[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] (Table 1). 8 Koiam-pay-bang Bandarban (Marma tribe) Rahmatullah et al. [18] Kakkhu Netrakona (Garo tribe) Bangladesh Rahmatullah et al. [19] Aksio Chittaggong (Chakma, Murong, Tonchonga tribes) Rahmatullah et al. [20]  This showy bush of small trees rapidly colonises wastelands as their seeds are dispersed by birds. The characteristics of M. malabathricum include its average height of 0.5-1(−5) m high but may occasionally grow up to 5 m long ( Figure 1). The stems are 4-sided to subterete, generally bristly, covered with small rough scales, and reddish. Branchlets are numerous, procumbent, densely covered with appressed scales. The petiole is approximately 0.5-1.9 cm while the leaves are blade ovate, elliptic, or elliptic-lanceolate, 4-14  1.7-3.5(−6) cm, stiffly papery, abaxially densely strigose and puberulous, adaxially densely strigose, secondary veins 2 (or 3) on each side of midvein, tertiary veins numerous and parallel, base rounded to subcordate, margin entire, apex acuminate ( Figure 2). Inflorescences subcapitate corymbose, terminal, 3-7-flowered, with 2 leaflike bracts at base. Pedicel 2-8(−10) mm, strigose, apically 2-bracteolate, bracteoles lanceolate to subulate, 2-5 mm, abaxially densely strigose, margin ciliate. Hypanthium 5-9 mm, densely compressed strigose, margin fimbriate. Calyx lobes lanceolate to ovatelanceolate, apex acuminate, on both sides and along their margin squamosly strigose and pubescent. Petals reddish purple, 2-3(−4) cm, margin only ciliate, apex rounded. The flowers, which are short-lived and last only a day, grow in 5 to 10 clusters and have 5 petals [21]. The flower has ten stamens of two different kinds: five larger ones with yellow filaments and purple curved upper parts including the anther and five smaller ones with yellow and straight filaments and yellow anthers. On rare occasions, M. malabathricum consists of three varieties, having large-, medium-and small-size flowers with dark purple-magenta petals (Figure 3(a)), light pink-magenta petals (Figure 3(b)), and (the rare variety) white petals (Figure 3(c)) [22]. The calyx closely set with short chaffy and silky or silvery scale. Longer stamens with connective long extended at base, curved, apex bifid. Shorter stamens with anthers 2-tuberculate at base; connective not extended. Ovary half inferior, densely strigose, apically with a ring of setae. Fruit urceolate-globular, 6-15 × 6-12 mm, succulent, densely squamose strigose [10]. The fruits are technically classified as berries, and, when they are ripe, they break open irregularly to reveal the soft, dark purple, sweet but rather astringent-tasting pulp and numerous orange seeds (Figures 4(a) and 4(b)). The seeds are dimorphic: with or without embryos. Fertile seeds are folded or spiral, tri-angular to D-shaped in outline, 0.45-0.8 mm long, 0.35-0.6 mm wide, 0.17-0.3 mm thick, with light yellow or pale to dark cream-coloured testa. Seeds without embryo are similar to the fertile seeds but smaller, 0.3-0.5 mm long, 0.2-0.3 mm wide, 0.2 mm thick, appear collapsed, dented, or wrinkled and with completely black or reddish-black testa. The seeds are tasteless and can be eaten, and they stain the tongue black. The name "melastoma" is Greek for "black mouth," a name appreciated by generations of children who have eaten the berries. M. malabathricum has evergreens and flowers throughout the year [23]. M. malabathricum has been claimed to possess various medicinal values according to the communities/tribes traditional beliefs and, interestingly, the whole plant could be used as herbal medicine. It is also a well-known herb in Malaysia, particularly, where its leaves, shoots, and roots are prepared in various ways for treatment of different diseases and ailments (Table 2(a)). Many reviews have appeared in the literature regarding M. malabathricum medicinal uses. However, none have described the complete chemical and pharmacological properties of this important ethnomedicinal plant. Therefore, we aimed to compile an up-to-date and comprehensive review of M. malabthricum that covers its ethnomedicinal uses, phytochemical contents, and scientifically proven pharmacological properties. Other than that, the powdered leaves and roots can be applied to wounds and pox scars to aid the healing process [7,24] or used to relieve the discomfort of hemorrhoids [7] with the former also used as astringent for dysentery [9]. The juice of leaves and roots is used as a digestive aid [9]. Furthermore, the leaves and flowers are useful for the treatment of cholera, diarrhoea, prolonged fever, dysentery, leucorrhoea, wounds, and skin diseases and for the preparation of gargles [11,23,24,36]. The decoction of roots and leaves or roots alone are also traditionally used to tone up the uterus after childbirth in order to strengthen the womb and accelerate wound healing. Other than that, women also use this herb for excessive menstrual bleeding and cramps, to relieve postmenstrual syndrome, stomach ache, and white discharge, and to enhance fertility [23]. Its flowers, seeds, and leaves are used to reduce white vaginal discharge and indigestion [9]. The flowers of M. malabathricum are also used as a nervous sedative and for hemorrhoidal bleeding [9]. The combination of leaves and flowers is used as astringent in leukorrhea and chronic diarrhea [9]. Despite being a traditional medicinal herb that is widely used, particularly, in Malay culture, there is not much scientific study carried out on M. malabathricum.

Reports on the Uses of M. malabathricum by Various
Communities/Tribes. The list of medicinal uses of M. malabathricum according to the different communities or tribes reported around the world is shown in Table 2(b). According to Elliott and Brimacombe [37] the cold infusion of M. malabthricum flowers is an optional ingredient added to an oral remedy for anaemia associated with gastrointestinal bleeding and epigastric pain. The Talang Mamak peoples of Riau province, Sumatra, Indonesia, used the ground leaf and applied it as a compress to cuts and wounds [8]. A survey of the Malay ethnomedico botany in the Machang district, Kelantan, Malaysia, revealed the application of M. malabathricum fruits juice on dry lip [38] while in the Gemenceh Evidence-Based Complementary and Alternative Medicine 5 Gemenceh, Negri Sembilan, Malaysia The pounded leaves are applied onto wounds to accelerate healing Ong and Nordiana [39] Jah Hut Jerantut, Pahang, Malaysia The roots are applied as decoction to treat diarrhea Lin [33] Lakher and Pawi Mizoram, India The decoction of the leaves or its juice is taken orally to treat diarrhoea and dysentery Sharma et al. [11] Meitei Manipur and Mayurbhanj, Orissa, India The bark and leaves are used for treating skin troubles, leukorrhea, diarrhea, and dysentry Thatoi et al. [14] Didayi Malkangiri, Orissa, India The leaves are applied externally as paste to treat cuts and wounds Pattanaik et al. [13] Sundanese Bogor, West Java, Indonesia The leaves is used as topical application or oral ingestion to treat toothache and for postpartum remedy Roosita et al. [40] Marmas Bandarban, Bangladesh The root juice is used to treat jaundice Rahmatullah et al. [18] Garo Netrakona, Bangaldesh The leaf juice is used as a diuretic and to treat various urinary problems Rahmatullah et al. [19] Murong Rangamati, Bangladesh The root juice or water extract of boiled roots are used orally to treat leukorrhea Rahmatullah et al. [20] Naga Manipur, India The fresh and dry leaves are used to treat cuts and wounds, stomach disorder, and fever Ringmichon et al. [15] Tahiti The plant is used to treat diarrhea and dysentery with its bark decoction used as gargle Umali-Stuart and Stiuart-Santiago [9] district, Negri Sembilan, Malaysia, the Malays applied M. malabathricum pounded leaves onto wounds to accelerate healing [39]. The Jah Hut tribe in Jerantut district, Pahang, Peninsular, Malaysia, used the roots of M. malabathricum as decoction to treat diarrhea [33]. The native peoples of Mizoram, India, used the decoction of the leaf or its juice, which is taken orally, in the treatment of diarrhoea and dysentery [11]. The Meitei community living in Manipur district in India and other tribes living in the Similipal Biosphere Reserve, situated in Mayurbhanj district of Orissa, India, used the bark and leaves of M. malabathricum for treating skin troubles, leukorrhea, diarrhea, and dysentery [14] while the Didayi tribe of Malkangiri district of Orissa, India, used the leaf externally as paste to treat cuts and wounds [13]. The Sundanese community of the West Java, Indonesia, uses the leaf of M. malabathricum as topical application or oral ingestion to treat toothache and for postpartum remedy [40]. The Marmas community of Naikhongcharri, Bandarban district, Bangladesh, used the root juice to treat jaundice [18]. The Garo tribal community living in Netrakona district, Bangladesh, used the leaf juice as diuretic and to treat various urinary problems [19]. The Murong tribes residing in the Rangamati district in the Chittagong Hill Tracts region, Bangladesh, used the squeezed juice from the roots or water extract of the boiled roots orally to treat leukorrhea [20]. In Tahiti, M. malabathricum is used to treat diarrhea and dysentery with its bark decoction used as gargle [9]. The Naga tribe of Manipur district, India, used the fresh and dry leaves of M. malabathricum to treat cuts and wounds, stomach disorder, and fever [15].
C Chemical structure adopted from http://www.wikipedia.com/. D Chemical structure adopted from http://www.guidechem.com/. E Chemical structure adopted from the respective reference.
Evidence-Based Complementary and Alternative Medicine

Pharmacological Activities
Various parts of M. malabathricum have been claimed to possess medicinal values, which is supported particularly by the Malay and Indian traditional uses of the plants in the treatment of a number of diseases as described earlier.
Scientifically, M. malabathricum, prepared as extracts using different types of solvents and tested using a range of in vitro and in vivo test models, demonstrated various pharmacological potentials that required in-depth studies ( Table 4). The plant, regardless of the parts used, has been shown to exert antibacterial, antiviral, antiparasitic, antioxidant, cytotoxicity, anticoagulant, platelet-activating factor inhibitory, wound healing, antiulcer, antidiarrheal, antivenom, anti-inflammatory, antinociceptive, and antipyretic activities at different doses/concentrations. The following subchapters will discuss in detail those scientific findings related to pharmacological properties of M. malabathricum.

Acute Toxicity Study.
Sunilson et al. [61] have determined the acute toxicity LD 50 level of water extract of M. malabathricum leaves (WMML) collected in the State of Selangor, Malaysia. The extract, in the doses of 62.5, 125, 250, 500, 1000, and 2000 mg/kg, was administered orally in mice. The authors recorded general signs and symptoms of toxicity, intake of food and water, and mortality for 48 h. The acute toxicity study showed that the WMML administered up to 2000 mg/kg dose to the mice showed neither mortality nor any visible clinical signs of general weakness in the animals. This indicates that the WMML is safe for consumption even at the highest dosage (2000 mg/kg) tested.

Antibacterial Activity.
Grosvenor et al. [62] studied the antimicrobial property of 70% methanol extract of combined M. malabathricum leaf, stem, and flower (MMMClsf), collected from the Riau province, Sumatra, Indonesia, against Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae, and Fusarium oxysporum using the agar diffusion assay. The extract was effective only against S. aureus, S. cerevisiae, and F. oxysporum. Standard antimicrobial references used were 1 mg/mL chloramphenicol, 1 mg/mL tetracycline, and 10000 units/mL nystatin suspensions. The chloramphenicol and tetracycline exerted antimicrobial activity against S. aureus and E. coli, while nystatin suspension exhibited antimicrobial activity against S. cerevisiae and F. oxysporum.
Wiart et al. [63] studied the antibacterial and antifungal properties of MMML, collected from Kuala Kangsar in the State of Perak, Malaysia, against Bacillus cereus, B. subtilis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans using the disc diffusion method. Standard antibiotics used were 10 μg gentamycin and 20 μg nystatin for antibacterial and antifungal comparison, respectively. The 1000 μg MMML exerted poor antimicrobial activity with zone of growth inhibition recorded at 7 mm only against B. subtilis. In comparison, gentamycin was effective against all microorganisms tested, except C. albicans, with zone of inhibition ranging between 14 and 20 mm while nystatin was effective against C. albicans with zone of inhibition recorded at 11 mm.
Thatoi et al. [14] reported the antimicrobial activity of the aqueous extract of different parts of M. malabathricum (e.g., AMML, stem (AMMS), bark (AMMBk), bulb (AMMBb), fruit (AMMFr), and root (AMMR)), collected from Simlipal Biosphere Reserve, Orissa, India, against a panel of Gram-positive and Gram-negative human pathogenic bacteria (e.g., , and Shigella flexneri (lab isolate)) and a fungal (e.g., C. krusei (lab isolate)). All extracts, in the volume of 200 μL and in the concentration of 200 mg/mL each, were tested using the agar cup method. From the results obtained, AMML was considered to have an outstanding antimicrobial activity as indicated by the inhibition zones produced that are more than 20 mm. The AMML was effective against B. brevis, V. cholerae, C. krusei, and B. subtilis, while the AMMBk was effective against S. aureus, B. brevis, and V. cholerae.

Antiviral Activity.
Lohézic-Le Dévéhat et al. [32] investigated the antiviral activity of ten methanolic extracts from eight Indonesian medicinal plants, which included M. malabathricum, against HSV-1 and Poliovirus. The virus titre was estimated from cytopathogenicity and expressed as 50% tissue culture infectious doses per millilitre (TCID 50 /mL). The antiviral activity was assayed using the cytopathic effect inhibition assay, which was expressed as EC 50 . The effect of extract on uninfected Vero cells was given by cytotoxic concentration 50% (CC 50 ; μg/mL). The MMML together with several species from the Indonesian Loranthaceae, which was collected from Padang in the State of Andalas, Indonesia, was reported to exert moderate anti-HSV-1 activity with significant activity against Poliovirus. The MMML exhibited CC 50 against HSV-1 and Poliovirus with value greater than 1000 μg/mL (>1000 μg/mL) or equal to 1000 μg/mL, respectively. In terms of EC 50 , the value recorded for treatment of extract against HSV-1 virus at 20 TCID 50 and 200 TCID 50 was 192 and 706 μg/mL, while, for treatment against Poliovirus, the EC 50 value obtained at 20 TCID 50 and 200 TCID 50 was 111 and 225 μg/mL, respectively.
Nazlina et al. [55] have studied the antiviral activity of MMML. The extract earlier subjected to the TLC assays resulted in the isolation and identification of rutin, quercitrin, and quercetin. The antiviral activity was screened against HSV-1 and vaccine strain of measles (Schwarz) wherein three methods of treatment to detect antiviral activity in each of the fraction were used (see Table 4). For the antiviral tests, the extract was diluted at 1.0 LC 50 , 0.1 LC 50 , and 0.01 LC 50 . The MMML exerted antiviral activity with different modes of action against HSV-1 or measles viruses. The extract was effective in inhibiting cell death by 0.01 LC 50 in HSV-1-inoculated cells using treatment mode ((C + V) + E) suggesting that virus-inoculated cells were able to overcome viral infection when treated with the extract. Cells treated with simultaneous addition of measles virus and the extract at 0.1 and 1.0 LC 50 were found to survive from viral infection. The effect of MMML is probably due to the quercetin content that can inhibit reverse transcriptase which is the early part of the measles' replication process. Another possibility is that the MMML is capable of acting directly on viral particle such as modification of free viruses surfaces that inhibits viral attachment to host cells. This belief was supported by the fact that MMML was not capable of inhibiting virus-inoculated cells in treatment mode I. However, the most probable mode of infection can only be further confirmed by transcriptomic or proteomic studies. The extract was found not to have prophylactic effect on both test viruses as demonstrated in treatment mode ((C + E) + V). As for control, guanidine hydrochloride is seen in this study to inhibit viral capability of infecting host cells when added simultaneously, which is believed to happen via blocking of the initiation step of viral RNA synthesis.

Antiparasitic Activity.
Alen et al. [67] have screened 65 methanolic extracts of Sumatran rain forest plants, including M. malabathricum, for their nematocidal activity against Bursaphelenchus xylophilus using the in vivo cotton ball-(bl-) fungal mat assay. The authors reported that the MMML, together with that of Allamanda cathartica, Ervatamia corymbosa, Hoya diversifolia, Bischofia javanica, Derris malaccensis, Ophiorrhiza konsteleary, and Brucea sumatrana, exhibited strong nematocidal activity with the recorded minimum effective dose (MED) of approximately 5 mg/bl. However, the extracts of Bischofia javanica, Knema hookeriana and Areca catechu were considered to be the most effective nematocidal agents with the recorded MED of approximately 0.7 mg/bl.
In addition, Faravani [58] also reported the antioxidant activity of crude MMMR and methanol extracts of M. malabathricum shoots (MMMSt) investigated using the DPPH assay. The MMMSt exhibited their radical scavenging activity as indicated by their ability to reduce the stable free-radical DPPH to the yellow-colored diphenylpicrylhydrazine with an IC 50 recorded at approximately 141.9 μg/mL and 154.5 μg/mL, respectively. However, the extracts antioxidant activity was considered to be lowered than that of the positive control, ascorbic acid, which produced an IC 50 of approximately 28.6 μg/mL.

Cytotoxic
Activity. An attempt to determine the cytotoxic activity of MMML together with nine methanolic extracts from seven Indonesian medicinal plants was also made by Lohézic-Le Dévéhat et al. [32]. All plants were collected from Padang in the State of Andalas, Indonesia, and subjected to the cytotoxic study on two murine cancer cell lines (e.g., 3LL (Lewis lung carcinoma cells) and L1210 (leukaemic cells)) and four human cancer lines (e.g., K562 (chronic myeloid leukaemia), U251 (glioblastoma), DU145 (prostatic adenocarcinoma), and MCF-7 (mammary carcinoma)). The MMML exhibited cytotoxic activity against 3LL, L1210, K562, DU145, MCF-7, and U251 at the IC 50 values of 19, 21, 67, 113, >400, and 30 μg/mL. Interestingly, MMML exhibited cytotoxic activity with IC 50 value of <25 μg/mL against both murine cell lines while the cytotoxicity activity against all human cancer cell lines was observed at an IC 50 value that was >25 μg/mL.
Susanti et al. [22] have investigated cytotoxic activity of the crude EAMMFw and MMMFw and several compounds isolated from the respective crude extract (e.g., naringenin, kaempferol and kaempferol-3-O-D-glucoside, kaempferol-3-O-(2 ,6 -di-O-p-trans-coumaroyl)-β-glucopyranoside, and kaempferol-3-O-D-glucoside) against a MCF-7 cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. DMSO (0.1%) and tamoxifen were used as negative and positive controls, respectively. The 500 μg/mL EAMMFw caused a change in the cell morphology of MCF-7 cells line, while MMMFw, at the same concentration, did not display any activity. Naringenin and kaempferol-3-O-(2 ,6 -di-O-p-trans-coumaroyl)-β-glucopyranoside demonstrated a significant anticancer effect against MCF-7 in a dose-dependent manner with IC 50 values of 1.30 ± 0.002 μM and 0.28 ± 0.004 μM, respectively. The anticancer activity of those compounds was demonstrated to involve cell proliferation and changes in the cell morphology. Thus, naringenin was suggested to contribute to the anticancer activity of the EAMMFw. The failure of MMMFw to exhibit its cytotoxicity effect suggested that the antagonist effects of the compounds present in the extract play an important role in not affecting the cell proliferation. Interestingly, the IC 50 value of kaempferol-3-O-(2 ,6 -di-O-p-trans-coumaroyl)-β-glucopyranoside was lower than that of the positive control, tamoxifen (the IC 50 value   42 Evidence-Based Complementary and Alternative Medicine was 0.76 ± 0.005 μM). Based on the cell morphology, it was proposed that kaempferol-3-O-(2 ,6 -di-O-p-transcoumaroyl)-β-glucopyranoside was active against human breast cell cancer by inhibiting cell proliferation. Meanwhile, naringenin inhibited cell proliferation, followed by cell lyses. Kaempferol-3-O-(2 ,6 -di-O-p-trans-coumaroyl)-β-glucopyranoside has a free hydroxyl group at position 3 and a parahydroxyl group in ring B, which probably increase the activity of this compound.
Nazlina et al. [55] have also studied the cytotoxic activity of MMML against Vero cell line (African green monkey, Cercopitheus aethiops kidney cells) and L929 cells (mouse fibroblast) whereby the cytotoxicity test was carried out according to the microculture method in, at least, two independent experiments in triplicates at different concentrations of MMML using doubling dilutions from initial stock concentration of 1000 μg/mL. Cytotoxicity screening towards Vero and L929 cells showed that MMML was not cytotoxic to both cells with LC 50 values of 750 μg/mL and >1000 μg/mL, respectively. As for gunaindine hydrochloride that was used as positive control, it was found not cytotoxic in Vero cells with the LC 50 value of 100 μg/mL while the LC 50 value for L929 cells was not determined.

Anticoagulant Activity.
Manicam et al. [68] have reported on the anticoagulant property of the leaves of M. malabathricum collected from the area of Serdang in the state of Selangor, Malaysia. The hot-and cold-WMML and MMML were assayed for their anticoagulant property using the blood samples drawn from healthy volunteer donors (n = 36) of both genders (18-50 years old) after screening via questionnaire for familial history of cardiovascular diseases and other major coagulopathies. The coagulation parameters used to determine the extracts anticoagulant activity were the activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin time (TT). These parameters were carried out in a STA Compact coagulation analyzer with the maximum cut-off time recorded by the coagulation analyzer set at 180 s. Plasma samples were spiked with different concentrations of M. malabathricum extracts (ranging between 100 and 1000 μg/mL), heparin (used as positive control), or deionized water (used as vehicle control). Based on the preliminary study, the hot-WMML, in the concentration of 1000 μg/mL, significantly prolonged (P < 0.05) the three parameters, namely, aPTT, PT, and TT, in plasma when compared to the normal control plasma. The PT and TT measurements were 20.0 ± 1.3 s and 43.2 ± 0.1 s when compared to control group (13.3±0.5 or 20.1±0.2 s), respectively. Interestingly, the hot-WMML did not clot the plasma samples when tested for aPTT, as evidenced by the maximum cut-off time recorded at 180 s. The cold-WMML and MMML also prolonged aPTT in a significant fashion (P < 0.05) with 120.0 ± 0.9 s and 108.0 ± 0.7 s, respectively, in comparison to the 38.9 ± 0.5 s of control plasma. Similar to the hot-WMML, the PT of cold-WMML was prolonging significantly (P < 0.05) in comparison to the control. On the other hand, these extracts did not affect the TT significantly. Based on the anticoagulant activity demonstrated above, at the concentration of 1000 μg/mL, Manicam et al. [68] selected the hot-WMML for further study on its effect on clot-based assays. At the concentration ranging between 100 to 1000 μg/mL, the hot-WMML caused prolongation of aPTT in a concentration-dependent manner with significant anticoagulant activity recorded at the concentration beyond 400 μg/mL in comparison with vehicle control. Interestingly, hot-WMML prolonged aPTT beyond 300 s at 900 and 1000 μg/mL, which was comparable to that of 5-1000 μg/mL heparin. The cut-off time to measure clotting times was 300 s; beyond which the plasma samples were rendered noncoagulable. The control plasma was found to record an aPTT of 64.3 and 60.7 s for females and males, respectively. On the other hand, the aPTT of normal pooled plasma (NPP) was 41.0 and 40.5 s for females and males, respectively, and was significantly different (P < 0.05) from that of the control plasma. In contrast to aPTT, hot-WMML caused no significant changes for PT and TT at the tested concentration range. However, PT was significantly (P < 0.001) prolonged at the highest contration of heparin (1000 μg/mL) while TT was significantly (P < 0.001) prolonged at the highest concentration of hot-WMML (1000 μg/mL) or at the range of 5-1000 μg/mL. Overall, the PT assay recorded the lowest coagulation inhibitory activity for hot-WMML. Mazura et al. [70], in their quest for natural anti-inflammatory agents, assessed the potential of α-amyrin, betulinic acid, quercetin and quercitrin isolated from M. malabathricum to inhibit PAF binding to rabbit platelets using 3 1 μM), which is a known PAF receptor antagonist.
4.9. Wound Healing Activity. Sunilson et al. [66] reported the wound healing potential of MMML, collected from the State of Selangor, Malaysia, in the form of ointment when examined in two types of wound model in rats: (i) the excision wound model and (ii) the incision wound model. The extract, prepared as 5% ointment, exhibited a wound healing activity that was comparable with the standard drug, nitrofurazone, which was prepared as 0.2% ointment, in terms of wound contracting ability, wound closure time, tensile strength, and regeneration of tissues at the wound site. The time to wound closure of the nitrofurazone-and the extracttreated groups was the same (18.0 ± 2.0 days). In the incision wound studies, the extract ointment and nitrofurazone caused a significant increase in tensile strength of the 10day-old wound when compared with the control (418.0 ± 13.8 g). The tensile strength of the extract ointment-and the nitrofurazone-ointment-treated groups was almost the same (551.0 ± 16.9 g versus 576.0 ± 12.5 g). Interestingly, the extract ointment enhanced original tissue regeneration of the skin wounds much greater than nitrofurazone with the standard drug exerting more relative fibrosis of skin wounds when compared to the extract ointment. Although fibrosis was relatively less in the extract ointment-treated rats, the original tissue was regenerated much more in the animal wounds. The skin adrenal structures such as the Pilosebaceous glands and sweat glands were better presented in wounds treated with extract ointment compared to nitrofurazone-treated animal wounds.

Antiulcer Activity.
Hussain et al. [71] studied the antiulcer activity of the AMML, collected around the University of Malaya campus in Petaling Jaya, Selangor, Malaysia, against ethanol-induced gastric mucosal injuries in rats. The extract, in the dose of 250 and 500 mg/kg, and 20 mg/kg omeprazole, used as positive control, were administered orally followed 1 hour later by the oral administration of the ethanol. Macroscopically, the oral administration of the test solutions was found to significantly (P < 0.05) reduce the formation of gastric mucosal injuries in a dose-dependent manner when compared to the group that was pretreated with only distilled water. The ulcer area recorded for groups pretreated with distilled water, 250 and 500 mg/kg extract, and 20 mg/kg omeprazole was 845.00 ± 52.17 mm 2 , 210.00 ± 8.17 mm 2 , 70.00±8.27 mm 2 and 30.00±5.32 mm 2 , respectively. In terms of percentage of protection, the respective test solution was found to give approximately 75.15%, 91.72%, and 96.45% protection. Microscopically, distilled-water-pretreated rats exhibited severe damage of the gastric mucosa, and induced submucosal edema and leucocytes infiltration while extractor omeprazole-received rats exerted marked reduction of gastric mucosal damage, reduction of oedema, and less leucocyte infiltration of submucosal layer. There were no significant differences between the cytoprotective abilities of the animals treated with 20 mg/kg omeprazole compared to the animals treated with 500 mg/kg AMML. Overall, the 500 mg/kg AMML was found to provide the best protection to the gastric mucosa in rats against ethanol-induced gastric ulcers.
4.11. Antidiarrheal Activity. The antidiarrheal activity of WMML, collected from the State of Selangor, Malaysia, was investigated using four experimental models of diarrhea in mice [61]. In model 1, mice were given test solutions (e.g., 100, 200, and 500 mg/kg of extract or 5 mg/kg loperamide) and fecal materials were collected for 12 h after treatment, dried in an incubator, and weighed. The percentage reduction in the fecal output was determined. In model 2, an overnight fasted male mouse was induced with diarrhea by oral administration of castor oil (0.5 mL/mouse, p.o.) 1 hour after the test solutions administration. The percentage protection from diarrhoeal droppings was calculated. In model 3, overnight fasted mice were subjected to the enteropooling assay method wherein the animals received the test solutions and, 1 hour later, administered orally with a diarrheal agent, 10% aq MgSO 4 (0.5 mL/mouse). Thirty min later, the animals were killed and the small intestines were collected and weighed to find out the accumulation of intestinal fluid secretion evoked by MgSO 4 . In model 4, the overnight fasted animals were subjected to the gastrointestinal transit test. The animals received test solutions, and, 5 min later, 0.5 mL of 3% charcoal suspended with tragacanth powder was administered orally to each mouse. Thirty min later, all the mice were killed by cervical dislocation and the distance travelled by the charcoal plug from pylorus to caecum was determined and expressed as a percentage of the total length of the small intestine. From the data obtained in model 1, 100, 200, and 500 mg/kg WMML significantly (P < 0.05) reduced the dried fecal output of the mice (dried fecal output per 100 g of mice) by 0.364 ± 0.012 (30.13% reduction), 0.314±0.046 (39.73% reduction), and 0.296±0.023 (43.19% reduction), respectively, while the reduction in the fecal output by loperamide (5 mg/kg) was noted to be 0.222 ± 0.015 (57.39% reduction) when compared to the control group (0.521±0.083). In model 2, the WMML, at its respective dose, significantly (P < 0.05) protected the mice against castor-oil-induced diarrheal droppings by 60, 80 and 80%, while loperamide produced the 100% protection. In model 3, the 100, 200, and 500 mg/kg WMML significantly (P < 0.05) and dose-dependently reduced the intestinal fluid secretion induced by MgSO 4 , with the weight of the small intestine per 100 g of mice recorded at 8.413 ± 0.431, 7.620 ± 0.469, and 7.314 ± 0.261 when compared to the control group (9.362 ± 0.518). Interestingly, 500 mg/kg WMML caused reduction in the intestinal fluid secretion that was almost comparable with that of 5 mg/kg loperamide (6.416 ± 0.514). In model 4, the 100, 200 and 500 mg/kg WMML significantly (P < 0.05) inhibited the small intestinal motility of the charcoal marker in mice in a dose-dependent manner by 8.10%, 25.15%, and 32.97% inhibition in comparison to 5 mg/kg loperamide (57.42% inhibition). The respective distance travelled by charcoal marker for each dose of extract presented as percentage of total length of small intestine was recorded at 63 [30] reported on the anti-inflammatory activity of AMML, collected from Shah Alam, Selangor, Malaysia. The anti-inflammatory activity was determined using only the carrageenan-induced paw edema wherein the extract, at the concentration of 10%, 50%, and 100%, was administered via subcutaneous route and measurement of paw thickness was carried out for 8 hours following the extract administration with 1-hour interval. The concentration of AMML used was equivalent to the doses of 4.87, 24.35, and 48.7 mg/kg, respectively. The AMML was found to show significant (P < 0.05) anti-inflammatory activity in a concentration-independent manner wherein the activity was seen only at the 50% and 100% concentrations. In terms of the onset of action, the AMML exhibited the anti-inflammatory activity 1 hour after its subcutaneous administration and this activity was seen until the end of experiments. Interestingly, 100 mg/kg acetylsalicylic acid (ASA), used as positive control, exerted similar strength of anti-inflammatory when compared to the extract as indicated by the statistically insignificant data at the respective time interval.
Susanti et al. [56] investigated the anti-inflammatory activity of pure compounds obtained from HMML, EAMML and MMML using the 12-O-tetradecanoylphorbol-13-acetate-(TPA-) induced mouse ear oedema assay. The pure compound (20 μL) was applied topically to the inner surface of the right ear of the mice with the left ear receiving only vehicle acting as negative control group. The pure compounds (0.5 mg/ear) and the standard drug indomethacin (0.5 mg/ear) were applied topically, simultaneous with TPA. From the results obtained, 0.5 mg/mL kaempferol-3-O-(2 ,6 -di-O-p-trans-coumaroyl) glucoside and α-amyrin demonstrated the strongest activities in the anti-inflammatory assay with the IC 50 of approximately 0.11 ± 0.4 and 0.34±1.1 mM/ear, respectively as compared to the 0.5 mg/mL indomethacin (2.10 ± 0.5 mM/ear). However, no data on the anti-inflammatory effect of those crude extracts were given for comparison with their pure compounds.
4.14. Antinociceptive Activity. The ethanolic extract of M. malabathricum stem barks and leaves (EMMSBL), collected from Serdang, Selangor, Malaysia, was earlier studied its antinociceptive activity using the acetic-acid-induced abdominal constriction and hot plate test in mice [73]. The extract, administered intraperitoneally in the doses of 30, 100 and 300 mg/kg, was found to exert potential (P < 0.05) antinociceptive activity in a dose-dependent manner in the former test with the percentage of nocicptive inhibition recorded at 23.0%, 50.0%, and 84.4%, respectively. The ED 50 recorded was approximately 100 mg/kg when given intraperitoneally. ASA, at 100 mg/kg dose, produced 79.0% nocicptive inhibition. In the latter test, the extract significantly (P < 0.05) increased the response latency period to thermal stimuli in mice also in a dose-dependent manner. The antinociceptive activity of EMMSBL reached its peak approximately 60 min after the extract administration. Morphine, at 5 mg/kg dose, significantly (P < 0.05) prolonged the response latency period with maximum effect obtained approximately 1 h after treatment. In an attempt to determine the role of opioid receptors in mediating the EMMSBL antinociceptive activity, 5 mg/kg naloxone, a nonselective opioid antagonist given via intraperitoneal route 15 min before the extract, was found to inhibit the antinociceptive activity of the extract in both tests.
Zakaria et al. [30] reported on the antinociceptive activity of the AMML, collected from Shah Alam, Selangor, Malaysia. The antinociceptive activity of subcutaneously administered extract, at the concentration of 10%, 50%, and 100% (which is equal to the dose of 4.87, 24.35, and 48.7 mg/kg, resp.), was determined using the acetic acid-induced abdominal constriction, hot plate and formalin test. The AMML was found to show significant (P < 0.05) antinociceptive activity in all the three tests. In the acetic-acidinduced abdominal constriction test, all concentrations of AMML caused significant (P < 0.05) reduction in the number of abdominal constriction which occur in a concentration-independent manner. The 10%, 50%, and 100% AMML exerted similar strength in antinociceptive activity when compared together or against 100 mg/kg ASA. In the hot plate test, all concentrations of AMML also exhibited a significant (P < 0.05) antinociceptive activity in a concentration-independent manner with only the 50% and 100% concentrations of AMML exerting an antinociceptive activity that lasted until the end of the experiment. In this test, 5 mg/kg morphine was used as a positive control and was more effective in increasing the latency to feel thermalinduced pain when compared to the extract. In the formalin test, the AMML at all concentrations used exerted antinociceptive activity in both the early and late phases of the test. The concentration-dependent activity was observed only in the late phase of the test.

Antipyretic Activity.
Zakaria et al. [30] also reported on the antipyretic activity of AMML, collected from Shah Alam, Selangor, Malaysia. The antipyretic activity of subcutaneously administered AMML, at the concentration of 10%, 50% and 100%, was determined using the Brewer's Yeast-(BY-) induced pyrexia test. The ability of M. malabathricum extract to reduce temperature of pyrexia induced rats was determined for 8 hours with 1-hour interval. The AMML was found to show significant (P < 0.05) antipyretic activity at all concentrations tested for the first 6 hours after BY administration. A rapid decrease in the antipyretic activity of the 100% concentration of AMML 3 h after BY administration when compared with the other concentrations of AMML, with completely diminished activity observed for the last 2 h of the experiment. The 100 mg/kg ASA demonstrated antipyretic activity only for the first 4 hours before the activity was gradually lost until the end of experiment.

Discussion and Conclusion
According to Mitchell and Ahmad [74], the wealth of a country resides to a large extent in its plant inheritance, regardless of whether the plants are endemic, naturalized, or recent introductions. Out of a total of more than 4000 species of Melastomataceae plants in the world, M. malabathricum has been one of the 22 species found in the Southeast Asian region alone [1] and one of the 12 species found in Malaysia. Considered as native to tropical and temperate Asia and the Pacific Islands [4], this commonly found small shrub has gained herbal status in the Malay folklore belief as well as the Indian, Chinese, and Indonesian folk medicines. Despite claims that this plant was one of the important herbs within the traditional Malay, Indian, and Chinese medicine, no proper documentation could be found to support them. For example, no documentation that provides proofs for the claims of the importance of M. malabthricum in traditional Chinese medicine could be found when the authors carried out thorough search via university or public libraries, as well as the internet. Various parts of this shrub have been claimed to be used in the treatment of various types of ailments and diseases (e.g., diarrhoea, dysentery, leucorrhoea, hemorrhoids, cuts and wounds, infection during confinement, toothache, stomachache, flatulence, sore legs, and thrush) with most of them not yet proven via clinical studies [23]. Despite the lack of clinical studies related to this plant, several attempts have been made to elucidate the pharmacological properties of M. malabathricum using the standard and scientific in vitro and in vivo techniques of biological evaluations and to finally confirm those folklore claims.
Present interests towards the medicinal benefits of herbal medicines have been increasing worldwide as can be seen with increased laboratory investigation into the pharmacological properties of various medicinal plants. Scientists have also been involved in the isolation, identification, and determination of the bioactive ingredients with specific ability to treat various diseases [75]. Various drugs have entered the international market as a result of scientifically in-depth and systematic exploration of ethnopharmacology and traditional medicine. Despite the increase in scientific study towards medicinal plants all over the world, a smaller number of phytochemical entities or drugs have entered the local or international market due to their evidence-based therapeutics [74]. Due to the latter market trend, efforts are needed to ascertain and confirm evidence regarding safety and practices of plant-based medicines. Furthermore, plantbased medicines are erroneously considered safe because they are regarded as naturally occurring and of plant origin. According to Yob et al. [76], the lack of scientific and clinical data has led to poor understanding of the efficacy and safety of the herbal drugs, which in turn contributes to major impediment in the use of plant-based medicinal pre-parations. The only attempt to establish acute toxicity profile of M. malabathricum was performed by Sunilson et al. [61]. The authors reported the nontoxic effect of WMML up to the dosage of 2000 mg/kg when given to mice. This indicates that the WMML is safe for consumption even at the highest dosage (2000 mg/kg) tested and justifies the traditional uses of M. malabathricum.
The dosage range used for in vivo study was very important to corroborate with the dosages used in traditional medicine and the selection of dosage range should not exceed the maximum tolerated dose (MTD) of 1000 mg/kg/day suggested for in vivo studies [77]. Based on MTD, the dosage range used for in vivo antiulcer, antidiarrheal, antinociceptive, anti-inflammatory, and antipyretic activities, which is between 4 and 500 mg/kg, was considered acceptable. In the in vitro studies, the pharmacological activities shown have to be interpreted cautiously depending on the EC 50 or IC 50 value obtained for the respective study [76]. According to Meyer et al. [78] for any compounds/extracts to be considered active, they need to exhibit the respective activity at EC 50 or IC 50 value of less than or equal to 30 μg/mL (≤30 μg/mL). Based on this suggestion, the antibacterial, antiviral, antiparasitic, and antivenom activities were observed at EC 50 or IC 50 values that are unrealistic and greater than 30 μg/mL, and, thus, should be ignored. Some of the potential activities of M. malabathricum, as indicated by EC 50 or IC 50 value of ≤30 μg/mL, include antifungal, antioxidant, and cytotoxic activities. The MMML, CMML, and AcMML exhibited antifungal activity against C. gloeosporioides with MIC of 20 μg/mL [64]. The MMMFw and EAMMFw exerted antioxidant activity in the DPPH assay at the IC 50 of 6.59 ± 0.8 μg/mL and 7.21 ± 0.5 μg/mL, respectively [22]. The MMMFw was also reported to show cytotoxic effect against 3LL, L1210, and U251 cell lines at the IC 50 values of 19, 21, and 30 mg/mL, respectively [32]. Another major flaw in some of the reports cited above was failure of the authors to provide proper comparison with reference drug [76]. The importance and reason for choosing certain routes of administration and in vitro rather than in vivo assays in some of the studies have been discussed by Yob et al. [76].
In this paper, we intended to briefly summarize the in vitro and in vivo assays applied in the discovery of possibly new pharmacological agents from M. malabathricum. In addition, various literatures pertinent to the pharmacological investigation of M. malabathricum were reviewed to gather all information related to the ethnobotanical, phytochemical, and pharmacological properties of M. malabathricum. Although various scientific papers were published on pharmacological properties of M. malabathricum, detailed and careful analysis revealed that M. malabathricum only exhibited promising antiulcer, antidiarrheal, antinociceptive, antiinflammatory, and antipyretic activities as measured via the various in vivo assays and antifungal, antioxidant, and cytotoxic activities as measured by the in vitro assays.
Despite the various medicinal uses of M. malabathricum as described in the ethnobotanical section, the therapeutics efficacy of this plant has not been fully studied indepth. Even though there are various types of bioactive compounds isolated and identified from M. malabathricum as 46 Evidence-Based Complementary and Alternative Medicine highlighted in the phytochemical section, their contribution towards the plant claimed medicinal uses or demonstrated pharmacological activities were also not fully studied. Thus, the quest for new compounds from M. malabathricum with specific pharmacological activity remains unsolved. It is suggested that researches should be increased to isolate, identify, and collect most of the reported new compounds from M. malabathricum so that their pharmacological potential could be investigated thoroughly if they were to be developed as candidates for new drug development in the future. In conclusion, it is hoped that this paper will serve as an encouragement for others to further explore the pharmacological potentials of M. malabathricum with hope of developing it as a new therapeutic agents as it is considered as one of the important herbs, particularly in the Malay folklore medicine.