Asian Pacific Journal of Tropical Biomedicine

Ethnobotanical review and pharmacological properties of selected medicinal plants in Brunei Darussalam: Litsea elliptica, Dillenia suffruticosa, Dillenia excelsa, Aidia racemosa, Vitex pinnata and Senna alata

3. D. suffruticosa

Locally known as “Simpor bini”, D. suffruticosa is a medium sized tree characterized by its large bright flowers with five thin yellow petals around its white stamen, and dark-pink star-shaped fruits all of which are surrounded by large oval leaves (Figure 2). This plant species typically grows in wastelands, swamps, poor soil, white sands, secondary forests, along roadsides or the edge of forests [11–13]. Its leaves have been traditionally used for different treatments such as to promote wound healing, relieve rheumatism and treat fever while the fruit was claimed to be able to treat cancerous growths [12–16].

The methanolic extract of the roots of this plant have displayed significant antioxidant and cytotoxic activities particularly towards the HeLa cervical cancer cell line [17]. Studies conducted by Armania et al. have indicated that the phenolic content was an important contributor to the high antioxidant activity observed in the methanolic root extract of this plant species [17,18]. Although this extract showed the highest antioxidant and cytotoxic activities in the Hela cell line, it was found that the dichloromethane and ethyl acetate extracts exhibited higher cytotoxicity in the breast cancer cell lines, MCF7, MDA-MB-231, the A549 lung cancer cell line and the HT29 colon cancer cell line [17]. Further mechanistic investigation demonstrated that the plant extract inhibited the proliferation of the HeLa cervical cancer cell line as well as the MCF7 and MDA-MD-231 breast cancer cell lines via the

induction of apoptosis and the G2/M cell cycle arrest [17,18]. An in vivo study conducted by Yazan et al. showed that oral intake of aqueous root extracts have successfully reduced breast cancer induced in rats and also inhibited metastasis of the cancer to the heart [19]. The study further demonstrated that the extract was not toxic at the acute toxicity level up to a high dose of 500 mg/kg, however, mild focal hemorrhage was observed when a dose of 1000 mg/kg of the extract was used for treatment [19]. The cytotoxic activities of this plant species could be attributed to the presence of phytochemicals such as saponins, triterpenes, sterols, and polyphenolic compounds [17,20–26].

Saponins is a collective term for triterpenoid and steroidal glycosides [27], which consists of at least 150 kinds of natural saponins that have displayed significant anti-cancer properties [28]. Moreover, they have been recognized for their ability to reduce cholesterol level in the blood [29]. Dietary and endogenous cholesterols pass through the bile or desquamatedintestinal cells and reach the intestine before they are absorbed into the blood stream [29]. Saponins, being poorly absorbable from the intestine into the blood stream, interact with cholesterols and other sterols, and thus interfere and prevent them from being absorbed into the blood stream [29]. Additionally, saponins have the ability to stimulate the immune system and enhance antibody production [30].

Interestingly, multiple studies have reported reduction in bone loss with diets that are high in saponins [31,32]. One of the many saponins in particular, called asperosaponin VI, was able to induce the differentiation and maturation of osteoblasts and thus increase bone formation via the bone morphogenetic protein-2/p38 synthesis, and activation of the extracellular signal-regulated kinase 1/2 pathway [32].

On the other hand, polyphenols, which include phenolic acids, flavonoids, stilbenes and lignans, have been recognized for their multiple health-benefiting properties [33]. Polyphenols such as resveratrol and quercetin were found to exhibit significant cardio-protective effects by preventing platelet aggregation, disrupting atherosclerotic plaques and inhibiting protein expressions [33]. Other than that, anti-cancer, anti-viral, anti-diabetic, anti-aging and neuro-protective effects were also noted for the phytochemicals in the polyphenol group. They also showed beneficial effects towards asthma, osteoporosis, bone loss, skin damage and mineral absorption in intestines [33].

The phytochemical contents within D. suffruticosa play a significant role in relieving and alleviating illnesses. Perhaps it is a combination of their effects that promote wound healing, relieve rheumatism, and treat fever and cancerous growth traditionally. However, more studies should be performed to fully validate their traditional uses for such diseases.

ABSTRAK

Nama                   :    Suyatno

Program Studi     :    S1 Farmasi

Judul                    :    Uji Aktivitas Antibakteri Ekstrak Etanol 70% Daun Sempur (Dillenia suffruticosa (Griff.) Martelli)  Terhadap Bakteri Staphylococcus aureus Dan Escherichia coli.

Telah dilakukan penelitian tentang uji aktivitas antibakteri ekstrak etanol 70% daun sempur (Dillenia suffruticosa (Griff.) Martelli) terhadap bakteri Staphylococcus aureus dan Escherichia coli. Penelitian ini bertujuan untuk mengetahui aktivitas antibakteri dan nilai konsentrasi hambat minimun dari ekstrak etanol 70% daun sempur. Bahan uji yang digunakan adalah daun sempur yang diperoleh dari Desa Pedindang, kecamatan Pangkalanbaru, kabupaten Bangka Tengah, Propinsi Kepulauan Bangka Belitung untuk selanjutnya diproses menjadi serbuk halus. Pembuatan ekstrak dilakukan secara maserasi dengan etanol 70%, kemudian di evaporasi menjadi ekstrak kental dengan hasil rendemen sebesar 24,74%, hasil ekstrak dilakukan pengujian aktivitas antibakteri dengan konsentrasi 5%, 10%, dan 15% terhadap bakteri uji. Selanjutnya dilakukan pengukuran diameter daya hambat, berdasarakan pengukuran DDH  terhadap Staphylococcus aureus dengan hasil 5%  10,52 mm , 10% 11,73 mm, dan 15% 12,67 mm sedangkan terhadap Escherichia coli 5% 11,29 mm, 10% 12,89 mm, dan 15% 13,84 mm. Nilai KHM pada Staphylococcus aureus 3% dan pada  Escherichia coli 5% .

Kata Kunci : Ekstrak Etanol 70% Daun Sempur, Staphylococcus aureus, Escherichia coli, DDH, KHM.

Dilleniae radix (Dillenia suffruticosa, L. Dilleniaceae) the cytotoxic effect of dichloromethane extract at the maximum concentration of 100 μg/ml was applied to the breast cancer cell line (MCF-7). The experiment was conducted using the MTT assay in the presence of tamoxifen as the positive control. The extract showed the time and dose dependent strong cytotoxic activity with an IC50 value of 15.5 ± 0.5 μg/mL after 72 hours of exposure. This significant result indicates that the Dilleniae radix extract has a potential for developing a new cytotoxic agent. In addition, the authors explained how the investigated extract exhibited the cytotoxic effect. The dichloromethane extract of Dilleniae radix induced the G0/G1 and G2/M phase cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 cells probably via the up-regulation of NF-κB, JNK1 and down regulation of the anti apoptotic genes AKT1 and ERK1 (70). Additionally, other studies confirmed the signalling pathway responsible for cytotoxicity of Dillenia suffruticosa extract on breast cancer cells (71, 72). Qualitative phytochemical screening of Dillenia suffruticosa extracts indicated the presence of saponins, triterpenes, sterols, and polyphenolic compounds, which contribute to the cytotoxic activities (72).

Journal of BIOLOGICAL RESEARCHES

ISSN: 08526834 | E-ISSN:2337-389X

Volume 22| No. 2| June | 2017

http://dx.doi.org/10.23869/bphjbr.22.2.20177

Published by © PBI East Java. Open Access  www.berkalahayati.org 74

 Corresponding Author:

Ridesti Rindyastuti

Purwodadi Botanic Garden, Indonesian Institute of Sciences.

Jl. Raya Surabaya-Malang Km. 65, Purwodadi, Pasuruan, Jawa Timur Phone: 0343-615033 Fax: 0341-426046

e-mail: ride17@gmail.com

Original Article

Carbon storage of medium-sized tree: a case study on Dillenia collection in Purwodadi Botanic Garden

Ridesti Rindyastuti

Purwodadi Botanic Garden, Indonesian Institute of Sciences Abstract

Abstract

Dillenia is a medium-sized tree which has high species diversity in tropical regions especially in Southeast Asia. Dillenia in Purwodadi Botanic Garden are collected from native habitats in Java, Kalimantan, Sulawesi and Papua which planted on the area of 17 x 55 m2. The purpose of this re-search is to study the above ground carbon storage in Dillenia collection in Purwodadi Botanic Garden. Carbon storage estimation was established by measuring stem carbon stocks from plant collections with plant age ranging between 12-30 years. Twelve years old collection contributed carbon storage of 7.35 tonnes/ha for D. sumatrana. Twenty years old species had the lowest carbon storage of 2.17 kg/plant for D. serrata and the highest of 51.9 kg/plant for D. auriculata with a range of carbon storage of 3.47 to 41.072 tonnes/Ha. Thirty years old plant contributed 39.465 kg/plant and carbon storages of 63.14 tonnes/ha for D. serrata and 135.59 kg/plant and 216.94 tonnes/ha for D. philipinensis. Overall, Dillenia collections in Pur-wodadi Botanic Garden contributed 793.94 kg carbon storages, store carbon on average of 30.54 kg/plant and 46.46 tonnes/ha. The increase of carbon storage in the second 10 years was higher than in the first 10 years. It indicated that Dillenia had growth strategy in the early growth then alocated more mass after 10 years. Carbon storage of Dillenia was high and different in age. D. serrata, D. papuana and D. auriculata are recommended species as a priority in planting trees based on carbon sink.

Keywords: Dillenia, carbon storage, medium-sized, tree, Purwodadi 

Received: 09 March 2017 Revised: 28 April 2017 Accepted: 08 June 2017

Introduction

According to Hairiah et al. (2011), carbon sequestra-tion is the ability of a system to store carbon from the atmosphere during a certain period. Plants through the process of photosynthesis have a function as an absorber of carbon emissions in the atmosphere and store it in the form of biomass. Generally, there are two kinds of carbon sequestration, which is below ground such as by soil microbes and roots, and above ground plant biomass mainly by tree stems (De Jong dkk., 1995; Cannell, 2003; IPCC, 2006). Climate change mitigation innitiatives recommend the reforestation programs and diversification of plant species for improving the ecosystems quality of degraded areas and for greening urban areas (IPCC, 2006; UN-FCCC, 2007). Selection of plant species in agroforestry programs based on carbon sink is important because plants could run various ecological functions and increase plants diversity as well (Diaz, 2009). Native species was proved of running more diverse environmental services compared to non-native species. In addition to its function as a carbon sink, native species are able to maintain the hydrology of an area, restore the food chain and native vegetation by associating with other local species.

Researches on carbon stocks of diverse plant species or groups, both native and non-native species in their nat-ural habitat have been carried out. Siregar and Darmawan (2011) examined the carbon sequestration of Dipterocar-paceae in Central Kalimantan, and argued that the dipter ocarp forests can store carbon 928.86 tonnes C/ha or 20.64 tonnes/plant. In the dipterocarp family, the varia-tions of their carbon storages are quite high. This can be caused by different species constituent, genetic factors that influence the allocation of nutrient storages and allocations, plant age and environmental factors (Diaz, 2009). Imiliyana et al. (2012) reported that mangrove is capable of storing 232.59 tonnes C/ha while according to Pramudji (2011) Acacia is capable of storing 56.05 tonnes C/ha.

Dillenia is one of medium-sized tree which has high species diversity in the tropics. This plant group has about 60 species distributed from Madagascar to Australia and is one of a vegetation component of tropical forest in low land areas. The leaves are oval to elliptical with promi-nent leaf midrib. The flowers are large with five petals and many stamens (Lemmens and Wong, 1995). Dillenia is utilized for many economic purposes such as wood products (most of Dillenia species), craft, and medicine such as Dillenia suffruticosa for anti-inflamatory (Shah et al., 2015), D. philiphinensis and D. indica for antimicrobial (Ragasa, 2009; Apu et al., 2010). Many previous studies on tree carbon storage did not use the growth size as a diagnostic characters to distinguish carbon stored in plants. Growth size of trees is controlled by gene and it influences common maximum tall and large of tree stem in which carbon stored. A group of trees which have a characteristic as medium-sized tree commonly grow to around 10-40 m tall, while group of large trees can grow up to 50 m tall even hundred m tall (Lemmens and Wong, 1995). The study on carbon storages of local plant species based on its growth size can increase the understanding of biology of trees and reveal the significant contributions of diverse plant species to carbon sequestration in reducing global carbon rising in the atmosphere.

Heng and Onichandran (2014), reported that Dille-nia suffruticosa in degraded area in Sarawak, Malaysia has slightly low biomass and carbon storage in the early forest formation (5.2 tonnes/ha). However, the highest proportion of biomass on this species was on the stem, and it increased with tree size and age. Dillenia can be a good recommendation for afforestation for both conserva-tion and tree planting in urban areas, but its carbon storage have not been studied well. Therefore, the aim of this research is to study 1) carbon storage of a medium-sized tree in a case of Dillenia collection in Purwodadi Botanic Garden and 2) the effect of different age on the carbon storage of Dillenia collection in a garden system, especially in Purwodadi Botanic Garden.

Method

Plant Materials

Dillenia collection examined in this study comprised of 10 species. The collections were planted on an area approximately of 17 x 55 m2. List of plant species studied together with the origin of the collection and distribution of the species are presented in Table 1.

Table 1. Species list, origin of plant collection and plant distribution of Dillenia collection in Purwodadi Botanic Garden.

(Source : Catalog of plant collection in Purwodadi Botanic Garden-LIPI, 2012; Lemmens and Wong, 1995)

Biomass measurement

Biomass was measured using allometrics method which can be estimated from stem diameter at breast height of adult trees so called DBH and trees height of each Dillenia collection in Purwodadi Botanic Garden-LIPI. Three replications were used in the measurement for each species. Biomass was obtained by calculating using the formula of biomass for plants in dry climates habitat and allometric equation for Dillenia as follows:

(Hairiah et al., 2010)

Biomass (kg) = 0,122 (rD2H)0.916

Where r = wood density (Zanne et al., 2009)

D = diameter (cm)

H = plant height (m)

Carbon stock values were obtained by multiplying the values of biomass with allometric values for carbon stock i.e. 0.46 (Hairiah et al., 2010). Carbon storage per hectare was converted using the total of area and plant spacing of collection in Purwodadi Botanic Garden i.e. 2.5 x 2.5 m.

Data Analyses

Biomass and carbon stocks of 10 Dillenia species were analyzed using the variance test (ANOVA) at the 95% confidence level to determine the variation of bio-mass and carbon stocks among species in Dillenia collec-tions. The data analyses were also conducted on the bio-mass and carbon stocks among species in different age level (12, 20 and 30 years).

Results

Carbon Storage in Dillenia

Table 2 showed the carbon storage of Dillenia collection in Purwodadi Botanic Garden estimated from their DBH and biomass. The statistical analyses are also conducted to show the differences of carbon stocks among Dillenia species. Based on the ANOVA test on a 95% confidence level, DBH of 10 species of Dillenia were significantly different amongs species with a P value of 0.004 (P <0.05). The statistical tests were conducted on the DBH of individuals in various age ranges (12-30 years). The data of biomass and carbon stocks were ab-normal based on the normality test on a confidence level of 95%. Therefore, the data only can be read descriptive-ly. The 30 years old D. philipinensis have the highest DBH while the twelve years old D. sumatrana has the lowest DBH. The species which are 20 years old have a range of DBH between 18.15 cm for D. ovalivolia to 54 cm for D. papuana. It showed that in the same age, plant species within genus may have different DBH and carbon storage.

Table 2. The value of DBH, biomass, per plant and per ha carbon stocks of 10 species of Dillenia in Purwodadi Botanic Garden.

*) Statictical analyses using ANOVA in the confidence level of 95%. Same letters in one column showed no significant  ifference among species 

**) These data are not normal and not random based on the normality and randomity test, thus the variance analysis can not be performed.

D. philipinensis has the highest biomass (29.47 kg) and contribute the total carbon storage of 135.58 kg C/plants or 216.94 tonnes C/ha. Twelve years old D. su-matrana has DBH 21.93 cm with 0.99 kg of biomass and carbon stocks of 4.59 kg/plant or 7.35 ton C/ha. This size is quite large compared to other 20 years old species (Fig 1 and Table 2). Figure 3 showed the carbon stored per hectare of Dillenia collection in Purwodadi Botanic Garden. Carbon storage of D. ovalifolia is the lowest among other species studied especially compared to other species at the same age (20 years old). At 20 years old, D. papuana has the highest carbon storage of 51.9 kg C/plant.

Medicinal uses, chemistry and pharmacology of Dillenia species (Dilleniaceae)

1. Introduction

The genus Dillenia belongs to the Dilleniaceae family and contains approximately 100 known species (Lim, 2012). According to The Plant List (2013), as many as 175 scientific plant names from the genus Dillenia have been recorded with 58 accepted names (low and medium confidence levels) and 71 names of synonym species.
The name Dillenia is derived from Joannes Jacobus Dillenius, a British botanist who dedicated his efforts in the field of taxonomy of this genus (Quattrocchi, 2012). Dillenia species are monoecious plants which produce attractive flowers and yellow fruits. D. indica is known for its lemon-flavored fruits that are use to make jellies and curries. These species are evergreen and deciduous trees or shrubs of disjunct distribution in the seasonal tropics of Madagascar through South and South East Asia, North Australia, and Fiji (Dickison, 1979; Horn, 2007; Kerrigan et al., 2011; Lim, 2012). They grow from sea level to an elevation of about 2000 m. The plants also grow in forests, and several species show an adaptation to temporary flooded situations. They are mostly trees that form large leaves and flowers in few-flowered inflorescences (Dickison, 1979). Their barks are unique in fine colors of red, grayish, and reddish brown that are used in furniture making (Hoogland, 1952). Several species of this genus produce sweetishsour and astringent edible fruits (Hoogland, 1952; Jansen et al.,
1992; Kerrigan et al., 2011; Lim, 2012; Saha and Sundriyal, 2012) and are cultivated as ornamental plants (Hoogland, 1952; Kerrigan et al., 2011).
Based on our extensive search regarding medicinal uses, chemical constituents, and biological activities of the species from the genus Dillenia, only very few species have been described thus far. Starting from 1962 to the present, 19 species of the genus Dillenia have been reported for their medicinal uses and their phytochemistry. These 19 species are Dillenia andamanica C.E.Parkinson, D. aurea Sm., D. bracteata Wight, D. excelsa (Jack) Martelli ex Gilg., D. indica L., D. ovata Wall. ex Hook.f. & Thomson, D. papuana Martelli, D. parviflora Griff., D. pentagyna Roxb., D. philippinensis
Rolfe, D. pulchella (Jack) Gilg., D. reticulata King, D. retusa Thunb., D. scabrella (D.Don) Roxb. exWall., D. eximia Miq., D. serrata Thunb., D. suffruticosa (Griff.) Martelli, D. sumatrana Miq., and D. triquetra (Rottb.) Gilg. However, out of these, only 7 species have been evaluated for their biological activities. As part of our search for natural anti-inflammatory compounds, three triterpenes have been isolated from D. serrata and they performed pronounced inhibitory activity on the production of prostaglandin E2, which is known as a predominant inflammatory mediator (Jalil et al., 2015). This review describes the current state of their medicinal properties, chemistry, and pharmacological aspects.

4.1. Antimicrobial activity

Some of Dillenia species were investigated for antibacterial, antifungal, and antiviral activities. The extracts and fractions of D. indica, D. papuana, D. pentagyna, D. suffruticosa, and D. sumatrana were reported to possess growth inhibition against Gram positive and negative bacteria (Table 5). However, they were found to exhibit weak growth inhibition against tested fungi, including Aspergillus fumigatus, A. niger, Candida albicans, C. arriza, C. crusei, Penicillium sp., Rhizopus oryzae, Saccharomyces cerevisiae, and Trichoderma viride (Nick et al., 1995a; Wiart et al., 2004; Haque et al., 2008; Apu et al., 2010; Smitha et al., 2012). Seven triterpenoids (37, 42e45 and 47e48) from Dillenia plants were proven to have antimicrobial action (Nick et al., 1994, 1995b; Ragasa et al., 2009).

Strong growth inhibition against Escherichia coli and Bacillus subtilis has been shown by 45 that was assayed using the bioautographic method on TLC plate. Meanwhile, 48 was the most active against the growth of Micrococcus luteus (Nick et al., 1995b). Similar pronounce growth inhibition of 48 also reported towards E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and B. subtilis (Ragasa et al., 2009). These findings suggested that Dillenia plants traditional uses for the therapeutic remedies of microbial infectionrelated diseases such as diarrhea, dysentery, septicaemical infection and skin-related diseases had a potential as antimicrobial agent, which supported their traditional uses for the therapeutic remedies of microbial infectionrelated diseases such as diarrhea, dysentery, septicaemical infection and skin-related diseases

Antibacterial and antifungal

The leaves extract inhibited the growth of B. cereus, B. subtilis, P. aeruginosa, and C. albicans with zones ranging from 7 to 9 at 1 mg/disc as compared to gentamycin (10 mg/disc; 18e20 mm for B. cereus, B. subtilis, P. aeruginosa) and nystatin (20 mg/disc; 11 mm for C. albicans).

The effect of herbal plant extracts on the growth and sporulation of Colletotrichum gloeosporioides

Johnny et al. .J. Appl. Biosci. 2010.                                                                                    The effect of herbal plant extracts on Colletotrichum gloeosporioides

Journal of Applied Biosciences 34: 2218 - 2224

ISSN 1997–5902

The effect of herbal plant extracts on the growth and

sporulation of Colletotrichum gloeosporioides

Lucy Johnny*, Umi Kalsom Yusuf, and Rosimah Nulit

Department of Biology, Faculty of Science, University Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan,

Malaysia.

*Author for correspondence: lucyjohnny13784@yahoo.com, Tel. +6017-3434717; Fax: +603-86567454

Original submitted on 22th July 2010. Published online at www.biosciences.elewa.org on October 7, 2010

ABSTRACT

Objectives: The antifungal activities of the leaf extracts of fifteen selected medicinal plants; Alpinia galanga L., Alstonia spatulata Blume., Annona muricata L., Blechnum orientale L., Blumea balsamifera L., Centella asiatica L., Dicranopteris linearis, Dillenia suffruticosa, Litsea garciae Vidal., Melastoma malabathricum L., Momordica charantia L., Nephrolepis biserrata (Sw.)., Pangium edule Reinw., Piper betle L., and Polygonum minus Huds., were evaluated on the plant pathogenic fungus; C. gloeosporioides isolated from mango.

Methodology and results: Different antifungal assays were employed, i.e. Agar-Disc Dilution assay as

primary screening assay, followed by determination of Minimum Inhibition Concentration (MIC), and the rate of sporulation assay. The antifungal assay was carried out in Potato Dextrose Media in five different treatments, i.e.; distilled water as negative control, crude extract of leaves in methanol, chloroform, acetone and Benomyl as positive control. A. galanga extracts were most effective and exhibited highest antifungal activities against C. gloeosporioides. Methanol crude extract reduced radial growth of C. gloeosporioides by 66.39%, followed by chloroform crude extract 63.26%, and 61.56% for acetone crude extracts. The exact concentrations that have definite potential to fully restrict the growth of C. gloeosporioides (MICs) for A. galanga is 15.00 mg/mL in methanol, 17.50 mg/mL in chloroform, and 17.50 mg/mL in acetone. The sporulation assay also revealed that A. galanga leaves crude extracts showed highest inhibition of spore germination of C. gloeosporioides overall at concentration of 10 mg/mL; with 68.89% inhibition by methanol extracts, 64.13% by chloroform extracts, and 62.86% by acetone extracts.

Conclusion and application of findings: Numerous natural products of plant origin are pesticidal and have the potential to control fungal diseases of crops. Thus, considerable effort should be devoted to screening plants in order to develop new natural fungicides as alternative to existing. In this study, the leaf crude extracts of A. galanga exhibited effectiveness against C. gloeosporioides and should be considered for further evaluation.

Key words: Medicinal plants, crude extracts, anti-fungal growth, C. gloeosporioides

INTRODUCTION

Colletotrichum gloeosporioides are one of the most important pathogens affecting the flowers and fruits of mango trees causing anthracnose worldwide. In areas where rain is prevalent during flowering and fruit set, anthracnose can cause destruction of the inflorescences and infection and drop of young fruit where this can obviously lead to serious losses, reaching up to 35% of the harvested fruit (Martinez et al., 2009). Excessive use of benomyl, thiophanate-methyl and thiobendazole as pre- and post-harvest sprays has led to a reduction in effectiveness in certain areas where pathogen resistance to fungicides has been reported (Spalding, 1982). Indiscriminate use of the chemicals is not only hazardous to people but also disrupt the natural ecological balance by killing the beneficial soil microbes (Ansari, 1995). So, alternatives have to be developed to control anthracnose in order to guarantee safe food production as well as reduce environmental pollution. The integration of a number of practices aiming to reduce or eliminate negative side effects caused by chemicals used for controlling major mango diseases is the most realistic option for solving the problem (Chowdury and Rahim, 2009). Research work in relation to anthracnose disease management of mango is yet to develop effective alternative/options.

Hence, this study was carried out with the aim of providing broader options by evaluating the antifungal activity of crude leaf extracts from selected medicinal plants against phytopathogenic fungi Colletotrichum capsici and Colletotrichum gloeosporioides.

MATERIALS AND METHODS

The leaves samples were collected from Sarikei, Sarawak. Samples were dried in the oven at 50ºC until the leaves become crunchy and were weighed. 100 g of leaves samples were then pounded using mortar and pestle into coarse powder. Leaves of the plants were extracted in polar solvent (methanol), semi-polar solvent (chloroform), and non-polar solvent (acetone) by soaking in each solvent for at least 48 hours. The extracts solution was filtered with filter paper (Whatman No. 1), transferred into pre-weighed 250mL round bottom flasks and evaporated using Rotavapor. The extracts and the round bottom flask were weighed again after solvent evaporation.

Pathogen culture: The culture of Colletotrichum gloeosporioides from Mangifera india L. were obtained from Faculty of Agriculture, UPM. Pure cultures were maintained on Potato Dextrose Agar slants.

Antifungal assay: The antifungal assay was carried out according to Alam (2004) with a slight modification.

A volume of 19 mL of molten PDA was poured in sterilized Petri dishes along with 1 mL of plant extract and plated. Mycelial discs (10 mm diameter) made using the cork borer were inoculated at the centre of the medium. The antifungal assay was carried out in PDA in five different sets: negative control, crude extract of leaves in methanol, chloroform, and acetone, and positive control (commercial fungicide). Colony growth was measured on the basis of linear dimensions. Minimum inhibitory concentration (MIC) and sporulation were determined.

Sporulation was determined by adding 10ml sterile distilled water to each seven days old plate that were obtained from agar disk method and gently scraping the mycelia with a sterile glass rod to dislodge the spores.

The spore suspensions obtained were filtered through sterile cheesecloth into a sterile 50 mL glass beaker and homogenized by manual shaking. The spores were then counted using a haemocytometer.

The percentage reduction (Sr) or stimulation (Ss) of sporulation by each extract was determined using the following formula, (Nduagu et al., 2008):

Sr = (S1 – S2) x 100

              S1

Where;

Sr = Percentage of reduction in sporulation;

S1 = Sporulation on the untreated medium (control);

and

S2 = Sporulation on the treated medium.

Ss = (S2 – S1) x 100

              S2

Where;

Ss = Percentage of stimulation in sporulation;

S2 = Sporulation on treated medium; and

S1 = Sporulation in untreated medium.

RESULTS

Inhibition of radial growth of Colletotrichum gloeosporioides: Among the plants screened, only 5 species showed 50% or more antifungal activity against C. gloeosporioides at varying concentrations (Table 1).

Methanol crude extracts of A. galanga, P. betle, M. malabathricum, B. balsamifera, and P. minus were

most effective. A. galanga exhibited highest antifungal activity of 66.78% at 10.00 Hg/mL, 66.27% at 1.00 Hg/mL, 61.57% at 0.10 Hg/mL, and 60.55% at 0.01 Hg/mL against C. gloeosporioides. This was followed by M. malabathricum and P. Betle, whose inhibition activity increased as the concentration of plant extracts increased (Table 1).

Table 1: Inhibiton of radial growth (mm) of Colletotrichum gloeosporioides by varying concentrations of leaf extracts in methanol.

Extracts of A. galanga in chloroform also showed high inhibition against C. gloeosporioides of 63.69% at 10.00 Hg/mL, 61.43% at 1.00 Hg/mL, 60.78% at 0.10 Hg/mL, and 58.62% at 0.01 Hg/mL. This was followed by chloroform crude extract of P. betle, M. malabathrichum, and B. balsamifera (Table 2). Extracts of A. galanga in acetone inhibited growth of C. gloeosporioides 62.60% at 10.00 Hg/mL, 60.50% at 1.00 Hg/mL, 57.05% at 0.10 Hg/mL, and 54.67% at 0.01 Hg/mL (Table 3).

Minimum Inhibition Concentration (MIC) of plant crude extracts to C. Gloeosporioides: Crude extracts of Alphinia galanga in all three solvents; methanol, chloroform and acetone exhibited the lowest MIC value against C. gloeosporioides of 15.0 Hg/mL in methanol, 17.50 Hg/mL in chloroform and acetone. This was followed by crude extracts of both P. betle and M. malabathricum which exhibited no visible growth of C. gloeosporioides at 17.50 Hg/mL and 20.00 Hg/mL. Crude extracts of C. asiatica, D. suffruticosa, A. muricata, D. linearis, A. spatulata, L. garciae, P. edule, and N. bisserrata in all three solvents exhibited MIC values that were out of the range of the prepared concentrations. Crude extracts of B. orientale did not exhibit any antifungal activities against C. gloeosporioides.

Table 2: Inhibiton of radial growth of C. gloeosporioides by varying concentrations of leaf extracts in chloroform.

Sporulation : The highest inhibition was recorded in treatments of crude extracts of A. galanga which exhibited the highest antifungal activities in inhibiting the sporulation of C. gloeosporioides among the 15 medicinal plants (Table 4-6). The methanol crude extract of A. galanga at 10.00 Hg/mL exhibited the highest inhibition overall of 68.89%. At the lowest concentration of 0.01 Hg/mL of acetone crude extract, A. galanga still inhibited sporulation by 62.86%.

Table 3: Inhibiton of sporulation (x105) of C. gloeosporioides by varying concentration of leaf extracts in acetone.

Table 4: Inhibiton of sporulation (x105) of C. gloeosporioides by varying concentration of leaf extracts in methanol.

Table 5: Inhibiton of sporulation (x105) of C. gloeosporioides by varying concentrations of leaf extracts in chloroform.

Table 6: Inhibiton of sporulation (x105) of C. gloeosporioides by varying concentrations of leaf extracts in acetone.

Each value represented the mean ± standard error; NI = No Inhibition, * represented crude extracts effectively inhibits growth

DISCUSSION

The results obtained from the different studies showed that the crude extracts of Alpinia galanga leaves exhibited the highest antifungal activities against C. gloeosporioides. The inhibition was expressed as reduced radial growth, dry mycelial weight, and sporulation germination rate of the pathogen. Inhibitory action of A. galanga crude extracts was recorded even at very low dose, which is a clear indication that the crude extracts contain active commencements that have antifungal properties.

Antimicrobial activity of extract from A. galanga could be attributed to its chemical constituents as reported by Saritnum and Sruamsiri (2003). Phongpaichit and Liamthong (2001) mentioned that chavicol acetate that has been identified as an antifungal component from A. galanga plays an antifungal role as in the Alpinia conchigera oil which has antifungal activity against C. gloeosporioides. The ethanol extracts from A. galanga have been found to have pronounced inhibitory activities against a wide variety of human pathogenic fungi, including strains resistant to the common antifungals amphotericin B and ketoconazole (Elfahmi, 2006). 1'-Acetoxychavicol acetate, the active compound from A. galanga has antifungal activity against Trichophyton mentagrophytes, T. rubrum, T. concentricum, Rhizopus stolonifer and Aspergillus niger at a concentration 14 mg/mL (Janssen and Scheffer, 1985). In agreement with this, Abad et al. (2007) mentioned that the chloroform extract of A. galanga has pronounced antifungal activity against Cryptococcus neoformans, Microsporum gypseum and Trichophyton longifusus. Many of the medicinal plants selected for this study have been reported to have novel bioactivities. Piper betle exhibited the best antifungal activities against C. gloeosporioides after Alpinia galanga. Johann et al. (2007) stated that the chemistry of Piper species has been widely investigated and phytochemical investigations from different parts of the world have led to the isolation of a number of physiologically active compounds such as alkaloids/amides, propenylphenols, lignans, neolignans, terpenes, steroids, kawapyrones, piperolides, chalcones, di-hydrochalcones, flavones and flavanones which exhibited high antimicrobial and antifungal properties. According to Lee et al. (2004), most Piper chemistry has been conducted to find potential pharmaceuticals or pesticides, and over 90% of the literature focuses on compounds that are cytotoxic, antifungal, antitumor, fragrant, or otherwise useful to humans.The leaves of Blumea balsamifera contain icthyothereol acetate and cryptomeridiol, lutein, and K-carotene (Ragasa, 2004). Antimicrobial tests indicated that icthyothereol acetate has moderate activity against Aspergillus niger, Trichophyton mentagrophytes and Candida albicans (Ragasa, 2004).

In conclusion, the crude extracts of leaves that exhibited good potential as fungicides of C. gloeosporioides should be evaluated further in in-depth to study the phytoextracts for their potentiality under in vivo condition.

ACKNOWLDEGEMENTS: Author acknowledges with gratitude the Graduate Research Fellowship (GRF) of Universiti Putra Malaysia.

REFERENCES

Abad MJ, Ansuategui M, Bermejo P, 2007. Active antifungal substances from natural sources. ARKIVOC. Vol. VII: 116-145.

Alam S, 2004. Synthesis, antibacterial and antifungal activity of some derivatives 2-phenyl-chromen-4-one. J. Chem. Sci. Vol. 116: 325-331.

Ansari MM, 1995. Control of Sheath blight of rice by plant extracts. Indian Phytopath. 48(3): 268-270.

Chowdury MNA. and Rahim MA, 2009. Integrated crop management to control anthracnose (Colletotrichum gloeosporioides) of mango. Journal of Agriculture and Rural Development 7(1&2): 115-120.

Elfahmi, 2006. Phytochemical and biosynthetic studies of lignans with a focus on Indonesian medicinal plants. PhD thesis. University of Groningen.

Janssen AM. and Scheffer JJC, 1985. Acetoxychavicol acetate, an antifungal component of Alpinia galanga. Planta Medica 6: 507-511.

Johann S, Pizzolatti MG, Donnici CL, Resende MA, 2007. Antifungal properties of plants used in Brazilian traditional medicine against clinically relevant fungal pathogens. Brazilian Journal of Microbiology 38: 632-637.

Khewkhom N, Shangchote S, Greger H, 2007. In vitro antifungal activity of some well-known spices against plant pathogenic fungi. Agricultural Sci. J. 38 (6) (Suppl.): 70-74.

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Martinez EP, Hio JC, Osorio JA, Torres MF, 2009. Identification of Colletotrichum species causing anthracnose on Tahiti lime, tree tomato and mango. Agronomia Colombiana. Vol. 27 (2): 211-218.

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Phongpaichit S. and Liamthong S, 2001. Antifungal activities of plant extracts against Colletotrichum gloeosporioides (Penz.) Sacc. J. Natl. Res. Council Thailand. 33 (1): 55-68.

Ragasa CY, 2004. Isolation, structure elucidation, and antimicrobial assay of secondary metabolites from five Philippine medicinal plants. The URCO Digest. VI (2): 3.

Saritnum O. and Sruamsiri P, 2003. Random amplified polymorphic DNA analysis of galanga (Alpinia spp.) accessions. CMU Journal 2 (3): 159-164.

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Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia indica Linn.

Apu AS, Muhit MA, Tareq SM, Pathan AH, Jamaluddin ATM, Ahmed M

Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka,

Dhaka 1000, Bangladesh

Address for correspondence: Mr. Apurba Sarker Apu; E-mail: apurba2sarker@yahoo.com

ABSTRACT

The crude methanolic extract of Dillenia indica Linn. (Dilleniaceae) leaves has been investigated for the evaluation of antimicrobial and cytotoxic activities. Organic solvent (n-hexane, carbon tetrachloride and chloroform) fractions of methanolic extract and methanolic fraction (aqueous) were screened for their antimicrobial activity by disc diffusion method. Besides, the fractions were screened for cytotoxic activity using brine shrimp (Artemia salina) lethality bioassay. Among the four fractions tested, n-hexane, carbon tetrachloride, and chloroform fractions showed moderate antibacterial and antifungal activity compared to standard antibiotic, kanamycin. The average zone of inhibition was ranged from 6 to 8 mm at a concentration of 400 mg/disc. But the aqueous fraction was found to be insensitive to microbial growth. Compared to vincristine sulfate (with LC50 of 0.52 mg/ ml), n-hexane and chloroform fractions demonstrated a significant cytotoxic activity (having LC50 of 1.94 mg/ml and 2.13 mg/ml, respectively). The LC50 values of the carbon tetrachloride and aqueous fraction were 4.46 mg/ml and 5.13 mg/ ml, respectively. The study confirms the moderate antimicrobial and potent cytotoxic activities of Dillenia indica leaves extract and therefore demands the isolation of active principles and thorough bioassay.

Key words: Antimicrobial activity, Artemia salina, brine shrimp lethality bioassay, Dillenia indica

DOI: 10.4103/0975-1483.62213

INTRODUCTION

The genus Dillenia has 60 species, of which Dillenia indica Linnaeus (Family: Dilleniaceae) is the most common edible species. Originally from Indonesia, this evergreen tropical tree is now found from India to China. The common names include Chulta (Bengali, Hindi), Bhavya (Sanskrit) and Elephant apple (English). It is a spreading tree and has beautiful white fragrant flowers, toothed leaves, and globose fruits with small brown seeds.[1] The leaf, bark, and fruit of this plant are used as traditional medicine. The juice of D. indica leaves, bark, and fruits are mixed and given orally (5-15 ml, two to five times daily) in the treatment of cancer and diarrhea.[2] The fruit juice of this plant has

cardiotonic effect, used as cooling beverage in fever and also employed in cough mixture.[3] The solvent extracts of fruits of D. indica are reported to have antioxidant activity. [4] CNS depressant activities in mice were found from the alcoholic extract of the leaves of D. indica.[5] Considering the traditional uses of D. indica plant parts, leaves can be the source of many modern medicines.

A survey of the published literature shows that there are a number of different methods used for the assessment of antimicrobial activity; however, there is no one method that is used by all researchers and no inclusive study to determine which one is the best method for in vitro assay.[6] Majority of the researchers uses one of the three following methods for the assessment of antimicrobial activity: Disc diffusion, agar dilution, and broth dilution/microdilution method. The disc diffusion method (also known the zone of inhibition method)[7] is probably the most widely used of all methods used for testing antibacterial and antifungal activity.[6] It requires only small amounts of the test substance (10-30 ml), can be completed by research staff with minimal training, and as such may be useful in field situations.[6] Several researchers have used the method to identify the antibacterial and antifungal activities of the plant extracts,[8] compounds isolated from plants,[9] and also to find out the antimicrobial resistant strains of microorganisms.[10,11] It is important to note that the disc diffusion method demonstrated activity in vitro does not always translate to activity in vivo.[6]

The brine shrimp lethality bioassay is rapid (24 h), simple (e.g., no aseptic techniques are required), easily mastered, inexpensive, and requires small amounts of test material (2‑20 mg or less).[12] The bioassay has a good correlation with cytotoxic activity in some human solid tumors and with pesticidal activity.[12,13] This test was proposed by Michael et al.[14] and modified by others.[15,16] Since its introduction, this in vivo lethality test has been successively employed for providing a frontline screen that can be backed up by more specific and more sophisticated bioassays once the active compounds have been isolated.

The objective of this research work was to investigate the antimicrobial and cytotoxic activities of the different solvent fractions of crude methanolic extract of D. indica leaves.

MATERIALS AND METHODS

Collection of plant material

The plant sample of D. indica was collected from Rangpur, Bangladesh, in the month of March 2007. The plant was identified and a voucher specimen (Accession number DACB 34359) representing this collection has been deposited in the Bangladesh National Herbarium, Dhaka, for further reference.

Preparation, extraction and fractionation of plant material

The freshly separated leaves of the plant were cut into small pieces, sun dried, and subsequently dried in the oven for 24 h at low temperature to grind these into coarse powder (40-mesh).

About 500 g of powdered leaves was taken in a 5 l round bottom flask and soaked in 2 l of methanol. The container with its content was sealed with cotton plug and aluminum foil and kept at room temperature for a period of 3 days accompanying occasional shaking and stirring. The extract was filtered through fresh cotton plug followed by Whatman No.1 filter paper. The filtrate was then concentrated and dried by a rotary evaporator (Heidolph, UK) at low temperature (398C). The weight

of the crude extract thus obtained from leaves was 7 g.

Solvent-solvent fractionation of the crude methanolic extract was conducted by using the protocol designed by Kupchan[17] and modified by Wagenen et al.[18] 5 g of the obtained methanolic crude extract was triturated with 90% methanol. The prepared solution was then fractionated successively using solvents of increasing polarity, such as, n-hexane (HX), carbon tetrachloride (CT), and chloroform (CF). The aqueous methanolic fraction was preserved as aqueous fraction (AQ). All the four fractions were evaporated to dryness by using rotary evaporator and then kept in beakers for further analysis (HX 820 mg, CT 550 mg, CF 665 mg and AQ 400 mg).

Antimicrobial screening

Antibacterial and antifungal activities of crude extracts were tested by the paper disc diffusion method.[7] Thirteen bacterial strains, which included 5 gram-positive and 8 gramnegative organisms, and 3 fungi collected from the Institute of Nutrition and Food Science (INFS), University of Dhaka, Bangladesh, as pure cultures were used. Microorganisms were maintained on the nutrient agar medium (Merck, Germany). 

The sterile Matricel (BBL, cocksville USA) 6.0 mm filter paper discs were impregnated with 400 mg of each of the sterile test substances and dried to evaporate the residual solvent (methanol). Standard kanamycin discs (30 mg/ disc) were used as positive control to ensure the activity of standard antibiotic against the test organisms. The sample discs, the standard antibiotic discs, and dried blank disc impregnated with methanol (negative control) were placed gently on the previously marked zones in the agar plates pre-inoculated with the test bacteria and fungi. The plates were then kept in a refrigerator at 4oC for about 24 h upside down to allow sufficient diffusion of the materials from the discs to the surrounding agar medium. The plates were then inverted and kept in an incubator at 378C for 24 h.

The antimicrobial activity of the test agents were measured by their activity to prevent the growth of the microorganisms surrounding the discs which gave clear, distinct zone of inhibition. The antimicrobial activity of the test agents was determined by measuring the diameter of zone of inhibition expressed in mm.[6]

Brine shrimp lethality bioassay

The brine shrimp lethality bioassay was used to predict the cytotoxic activity[15,19] of the n-hexane, carbon tetrachloride, chloroform, and aqueous fractions from methanolic crude extracts. For the experiment, 4 mg of each of the extracts was dissolved in dimethylsulfoxide (DMSO) and solutions of varying concentrations (400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 mg/ ml) were obtained by the serial dilution technique using simulated seawater. The solutions were then added to the pre-marked vials containing 10 live brine shrimp nauplii in 5 ml simulated seawater. After 24 h, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted.

The mortality endpoint of this bioassay was defined as the absence of controlled forward motion during 30 s of observation.[20] From this data, the percent of lethality of the brine shrimp nauplii for each concentration and control was calculated. An approximate linear correlation was observed when logarithm of concentration versus percentage of mortality[21] was plotted on the graph paper and the values of LC50 were calculated using Microsoft Excel 2003 [Figure 1].

Vincristine sulphate was used as positive control.

Figure 1: Plot of log concentration of n-hexane (— 􀌆 —), carbon

tetrachloride (— O —), chloroform (— Δ —) and aqueous (— 3 —) fraction

of methanolic extract versus percent shrimp mortality after 24 h of exposure

RESULT AND DISCUSSION

With the exception of aqueous fraction, all the other fractions of D. indica leaves were active against most of the tested organisms [Table 1]. The average zone of inhibition produced by the n-hexane, carbon tetrachloride, and chloroform fraction was ranged from 6-8 mm, 7-8 mm, and 6-7 mm, respectively, at a concentration of 400 mg/ disc. Against the Escherichia coli, only chloroform fraction was active (zone of inhibition was 7 mm) and carbon tetrachloride fraction exhibited highest antimicrobial activity compared to other solvent fractions. In both the cases of bacteria and fungi, the zone of inhibition was found to be 6-8 mm.

The LC50 values obtained from brine shrimp lethality bioassay [Tables 2 and 3] were 1.94, 4.46, 2.13, and

Table 1: Antimicrobial activity of chloroform, carbon tetrachloride, n-hexane, and aqueous fraction of methanolic extract of Dillenia indica leaves and positive control kanamycin

Table 2: Effect of n-hexane, carbon tetrachloride, chloroform and aqueous fraction of methanolic extract and positive control vincristine sulphate on brine shrimp

Table 3: The result of cytotoxic activity of n-hexane (HX), carbon tetrachloride (CT), chloroform (CF), and aqueous (AQ) fraction of methanolic extract and positive control vincristine sulphate (VS) on brine shrimp

5.13 mg/ml for n-hexane (HX), carbon tetrachloride (CT), chloroform (CF), and aqueous (AQ) fraction, respectively. Compared to positive control (vincristine sulphate, VS, LC50 0.52 mg/ml), all the fractions tested showed good brine shrimp larvicidal activity. Again the crude extracts resulting in LC50 values less than 250 mg/ml were considered significantly active and had the potential for further investigation.[22] The cytotoxic activity exhibited by the solvent fractions was promising and this clearly indicates the presence of potent bioactive compounds.[15]

CONCLUSION

The antimicrobial and cytotoxic activities of various fractions of D. indica leaves, found in this study, may explain some of the traditional medicinal uses of this plant. These could be of particular interest in relation to find out its unexplored efficacy and can be a potential source of chemically interesting and biologically important drug candidates.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the head of Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka, Bangladesh for providing facility and moral support to conduct the research.

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Source of Support: Nil, Conflict of Interest: None declared.