Asian Pacific Journal of Tropical Medicine (2010)769-774 769

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Document heading Pharmacological activity, phytochemical analysis and toxicity of methanol extract of (torch ) flowers Subramanion Jo Thy Lachumy1, Sreenivasan Sasidharan2*, Vello Sumathy1, Zakaria Zuraini1

1School of Distance Education, Universiti Sains , Minden 11800, Pulau Pinang, Malaysia 2Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia

ARTICLE INFO ABSTRACT

Article history: Objective: Etlingera elatiorTo elucidate (E. elatior) its pharmacologicalMethods: activities and medicinal potential of extract of Received 12 July 2010 . Phytochemical screening of the flower extract was Received in revised form 17 August 2010 done to determine the phytochemical in the extract. The pharmacological study included the Accepted 15 September 2010 determination of antimicrobial activity and minimum inhibitory concentration (MIC) of metabolic Available online 20 October 2010 flower extract. The antimicrobial activity of the extract was tested against medicallyE. elatior important bacterial, yeast and fungal strains. Apart from that, the methanolic extract of flower Keywords: in vivo was further tested toxicity using the brine shrimp lethality test. Moreover, the flower Etlingera elatior extract was qualitatively screened forResults: their free radical scavenging activity by 2, 2-diphenyl-1- Pharmacological activity picrylhydrazyl radical (DPPH) assay. The extract was effective on tested microorganisms MIC 1.563-50.000 ArtemiaPhytochemical salina analysis and values were in the range of mg/mL. TheArtemia brine salina shrimp lethalityE. elatior test exhibited no significant toxicity (LC50 = 2.52 mg/mL) against . The flower extract with high LC50 value signified that this is not toxic to humans. While the phytochemical screening of the flower extract revealed the presence of the following compounds: flavonoids, terpenoids, saponin, tannins and carbohydrates whereas, alkaloids, anthraquinone and reducing sugars were absent. The concentration of the flower extract required for 50% DPPH (IC50) 9.14 8.08 inhibition of radical scavengingConclusions: effect were mg/mL and mg/mL for E.butylated elatior hydroxytoluene 8.08 mg/mL. These findings indicate that the extract of flower possesses pharmacological properties and potential to develop natural products based pharmaceuticals products.

1. Introduction flowers, leaves, stem, fruits and roots extracts are used as powerful raw drugs possessing a variety of antimicrobial and healing properties. Discovery of new pharmaceutical agents Various edible have been used for cooking or have from medicinal plants can combat the drastic increase in been eaten raw in daily life as an important therapeutic infectious diseases in many countries especially in rural aid for various ailments. From ancient times, different areas and it has been used for economic reasons as well. parts of medicinal plants have been used to cure ailments The interest in medicinal plants reflects its recognition of caused by microorganisms such as bacteria, viruses and the validity of many traditional claims regarding the value fungi. Moreover, the potential of higher plants as a source of natural products in health care[1]. Therefore, in order for new drugs is still largely unexplored. There is an to determine the potential use of herbal medicine, it is abundant of medicinal plants thr=oughout the world but essential to intensify the study of medicinal plants that find [1, 2] only small amounts are investigated for its biological and placeEtlingera in folklore . pharmacological properties. Nevertheless, today there is is a genus of Indo-Pacific terrestrial and widespread interest in drugs derived from plants. There is perennial herbs in the ginger family, , a wide range of medicinal plant parts which includes the consisting of more than 100 different species native to , , , Malaysia and widely

*Corresponding author: Dr Sreenivasan Sasidharan, Institute for Research in cultivated and neutralizedEtlingera in South EastEtlingera Asia. elatior The Molecular Medicine (INFORMM), Universiti Sains Malaysia, Minden 11800, Pulau commonly known species of is Pinang, Malaysia. (E. elatior) Tel: +604653-4820 (Office) (torch ginger) is an edible plant which is E-mail:[email protected] Subramanion Jo Thy Lachumy et al./Asian Pacific Journal of Tropical Medicine (2010)769-774 770 E. elatior widely used as cooking herbs or eaten raw for its medicinal entire extract of flower was evaporated under properties and also used as an ingredient for localEtlingera products reduced pressure using rotary evaporator. The solvent was like soap, shampoo and perfume. A total of 15 then evaporated at 50 曟 in oven to get a paste form. Then it species have been recorded in Peninsular Malaysia[3]. was sealed in Petri plates and stored at room temperature for The height of the plant is 3.6-4.7 m while all plant parts further studies. The filtrate was then re-dissolved again in including flower bud, , stem and leaves have diverse 80% methanol (v/v). biological and pharmacological properties with the potential 2.3. Test microorganisms and growth media to heal a wide range of illness,E. infections elatior and other ailments. The showy pink flowers of are very attractive and widely cultivated throughout the tropics as a spice for The following Gram-positive and Gram-negative bacteria, food flavouring and for ornamental purposes. In Peninsular E. elatior yeasts and molds were usedStaphylococcus for antimicrobial aureus, Bacillusactivity Malaysia,bunga kantanthe flower of the bungais commonly kecombrang known studies: bacteria included as , in Indonesia as or thuringiensis, Escherichia coli, Salmonella, Proteus mirabilis honje kaalaa Micrococcus Bacillus subtilis and in Thailand as . laksaThey areasam, commonly nasi kerabu used sp. and ; yeast included as ingredients of dishes such as , Candida albicans Aspergillus niger nasi ulam [4] ; molds included . The and in Peninsular Malaysia . In ariskNorth ikanSumatra, bacterial strains were grown in Nutrient Agar (NA) plates at the flower buds are used for a dish called mas 37 曟, whereas the yeast and molds were grown in Sabouraud (Szechuan pepper Spiced Carp). In Karo, it is known as asam cekala Dextrose Agar (SDA) media, respectively, at 30 曟. Then , and the flower buds are an essential ingredient sayur asam stock cultures were maintained at 4 曟. of the Karo version of , and are particularlyE. elatior suited to cooking fresh fish. In Malaysia, fruit of 2.4. Antimicrobial disk diffusion assay are used traditionally to treat earache, while leaveset al are applied for cleaning wounds[5]. According to Chan [6], E. elatior The extract was tested for antibacterial and antifungal the leaves of together with other aromatic herbs in activities by the disk diffusion method according to the water are used to remove body odour. E. elatior [10] NA Apart from that, there are insufficient studies on National Committee for Clinical laboratory standards . (SDA) flower extract and studies must be conducted on this species and Sabouraud dextrose agar sterilized in a flask and 45-50 in order to determine its activity as medicinal plants with cooled to 曟 were distributed to sterilized Petri dishes with a diameter of 9 cm (15 mL). Then NA plates, containing antimicrobial agents against various microorganisms. The 6 an inoculum size of 10 colony-forming units (CFU)/mL of presentE. elatior study was designed to screen the methanol extract 5 of flower against several pathogenic bacteria and bacteria or 2 伊 10 CFU/mL yeast cells or mold spores on fungi in order to investigate new sources of antimicrobial SDA plates, respectively, were spread on the solid plates. agents and also to report on its antioxidant properties and The filter paper discs (6 mm in diameter), individually phytochemical screening test. This study also investigates impregnated with 25 毺L of extract at concentration of 100 the toxicity of the methanolic extract using the brine mg/mL, was placed on the agar plates previously inoculated shrimp assay. Toxicity studies also play an important role with test microorganisms. Similarly, each plate carried a in identification and isolation of new compounds from blank disc by adding methanol solvent alone in the center crude extracts[7]. The assay is considered as useful tool for to serve as a negative control and antibiotic discs (6 mm in preliminary toxicity assessment of plant extract[8,9]. diameter) of 30 毺g/mL of chloramphenicol (for bacteria), and 30 毺g/mL of Miconazole nitrate (for fungi) was used as a 37 曟 2. Materials and methods positive controls. All the plates were incubated at for 24 h for bacteria and 30 曟 for 48 h for fungi. The diameters 2.1. Plant collection of the inhibition zones were measured in millimeters. The sensitivity of the microorganisms to the extract was E. elatior determined by measuring the size of inhibitory zones on The flowers of (pink torch ginger) were collected the agar surface around the discs[10]. All the tests were from Gelugor, Penang Malaysia, in April 2010 and performed in triplicate. authenticated by the botanist of the School of Biological 2.5. Determination of minimum inhibitory concentration Sciences at Universiti Sains Malaysia where the herbarium was deposited. Then the flowers were washed under running (MIC) tap water and dried in oven at 50 曟. The dried flowers were homogenized to fine powder and stored in airtight bottles. A 16 h culture was diluted in 10 mL of distilled water with 0.5 2.2. Solvent extraction reference to the 6McFarland standards to achieve inocula of approximately 10 colony forming units. A serial dilution was carried out to give final concentration between 0.10- One hundred fifty grams of dried powder was extracted 100.00 mg crude extract per mL. The tubes were inoculated with 400 mL of 80% methanol (v/v) for one week. Then, it with 20 毺L of bacterial suspension per mL of nutrient broth, was filtrated through No.1Whatman filter paper and the homogenized and incubated at 37 曟. Then after incubation Subramanion Jo Thy Lachumy et al./Asian Pacific Journal of Tropical Medicine (2010)769-774 771 2.8. Phytochemical screening 50 毺L was withdrawn from each tube, inoculated on agar 37 24 MIC plates and incubated at 曟 for h. The value was E. elatior determined at the lowest concentration of crude extract The flower extract was further i.e.subjected to in the broth medium that inhibit the growth of the test determination of phytochemical constituents alkaloids, microorganism. E. elatior flavonoids, tannins, saponins, terpenoids, reducinget al sugar MIC The of the flower extract of Artemia niger was determined(A. niger) and anthraquinoneet al using the methods of Evans and by broth dilution method against . Parekh [17,18]. The flower extract was serially diluted in Potato Dextrose 2.9. Determination of antioxidant activity (PDB) 0.10-100.00 Broth to a final concentration of A. niger mg crude extract per mL. The spore suspensions of was 10 obtained from their respective days stock culture, mixed The quantitative measurement of free radical scavengingE. elatior with sterile distilled water to obtain a homogenous spore activities of the methanolic flower extract of 8 [19, 20] suspension of 1伊10 spore/ mL. Ten 毺L of spore suspension was carried out in a universal bottle . Each reaction was inoculated in the test tube containing PDB medium[10]. mixture contained 50 毺L of test sample with concentration The minimum concentration at which no visible growth was ranging from 0.031- 2.000 mg/mL and 5 mL of 0.004% (w/ observed were defined as the MICs, which expressed in mg/mL. v) of 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) radical solution in methanol. The commercial antioxidant butylated 2.6. Toxicity testing against brine shrimp hydroxytoluene (BHT, Sigma) was used as a positive control. The discolouration was measured at 517 nm after the 2.6.1. Hatching shrimp incubation for 30 min in the dark condition. The 80% (v/v) Artemia salina (A. salina) Brine shrimp eggs, were hatched methanol was used as a blank and DPPH (in 80% MeOH) used in artificial seawater prepared by dissolving 38 g of sea as control. Measurements were taken at least in triplicate. DPPH salt in 1 L of distilled water. After 24 h incubation at room The radical concentration was calculated using the temperature (22-29 曟), the larvae was attracted to one side following equation: of the vessel with a light source and collected by pipette. – DPPH 1 100 Larvae were separated from eggs by aliquoting them three scavenging effect (%) = Ao A /Ao伊 times in small beakers containing seawater. Where Ao was the absorbance of the control and A1 was the 2.6.2. Brine shrimp assay absorbance of the tested sample (crude extract) in DPPH. The degree of discoloration indicates the free radical scavenging Toxicity of the extract was monitored by the brine shrimp efficiency of the substances. The absorbance measurements lethality test[11]. Two mL of seawater was placed in all the were recorded on Spectrophotometer (HITACHI- U 1900 bijoux bottles. A two-fold dilution of methanol extract Spectrophotometer 200V). was carried out with the artificial seawater to obtain the concentrations ranging from 50 mg/mL to 0.098 mg/mL. Potassium dichromate used as a positive control and was 3. Results prepared by dissolving it in artificial seawater to obtain concentrations ranging from 0.1 to 0.9 mg/mL[12]. The 3.1. Antimicrobial activity last bottle was filled with sea salt water only to serve as a drug-free control or negative control. Ahundred 毺 The data obtained from disk diffusion method was shown L of suspension of larvae containing about 10 -15 larvae in TableStaphylococcus 1. Maximum aureus inhibitory activity was obtainedBacillus 24 23 was added into each bottle and incubated for h. The thuringiensisagainst Bacillus ( subtilis mm), followed by 21 19 bottles were then examined and the number of dead Salmonella ( mm),Proteus mirabilis ( mm) and finally 18 shrimp in each bottle was counted. The total number of Escherichia sp. coli and Micrococcus ( mm). In contrast, shrimp in each bottle was counted and recorded. The mean and sp. exhibited weak percentage mortality was plotted against the logarithm of inhibition zones respectively 16 mm and 12 mm. Apart from concentrations. Lethal concentration (LC50) was determined that, antimicrobialCandida activity albican(C.of this extract calbican) was also observed [13, 14] 22 from the graph . against the yeast A. niger ( mm) and filamentous fungi (20 mm). The inhibition zone of 2.7. Data analysis solvent control methanol (negative control) was zero so that it was not active against all of the tested microorganisms. The mean results of brine shrimp mortality against However, the two antibiotics 30 毺g/mL of chloramphenicols the logarithms of concentrations were plotted using the and miconazole nitrate (positiveE. controls) elatior were more Microsoft Excel computer program, which also presents effective than the flower extract of with zones of regression equations[15]. The regression equations were inhibition ranging between 28 and 31 mm. used to calculate the LC50 value. Extracts giving LC50 values Furthermore based on the initial antimicrobial screening greater than 1.0 mg/mL were considered to be nontoxic[16]. assay, the susceptible strains were selected for further studies to determine the concentration effect and MIC Subramanion Jo Thy Lachumy et al./Asian Pacific Journal of Tropical Medicine (2010)769-774 772 Table 1 E. elatior Antimicrobial activity (zone of inhibition and MIC) of flower extract compared with commercial antibiotic. Zone of inhibition (mm) MIC of the extract Microorganisms a Extract(100 mg/mL) Chloramphenicol(30 毺g/mL) Micanozole nitrate (mg/mL) Staphylococcus aureus 23 28 ND 1.563 Bacillus thuringienesis 21 31 ND 6.250 Escherichia coli 16 30 ND 12.500 Salmonella . sp 18 29 ND 12.500 Bacteria Microccocus . sp 12 32 ND 50.000 Bacillus subtilis 19 30 ND 25.000 Proteus mirabilis 18 30 ND 25.000 C. albican Yeast 22 ND 24 3.125 A. niger Fungi 20 ND 23 1.563 a n The values (average of triplicate) are diameter of zone of inhibition at 100 mg/mL extract and Broth dilution method, mean value = 3.

values. The MIC values against all the tested strains ranged 3.125- 50.000 from mg/mLStaphylococcus (Table 1) and aurues, were Bacillus active 120 y=123.46x+99.435 against bacteria such as 2 100 thuringiensis Candida albican(C. R =0.873 2 and fungal strains such as 80 albicans) A. niger 50 and with MIC values of 1.563 mg/mL to LC =0.4 mg/mL 60 6.250 MIC 12.500- (%) Mortaligy mg/mL. Meanwhile, the value rangedSalmonella from 50.000 40 Bacillusmg/mL subtilis, for Proteus bacterial mirabilis stains suchEscherichia as coli sp., and . 20 0 3.2. Toxicity study -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 -20 -40 Log10 Concentration The result of the brine shrimp lethality bioassay showed Figure 2. the extract to be none toxic to brine shrimp (LC50: 2.52 mg/mL The toxicity effects of the potassium dichromate using brine at 24 hours) (Figure 1). Potassium dichromate served as the shrimp lethality assay after 24 hours. positive control. The LC50 value for the positive control at 24 0.4 2 3 hours wasA. salina mg/mL (Figure ). Figure showed the extract treated . 3.3. Phytochemical screening

E. elatior The phytochemical screening of the flower extract of shows the presence of the flavonoids, terpenoids, saponins, tannins, cabohyrates and absence of alkaloids, reducing sugars, anthraquinone in the extract.

120 100 80 y=46.001x+31.599 Figure 3. E. elatior 2

Mortaligy (%) Mortaligy Light microscope micrograph of the flower extract 60 R =0.929 9 A. salina 40 LC50=2.52 mg/mL treated . 20 3.4. Radical scavenging (antioxidant) activity 0 -1.5 -1 -0.5 0 0.5 1 1.5 2 -20 The scavenging effects of the flower extract and BHT -40 on DPPH radicals was 76.26% and 86.77% at 1.0 mg/mL, Log10 concentration respectively. The methanol flower extract exhibited a Figure 1 E. elatior significant dose dependent inhibition of DPPH activity with . The toxicity effects of the flower extract using 50% inhibition (IC50) at concentration of 9.14 mg/mL and IC50 brine shrimp lethality assay after 24 hours. value of BHT was 8.08 mg/mL. Subramanion Jo Thy Lachumy et al./Asian Pacific Journal of Tropical Medicine (2010)769-774 773 or free radicals scavengers[31]. Apart from that, since the 4. Discussion flower has a strong- smelling and pungent tasting thus it has been widely used as main ingredient in cooking, traditional medicinal use and also in local products. Moreover, the Plants are an important source of potential useful bioactive presence of this chemical compound further confirmed that compounds for the development of new therapeutic agents. the flower extract use as a traditional remedy for headaches There are many reports available on the antibacterial, or stomach aches, and is commonly applied externally antiviral, antifungal properties of plants[21-23]. Thus, these to relieve itching and treat skin problems and also the observations have helped in developing new drugs for the biological activities observed in this study. therapeutic use in human beings. However, not many studies The DPPH test provides information on the reactivity of the DPPH areE. available elatiora on the antibacterial or antifungal property test compounds with stable free radicals. The assay is of E.. elatioraHence, here we report the pharmacological often used to evaluate the ability of antioxidants to scavenge activities of . the free radical from test samples. Whereby, the free radicals In the present study, we haveE. elatiorainvestigated the potential cause biological damage through oxidative stress and of the of flower extract of as a source for such process lead to many disorders like nuerodegerative AIDS[32] DPPH antimicrobial and antifungal agents. The results in thisE. elatior study diseases, cancer and . Therefore, assay is an revealed that thein methanolicvitro extract of flower of effective method to measure their scavenging power. The possesses great potential for antimicrobial activity to principle of the DPPH assay is based on the colour changes varying degrees against all microorganisms tested. The data from purple (DPPH solution) to yellow[33]. The colour changes obtained from disk diffusion method indicates that Gram- can be measured quantitatively by spectrophotometer positive bacteria are more susceptible than Gram-negative absorbance at 517 nm. There is no significant difference bacteria towards the flower extract. These differences could between the flower extract and the commercialE. elatior antioxidant be due to the nature and level of antimicrobial agents (BHT) thus, this further confirm that flower extract present in the flower extract, their mode of action and the is a comparable antioxidant to BHT. In addition, this typical differences in the cell wall between the strains[24]. finding will help to develop new drugs for medicinal uses Our results also showed that some of the susceptible strains such against aging and other diseases related to radical selected have a high range of MIC values. This could be mechanisms[34-37]. possible due to the fact that the compounds responsible The results of the presentE. elatior investigation clearly indicate for displaying their antimicrobial activities that present that the flower extract of possesses broad spectrum in the flower extract at different concentrations and were antibacterial and antifungal activity. Also revealed that, not enough to exhibit their antimicrobial action at a low it has good antioxidant properties and possessing all the [25] E. elatior concentration of extract . necessary phytochemicalA. salina constituents. In addition In toxicity evaluation of plant extracts by the brine flower is not toxic to . Moreover, this plant extract LC50 1 shrimp bioassay, an value of more than mg/mL is has been widely usedE. elatioras main ingredient in traditional considered non-toxic[11]. Therefore, they can be classified medicine. Hence, flower extract could be of as biologically safety compoundsE. elatior with pharmaceutical considerable interest to the development of new drugs. properties. In our study, the flower extract Conflict of interest statement exhibited no significant toxicity against brine shrimp with LC50 2.52 24 E.an elatior value mg/mL ( h). Thus,A. this salina. signified E. elatior that flower might not be toxic to We declare that we have no conflict of interest. flower extract was not toxic against brine shrimp therefore it Acknowledgments can be usedin vivo as an antimicrobial agent in known dosage with further evaluations. The brine shrimp bioassay can be useful as a quick, simple and low cost test for predicting S. Jo Thy Lachumy and V. Sumathy were supported by the toxicity of the plant extract in order to consider the Universiti Sains Malaysia fellowship from Institute for [26] safetyE. elatior of the therapeutic agents to human beings . 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