HerlinaRante1 ,Risfah Yulianti1, YayuMulsiani Evary1, , Elvira Hardiana1

  1. Pharmacy Faculty of Hasanuddin University, South Sulawesi, Indonesia
(Perintis Kemerdekaan KM.10, Tamalanrea, Makassar, 90245)

 

ABSTRACT

This study aims to identify endophytic fungi from medicinal plant Mellochiaumbellataand investigate  their  antibacterial  potencies. Twelve species of endophytic fungi weresuccessfully  isolated  from  Mellochiaumbellataleaves and twigs. Four of them showed an activity in the antagonist test against Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae and Shigelladysentriae. The active isolates were fermented in potato dextrose yeast (PDY) medium at 25oC for 21 days with agitation of 150 rpm. The secondary metabolites were extracted from the fermentation medium using ethyl acetate and the mycelia were extracted using methanol. The ethylacetate and methanol extracts  were  assessed  for their antibacterial  activity  against the pathogenic  bacteria and some of the isolates extract showed antibaterial activity with inhibition zone obtained were between 0 and 10,51 mm.From ethyl acetate extracts, We were obtained MUD5 94 mg  withinhibition zone 8,08 mm against E. coli, 10,51 against S.dysentriae, 7,81 mm against P. aeruginosa, 9,00 mm against V. cholerae, MUR4 116 mg with inhibition zone 8,61 mm against E. Coli, MUR5 was obtained 93 mg with inhibition zone 7,54 mm against E. coli, and  MUR6 96 mg with inhibition zone 7,44 mg against P.aeruginosa. While from methanol extracts were obtained MUR6 516 mg with inhibition zone 9,3 mm against P. aeruginosa. Phytochemical identification revealed that the active extracts contain alkaloids, flavonoids and steroids. From this present work, it can be concluded that these endiphytic fungi could be promisng source of bioactive compounds and can be used for further study.

Keywords: Antibacterial Activity; Endophytes;MellochiaUmbellata; Mycelia.

INTRODUCTION

The discovery of natural products  and novel bioactive  molecules have  played  major  role  in the  search  for  new  drugs.2Microorganisms present  in  living  tissues  of  various  plants  parts (root, fruit,  stem,  seed,  leaf, etc.) provide mutual relationship  without  causing any  symptom of diseases are called endophytes.1 The host is protected from infectious agents  and  adverse conditions by endophytic fungi. This protection held withthe secretion of bioactive secondary metabolites.Biologically  active  compoundsfound in endophytic fungi which resides asymptomatically in internal tissues beneath epidermal cell layers and lives within the intercellular spaces of the tissues and it seems that they may penetrate the living cells of all higher plants2. Endophytic  fungi  are  a  good and interesting  source  of antibiotics.  Natural  products  from  endophytic microbes have been observed to be able to inhabit or kill a wide variety  of  harmful  disease-causing  agents but  not  limited  to  phytopathogens,  as  well  as bacteria, fungi, viruses and protozoan that affect humans and animals1.

Isolation  of  endophytes  is  an important step, because it requires sensitivity to recover a maximum number of colonized endophytes and should be accurate enough to remove the epiphytic  microbes  which  are  present  on  the plant surface. Endophytes can be isolated from various  plant  parts such as seeds, leaves and stems2. The collected plants for studying endophyticcommunities  should look apparently healthy and disease-free plant to decrease the possibility of pathogenic and saprobic species contaminant, and to avoid the isolation of pathogenic endophytic  microorganisms around it2.

Studies about the endofotic fungi have been widely conducted in some countries.Recent study showed that Mellochiaumbellataleave extract triggered antibacterial activity against Staphylococcus aureus, andShigelladysenteriaewith inhibition zone of 10.5 mm (2500 ppm) and 9.36 mm (2500 ppm), respectively3. In this study, we focus on the isolation and identification of endophytic  fungi  of Mellochiaumbellataand the screening of their antibacterial activity and to identify the phytochemical compounds in the extracts of endophytic fungi.

MATERIALS AND METHODS

Sources for Endophytic Fungi

Leaves and twigs were  thoroughly  washed  with  mild  detergent  andrunning  tap  water  and  then  air–dried.  After  which  theywere  surface  sterilized  by  using 3 step surface sterilization start with submerging  them  in  75%ethanol  for 3  min.  Further sterilization was performed by using 5.3%  sodium  hypochlorite solution  for  5  min, and  75%  ethanol  for  0.5  min, sequentially.  Aftersterilization, samples were washed with sterile water to remove ethanol residues. Each leaf was cut into 1 cm in size and twigs were cut into two pieces. Samples were placed at the surface of sterile potato dextrose agar (PDA) medium and incubated for 3-5 days, at 25oC. Thefungal isolates were identified based on theirmorphological characters6,7,8,9.

Mass cultivation and extraction of metabolitesofendophytic fungi

The fungalendophytes were mass cultivated on potatodextrose yeast (PDY) by placing agar blocks of actively growingpure culture (3mm in diameter)  in  250ml  Erlenmeyerflasks that contain 100ml medium.  The flasks wereincubated at room temperature for 21 days with periodicalshaking  at  150  rpm.  After  incubation  period,  culture media and mycelia were separated by filtration and extracted with the method described by Dasaleet al (2013).The Fungi mycelia was extracted in 100 ml methanol using sonicator for 30 minutes. The mycelia extract was filtered and evaporated. The culture media was extracted by liquid-liquid extraction using ethyl acetate in the same volume with the media. Extraction was repeated three times and the organic solvent of collected extract was evaporated under reduced pressure. The crude extract was then disssolved in Dimethyl sulphoxide (DMSO) for antibacterial bioassay.

Antibacterial Activity

Test Organisms

There were 4 strains of patogenic bacteria which were used for the antibacterial activity of fungi. They wereE. coli, S. dysentriae, P.aeruginosa,and V.cholerae.

Antagonist test

Endophytic fungi cultures were transferred to petri dish which consisted of nutrient agar medium and patogenic bacteria. The dishes were incubated for 24 hrs, at 37oC.

Antibacterial activity test

The agar well diffusion assay method was used to examine the antibacterial activity of fungi. In this method, wells were aseptically made in seeded media using sterilecork borer and 20 µl bioactive metabolite was dropped in the previously prepared wells and incubated at 37oC in bacteriological incubator for 24 hrs. Finally, plates were observed for zones of inhibition and their diameter was measured with Antibiotic zone scale.

Phytochemistry identification

The active antibacterial isolate extracts were identified for their phytochemistry compounds. These were done by thin layer chromatography method. The extracts were eluted using hexane: ethyl acetate (2:1)and the results were observed under UV 254, 366 nm and visible (after sprayed with 10% H2SO4). For Alkaloid identification, the spots were sprayed with dragendorfragent and for flavonoid identification by using sitroborat reagents11,12.

Table :Zone of Inhibition produced by endophytic fungi on human bacerial pathogens

extract Endophytic Fungi Diameters of Inhibition zone (mm) Amount of extract (mg)
Escherichia coli Shigelladysentriae Pseudomonas aeruginosa Vibrio cholerae
Ethyl acetate MUD1
MUD2
MUD3
MUD4
MUD5 8.08±0.053 10.51±0.153 7.81±0.0781 9.00±0.097 94
MUD6
MUR1
MUR2
MUR3
MUR4 8.61±0.215 116
MUR5 7.54±0.262 93
MUR6 7.44±0.301 96
Methanol MUD1
MUD2
MUD3
MUD4
MUD5 257
MUD6
MUR1
MUR2
MUR3
MUR4 97
MUR5 614
MUR6 9.30±0.106 516

 

Results

Isolation of Endophytic fungi

In the present study, fungal strains were isolated from leaves and twigs of Mellochiaumbellata. A total of 12 fungi wasisolated.Four most active isolates from antagonist test which showed the biggest inhibition zone were fermented. The active endophytic fungi were fermented for 21 days using PDY to obtain the secondary metabolites. The fermentation broth or supernatant was extracted with ethylacetate and the biomass was extracted with methanol. Screening of endophytic fungi to determine antibacterial activity was executed by agar well diffusion method against 4 pathogenic human bacteria (Escherichia coli, Shigelladysentriae, Pseudomonas aeruginosa,and Vibrio cholerae).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.Endophytic Fungi Isolates

Extraction of isolates in 200 ml PDY medium resulted in 94 mg ethyl acetate extract from MUD5 isolates, 116 mg extract MUR4, 93 mg extract MUR5 and 96 mg extract MUR6. The methanol extract obtained were 257 mg from MUD5 isolates, 97 mg extract MUR4, 614 mg extract MUR5 and 516 mg extract MUR6.

In antibacetrial activity, MUD5 showed high inhibition zone, 8,08 mm against E. coli, 10,51 against S. dysentriae, 7,81 mm against P. aeruginosa, 9,00 mm against V. Cholerae. Diameter of inhibition zone of MUR4 was 8,61 mm against E. Coli, MUR5 was 7,54 mm against E. Coli, and  MUR6 was 7,44 mg against P. Aeruginosa. While from methanol extracts were obtained MUR6 with inhibition zone 9,3 mm against P. Aeruginosa.

 

 

 

 

 

 

 

 

 

 

Figure 2. Antagonist test result

  1. Antagonist test against Escherichia coli
  2. Antagonist test against Pseudomonas aeruginosa
  3. Antagonist test against Shigelladysentriae
  4. Antagonist test against Vibrio cholera

 

1-12 are the 12 endophytic fungi isolate examined in the antagonist test

1 : MUD1             7   : MUR1

2 : MUD2             8   : MUR2

3 : MUD3             9   : MUR3

4 : MUD4             10 : MUR4

5 : MUD5             11 : MUR5

6 : MUD6             12 : MUR6

Phytochemical identification were carried out for all the active extracts and showed that ethyl acetate exttract of MUD5, MUR4, MUR5, MUR6 contained alkaloid compound based on red spot formation on thin layer chromatography after spraying of Potassium Bismuth Iodidesolution. Flavonoid content of all active extracts showed by the light green spot on uv 366 after being sprayed with sitroborat. From the preliminary phytochemical screening also revealed the presence of steroids in ethyl acetate extract  of MUR6 based on the formation of red spot after the spot being sprayed with H2SO4 10%.(Figure.3).

 

 

 

 

 

 

 

Figure.3

  1. UV 254 Ethyl acetate extract of MUD5
  2. UV 366 Ethyl acetate extract of MUR4
  3. H2SO410% Ethyl acetate extract of MUR5
  4. Sitroborat Ethyl acetate extract of MUR6
  5. Dragendorf Methanol extract of MUR6

 

Discussion

Various species of endophytic fungi made an ecological niche in the inner space of plants. The fungi interact with their environment in a possitive manner in the role of improving plant defence and disease control. Endophytic fungi isolation from medicinal plantresults in the production of bioactive metabolites which has a great activity against microbes. Hence, scaling up the production of bioactive metabolites is necessary to fullfill the demand of agriculture and pharmaceutical industries. In this study, we have isolated 12 endophytic fungi from Mellochiaumbellata. Ethylacetate extract from fungi isolate (MUD5) shows activities against E. coli, S.dysentriae, P.aeruginosa, and V.cholerae.MUR6 produce active secondary metabolites againstP.aeruginosa, both in the supernatant and the biomass. Phytochemical identification  showed that all active extract consist of alcaloids, steroids and flavonoids compound.

Conclusion

The present investigation was an attempt to search for potential endophytic fungi from Mellochiaumbellata. The study revealed the active antibacterial extract which were extracted with ethyl acetate and methanol. Four active ethyl acetates and 1 active methanol extract were examined for their antibacterial activities based on their inhibition zone diameters. Therefore, further determination of active metabolites content in the extractneeds to be investigatedquantatively and completely. Beside that, TLC bioautography need to be developed in case to investigate the active antibacterial fraction.

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