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CATRINA (2006), 1 (2): 91-96
© 2006 BY THE EGYPTIAN SOCIETY FOR ENVIRONMENTAL SCIENCES
PROCEEDINGS OF THE FIRST INTERNATIONAL CONFERENCE ON CONSERVATION AND MANAGEMENT
OF NATURAL RESOURCES, ISMAILIA, EGYPT, JUNE 18-19, 2006
____________________________________________
* Corresponding Author: selshatoury@hotmail.com
Endophytic Actinomycetes of Some Medicinal Plants in Saint Katherine Area, Egypt
Sahar A. El-Shatoury*, Hesham M. Abdulla, Omnia A. El-Kraly,
Waleed M. El-Kazzaz, and Ahmed Dewedar
Botany Department, Faculty of Science, Suez Canal University, 41522 Ismailia, Egypt
ABSTRACT
A number of 41 morphologically different endophytic actinomycete isolates were recovered from
surface-sterilized medicinal plants; Artemisia herba-alba, Echinops spinosus, Mentha longifolia and
Ballota undulate. A simplified method for selective recovery of actinomycetes from internal plant tissues
is described. Successful recovery was achieved on the media: starch-casein agar, 1/10 diluted starch
casein agar, tap water-yeast extract agar and MG-plant extract agar. Preliminary description of the
isolates, according to microscopic examination and amino acid composition of the cell wall
hydrolystates, indicated prevalence of genus Streptomyces. The highest number of endophytic
actinomycetes was recovered from Mentha longifolia and represented 34% of the isolates. Organic
extracts of the growth metabolites from the cultures were tested for cytotoxicity against Artemia salina as
well as for antimicrobial activities against 4 reference bacterial strains, 6 clinical bacterial cultures and
two clinical fungal cultures. Variable activities were obtained with different actinomycete isolates; the
highest activity could be detected against Gardnerella vaginitis and Shigella boydii strain ATCC 9207;
being represented in 29% of the isolates in both cases. For the first time, the isolation of endophytic
actinomycetes from Saint Katherine wild plants is reported and their potential use as novel source of
bioactive compounds is discussed.
Key words: Antimicrobial activity, cytotoxicity, endophytic actinomycetes, medicinal plants, Saint
Katherine.
INTRODUCTION
Actinomycetes are a diverse group of filamentous
Gram-positive bacteria well known for their production
of an extensive array of chemically diverse and
medically important secondary metabolites. Only a few
recent studies have highlighted the bioactive importance
of endophytic actinomycetes, including biocontrol of
fungal plant pathogens (El-Tarabily, 2003; Coombs
et al., 2004), production of antimalarial and
antibimicrobial agents (Castillo et al., 2002), production
of anticancer compounds (Caruso et al., 2000),
production of plant growth regulators (Igarashi, 2004)
and production of enzymes (Stamford et al., 2002).
Plants growing in areas of great biodivesity usually
have the prospect of harbouring endophytes with great
biodiversity (Strobel and Daisy, 2003). The Saint
Katherine World Heritage Site (WHS No. 954,
UNESCO) of Sinai, Egypt, is one of the world's most
biodiverse area; it is characterized by high percent of
endimism in flora, fauna and related microbiota merging
from the existing altitude gradient. However, to the best
of our knowledge, microbiological studies on the
endophytes residing in their plants were not considered
yet. Thus, this habitat is one that deserves close
examination for novel microbes that produce
compounds with desired bioactivities. The present study
describes the isolation of actinomycetes from internal
tissues of some medicinal plants in the WHS of St.
Katherine and evaluates metabolites for antimicrobial
and cytotoxic activities.
MATERIALS AND METHODS
Site description and plant sampling
Plants were collected during the early summer of
2005 from six sites with characteristic granitic country
rocks, located in the ring dyke of the WHS. These were:
Wadi Shreiag (29.8° 33' 57'' E, 51.9° 28' 33'' N and
1545.3 m Altitude), Wadi El Arba'een (18.2° 33' 59'' E,
24.7° 28' 40'' N and 1306.1 m Altitude), Al-Kharazen
(53.2° 33' 57'' E, 12.2° 28' 43'' N and 1627.6 m
Altitude), El-Talaa (55.8° 33' 55'' E, 00.6° 28' 34'' N and
1627.6 m Altitude), Wadi El-Sheikh (08° 33' 59'' E,
35.6° 28' 40'' N and 1281.7 m Altitude) and Wadi El-
Raha (14.6° 33' 57'' E, 50.2° 28' 33'' N and 1544.7 m
Altitude). Four plant species were collected for
investigation: Artemisia herba-alba (Shieh) and
Echinops spinosus (Khasheer), belonging to the family
Compositae; Mentha longifolia (Habak) and Ballota
undulate (Ghassa), belonging to the family Labiatae.
These four plant species were selected for there
medicinal value due to their high content of functional
flavonoids, sesquiterpene, lactones and other essential
oils (Rimbau et al., 1999). Healthy, green aerial parts
were cut from these plants and placed in sealed plastic
bags. Collected plant samples were dried at room
temperature for 2 days and stored at 4o
C for further
processing.
Isolation and characterization of endophytic
actinomycetes
Plant materials were processed with modification of
the method described by Demain and Davies (1999) and
plated into a battery of ten media: Tap Water Yeast
Extract (TWYE) agar, Starch-Casein (SC) agar, 1/10
Starch-Casein agar, Tryptone Soya (TS) agar, MGA-PE
medium, Potato-Dextrose (PD) agar, CYC agar,
Arginine-Glycerol Salts (AGS) agar, Starch Nitrate
(SN) agar and Actinomycetes Isolation (AI) agar (Basil
et al., 2004; Demain and Davies 1999). Plants were
aseptically cut into 1 g pieces (ca. 3-5 cm in length) and
Endophytic actinomycetes of medicinal plants
92
immersed in 70% ethanol for 5 minutes for surface
sterilization, followed by rinsing in sterile distillated
water and air drying under a laminar flow hood. Surface
sterility check and processing of plant material for
endophytes isolation were performed as illustrated in
flow chart (Fig. 1). All plants were processed in
triplicates and all media were supplemented with
50µg/ml cycloheximide (antifungus) after sterilization.
Plates were incubated at 28o
C with regular monitoring;
counts were recorded after 4 weeks.
A total of 41 morphologically different actinomycete
isolates were purified from plates using starch-casein
agar medium and maintained as spore suspensions, in
20% glycerol, at 20o
C (Hopwood et al., 1985). Isolates
were characterized for their micromorphology and
diaminopimelic acid (DAP) isomer in whole cell
hydrolysate. Microscopic examinations of sporulated
mycelia were performed on cultures grown on ISP4
medium (Shirling and Gottlieb, 1966). The
determination of the DAP isomer in whole cell
hydrolysate was performed as described by Schaal
(1985) using dried mycelia obtained from cultures
grown in TS broth in baffled flasks on shaking
incubator (100 rpm), at 28 o
C for 4-7 days.
Enumeration of soil actinomycete
Soil samples (10-15 cm depth) were collected
aseptically from the target localities and kept in sterile
polyethylene bags at 4 °C until isolation. Actinomycetes
were isolated after serial dilution in phosphate buffer on
SC agar supplemented with cycloheximide (50 µg/ml)
using spread plate technique. Counts were recorded
after incubation for 2-3 weeks at 28 °C.
Fermentation procedure
Preserved spore suspensions of actinomycete isolates
were inoculated into 30 ml of SC liquid medium and
incubated in a shaker incubator (100 rpm), for 5-7 days
at 28 °C. Cultures were extracted using equal
concentrations of ethyl acetate, for three successive
times with vigorous shaking to thirty minutes. This
allowed any organic molecules to be suspended in the
less polar solvent. Ethyl acetate fractions were
evaporated under vacuum into a preweighed vial, and
then redissolved in ethyl acetate giving a final
concentration of 10 mg/ml.
Antimicrobial screening
The raw extracts were tested by disk diffusion using
0.1 mg per disk against a representative panel of human
pathogenic microorganisms as described by Castillo et
al. (2002). This panel included 4 reference bacterial
strains Salmonella typhimurium (NCMB 74),
Escherichia coli (NCMB 11943), Shigella boydii ATCC
9207 and Pseudomonas aeruginosa NCMB 8295; 6
clinical bacterial cultures Vibrio sp., Proteus vulgaris,
Klebsiella pneumonia, Gardnerella vaginitis,
Staphylococcus aureus, and Streptococcus sp.; and two
fungal cultures Candida albicans and Candida sp.
Brine shrimp cytotoxicity assay
The brine shrimp eggs were hatched in seawater (40
mg/l) as described by Svoboda and Hampson (1999).
Hatched eggs were supplemented with 6 mg/l dried
yeast and oxygenated for 48 hours at room temperature.
Crude organic extracts (1 mg in ethyl acetate) were
transferred into sterile microtubes and evaporated under
Plant
Sample
Surface sterilization
70 % ethanol / 5min
Aseptic Grinding
Dilution in phosphate
buffer plus 0.001%
tween 80
Direct
implanting
Plating on
SC, 1/10 SC, MGA-PE,
AI, AGS, PD, CYC, TS,
TWYE, SN
Moist heat
55o
C / 6min
Washing in phosphate buffer Surface sterility check
Plant pressing &
washing buffer
streaking
on TSA plates
Figure (1): Proposed strategy for selective isolation of endophytic actinomycetes from internal plant tissues.
El-Shatoury et al.
93
vacuum,to which 10-20 A. salina larvae in 1 ml of
seawater were added. Control blind sample was
accompanied by adding ethyl acetate and duplication
was performed. Cytotoxiciy was scored at intervals up
to 84 hours compared to the blind samples.
Statistical analysis
Significant differences among different cultivation
media and for different plant origins were studied by
one-way analysis of variance. The means were
separated at significant difference (p = 0.05).
RESULTS
Culturable endophytic actinomycetes
The endophytic actinomycete populations from the
investigated plants averaged between 102
-103
CFU/g
dry plants. It was noticed that, recovery of
actinomycetes was only successful using MGA-PE, SC,
1/10 SC and TWYE agar media, while the other media
plates were over-grown with fungi and unicellular
bacteria. Statistical analysis showed significant
differences (p = 0.049) for actinomycete counts using
the above mentioned four media. As shown in Figure
(2), higher numbers were recovered on 1/10 SC and
TWYE media compared to the other two media. The
maximum population recorded was from Ballota
undulata on TWYE agar.
Direct implanting of grinded plant samples has
recovered endophytic actinomycetes (data not shown);
however, better separation of individual colonies was
achieved by dilution and spread plate technique. In
addition, the heat treatment did not enhance the
recovery of actinomycetes from internal plant tissue as
shown in Figure (3), and difference in actinomycetes
counts between heat treated and untreated samples were
not significant (p = 0.072).
Tentative identification by microscopic examination
and analysis of amino acid composition of the cell wall
(Holt et al., 1994), indicated prevalence of genus
Streptomyces, representing 41.5% of the isolated
endophytic actinomycetes, in this study. Grouping of
the isolates, based on DAP analysis of the whole cell
hydrolysate revealed a number of 19 L-DAP containing
isolates and 22 m-DAP containing isolates.
As shown in Figure (4), high significant difference
could be noticed between soil actinomycete population
in the three investigated localities (p = 2.85 E-06). In
contrast, endophytic actinomycete counts from Mentha
longifolia collected from these localities were not
significantly different (p > 0.05).
Bioassay of antimicrobial and cytototoxic activities
Results indicated bioactivity of 81% of the isolates
(Fig. 5). Of the 41 endophytic actinomycete isolates, 30
exhibited microbial inhibitory effect. The highest
activity was against Gardnerella vaginitis and Shigella
boydii ATCC 9207, being represented in 29 % of the
isolates in both cases. All the isolates failed to give
activity against E. coli, P. Aeruginosa as well as the
clinical cultures Proteus vulgaris and Candida sp.
Cytotoxic effect against Artemia salina was positive for
27 endophytic actinomycetes. Of these, nine isolates
exhibited high mortality rate reaching to 100% death
after 12 hours. Those were mainly A. herba alba and
E. spinosus isolates (Table 1).
Figure (2):.Enumeration of endophytic actinomycetes on four
selected media. Counts are average of triplicates with bars
indicating Standard Error of the mean.
Figure (3): Effect of heat treatment on recovery of endophytic
actinomycetes from four plant species on 1/10 SC media.
Counts are average of triplicates with bars indicating
Standard Error of the mean.
Figure (4): Actinomycetes population from Mentha longifolia
and the surrounding bulk soil. Counts are average of three
replications.
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Count
cfu
/
g
of
dry
plant
W. ElSheikh AlKharazen AlTalaa
Sampling locality
Bulk soil Mentha
M. longifolia
W. El-Sheikh Al-Kharazen El-Talaa
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Artemisia Echinops Mentha Ballota
Count
cfu
/
g
of
dry
plant
MGA-P E SC 1/10 SC TWYE
A. herba alba E. spinosus M. longifolia B. undulata
0
500
1000
1500
2000
2500
3000
Artemisia Echinops Mentha Ballota
Count
cfu
/
g
of
dry
plant
heat treated untreated
A. herba alba E. spinosus M. longifolia B. undulata
Endophytic actinomycetes of medicinal plants
94
Figure (5): Total number of endophytic actinomycete isolates
showing bioactivities. Isolates from the four investigated
plant species are categorized into L-DAP and m-DAP
containing groups.
DISCUSSION
It is recently apparent that plants can serve as a
reserviore of endophytic microorganisms, meantime
evidence thus far indicates that, metabolites from these
sources hold pharmaceutical and agriculture promise
(Castillo et al., 2002; Kunoh, 2002). In the present
study, endophytic actinomycetes isolation was
significantly higher on the low nutrient-content media
1/10 SC and TWYE, compared to the high nutrient-
content media which allowed fast growing fungi and
unicellular bacteria to dominate the isolation plates.
These results were in agreement with Basil et al. (2004)
for isolation of soil actinomycetes using TWYE and
with May et al. (2003) for isolation of stone
actinomycetes using 1/10 SC. McCarthy and Williams
(1992) have described an autochthonous behaviour, i.e.
sustained growth at low nutrient concentrations amongst
actinomycetes, which may explain the successful
selectivity of these low nutrient-content media for
actinomycetes.
Although average endophytic actinomycetes
population was 102
to 103
cfu/g dry plants in the four
plant species studied, the bioactivities of these
endophytes varied widely. For example, 92% of
M. longifolia endophytes showed broad antimicrobial
activities and inhibited eight representatives of Gram
positive, Gram negative bacteria and yeast strains. In
contrast, only 31% of A. herba-alba endophytes
exhibited antimicrobial activities against six of the
tested microbial strains. Similarly, the cytotoxicity of
M. longifolia isolates was 33% compared to 62% for
A. herba-alba isolates. The high ratio of bioactive
isolates obtained from this study (81%) compares well
with other investigations, for example antimicrobial
activity was 14% for actinomycetes endophytic in Taxus
plant (Caruso et al., 2000) and 20% for endophytic
actinomycetes isolated from a variety of Japanese wild
plants (Igarashi et al., 2002). However, it should be
emphasized that attempts to compare and contrast
antimicrobial potential are susceptible to differences in
protocols used.
Although the available literature doesn't provide
knowledge on the endophytes of the currently
investigated plants, the four species are rich in
metabolites of high medicinal value (Hanafy et al.,
2000) and thus represent a promising source for
bioactive microbial products. The recent discovery of
different genera of actinomycetes and fungi that can
produce quinoline alkaloids and diterpenoids identical
to those originally characteristic of their host plants
(Caruso et al., 2000; Puri et al., 2005) have arisen an
interesting aspect that microbial endophytes may exhibit
genetic recombination with their host plant to produce
metabolites that facilitate the domination of its
biological niche within the plant or even provide
protection to the plant from harmful invading
pathogens.
While there was a significance difference in the
culturable actinomycetes numbers in bulk soil of the
studied localities, there were no qualitative differences
in numbers and bioactivities of endophytic
actinomycetes from M. longifolia collected from these
localities. Bills et al. (2002) described a metabolic
distinction between endophytes of the same plant origin
in tropical regions and suggested the importance of the
host plant in influencing the general metabolism of its
endophytic microbes.
Table (1): Source of actinomycetes and their bioactivities measured as diameter of the inhibition zone for antimicrobial activities
and as percentage of dead larvae for cytotoxicty. Results were scored after 24 hours of incubation.
Antimicrobial activity (avg ± SD) b
Actinomycetes group
(no. of isolates) a
S. typhimurium Vibrio sp. S. boydii K. pneumonia Streptococcus sp. S. aureus G. vaginitis C. albicans
Cytotoxicity
(Death %)
Artemisia herba-alba
L-DAP group (2) 9 ± 0.7 9 ± 0.7 12 ± 0.5 16 ± 0.6 6 ± 0.7 17 ±.01
m-DAP group (5) 9 ± 0.6 7 ± 1.4 27± 0.7 100 ± 25
Echinops spinosus
L-DAP group (3) 18 ± 0.3 7 ± 0.5 6 ± 0.6 100 ± 0.01
m-DAP group (1) 7 ± 0.3 100 ± 0.01
Mentha longifolia
L-DAP group (7) 11 ± 0.7 10 ± 4.6 6 ± 0.7 15 ± 0.6 13 ± 0.6 7 ± 0.6 7 ± .05 33 ± 0.7
m-DAP group (4) 13 ± 7 12 ± 0.6 18 ± 3 12 ±0.7 8 ± 0.5 7 ± .01 67 ± 33
Ballota undulate
L-DAP group (5) 7 ± 0.5 12 ± 0.5 7 ± 0.6 8 ± 0.7 9 ± 4 66 ± 40
m-DAP group (3) 15 ± 0.7 7 ± 0.6 0
a
: Number of actinomycetes isolates in each group. b
: Average (avg) of inhibition zone diameter ± Standard deviation (SD).
0
1
2
3
4
5
6
7
8
9
10
L-DAP m-DAP L-DAP m-DAP L-DAP m-DAP L-DAP m-DAP
Artemisia Echinops Mentha Ballota
Bioactive inactive
A. herba alba E. spinosus M. longifolia B. undulata
Number
of
isolates
El-Shatoury et al.
95
Overall, it is concluded that, actinomycetes could be
selectively isolated from the internal tissues of wild
medicinal plants in the WHS using a simplified method.
The bioactivities of these isolates varied with plant
origin and the higher percentage of wide-spectrum
antimicrobial strains were from M. longifolia. The
reason for this requires further investigation. We
hypothesize that actinomycetes which colonize internal
tissues of these plants are highly adapted to this unique
little-studied habitat and should be investigated for
detailed identification and potential use as source of
bioactive agents.
ACKNOWLEDGMENTS
We are grateful to Dr. Kamal Ghodeif, Geology
Department, Faculty of Science, Suez Canal University
for his invaluable guidance during the sampling trips.
We also thank Dr. Raafat Abd El-Wahab, Botany
Department, Faculty of Science, Suez Canal University
for his effort to identify the plants. This study was
funded by grant C/3801-1 from the International
Foundation for Science (IFS), Sweden.
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Received August 5, 2006
Accepted October 31, 2006
CATRINA (2006), 1 (2): 91-96
© 2006 BY THE EGYPTIAN SOCIETY FOR ENVIRONMENTAL SCIENCES
PROCEEDINGS OF THE FIRST INTERNATIONAL CONFERENCE ON CONSERVATION AND MANAGEMENT
OF NATURAL RESOURCES, ISMAILIA, EGYPT, JUNE 18-19, 2006
96
‫ا‬
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‫ا‬ ‫ﺏﺎﻟﻌﺰل‬ ‫اﻟﺪراﺱﺔ‬ ‫هﺬﻩ‬ ‫ﺗﺨﺘﺺ‬
‫ﻟﻤﻴﻜﺮوﺏﻰ‬
‫ﺔ‬‫ﻷرﺏﻌ‬ ‫ﺔ‬‫اﻟﺪاﺥﻠﻴ‬ ‫ﺴﺠﺔ‬‫اﻷﻥ‬ ‫ﻓﻰ‬ ‫اﻟﻤﺘﻮاﺟﺪة‬ ‫ﻟﻸآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫اﻟﺤﻴﻮى‬ ‫اﻟﻨﺸﺎط‬ ‫وﺗﻮﺹﻴﻒ‬
‫ﻰ‬‫وه‬ ‫ﺔ‬‫ﻃﺒﻴ‬ ‫ﺎت‬‫ﻥﺒﺎﺗ‬
:
Artemisia herba-alba
‫و‬
Echinops spinosus
‫و‬
Mentha longifolia
‫و‬
Ballota undulate
‫ﺪ‬‫ﺗﺤﺪی‬ ‫ﻦ‬‫أﻣﻜ‬ ‫ﺚ‬‫ﺡﻴ‬
‫اﻟﻨﺒ‬ ‫ﺗﻠﻚ‬ ‫ﺱﺎآﻨﺔ‬ ‫ﻟﻸآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫اﻹﺥﺘﻴﺎرى‬ ‫ﻟﻠﻌﺰل‬ ‫ﻣﺒﺴﻄﺔ‬ ‫ﻃﺮیﻘﺔ‬
‫اﻟﻐﺬاﺋﻴﺔ‬ ‫اﻷوﺱﺎط‬ ‫ﻋﻠﻰ‬ ‫اﻟﻌﺰل‬ ‫أﻓﻀﻠﻴﺔ‬ ‫اﻟﻨﺘﺎﺋﺞ‬ ‫وأوﺿﺤﺖ‬ ،‫ﺎﺗﺎت‬
:
‫ﺎر‬‫ﺁﺟ‬
‫إﻟﻰ‬ ‫اﻟﻤﺨﻔﻒ‬ ‫واﻟﻜﺎزیﻦ‬ ‫اﻟﻨﺸﺎ‬ ‫وﺁﺟﺎر‬ ،‫واﻟﻜﺎزیﻦ‬ ‫اﻟﻨﺸﺎ‬
‫اﻟﻨﺒﺎﺗﻰ‬ ‫اﻟﻤﺴﺘﺨﻠﺺ‬ ‫وﺁﺟﺎر‬ ،‫اﻟﺨﻤﻴﺮة‬ ‫ﻣﺴﺘﺨﻠﺺ‬ ‫وﺁﺟﺎر‬ ،‫اﻟﻌﺸﺮ‬
.
‫ﻋﺪد‬ ‫ﻋﻠﻰ‬ ‫اﻟﺤﺼﻮل‬ ‫ﺗﻢ‬ ‫وﻗﺪ‬
41
‫ﻟﻬ‬ ‫ﺪﺋﻰ‬‫اﻟﻤﺒ‬ ‫اﻟﺘﻌﺮیﻒ‬ ‫أﻇﻬﺮ‬ ،‫اﻟﻈﺎهﺮى‬ ‫اﻟﺸﻜﻞ‬ ‫ﻓﻰ‬ ‫ﻣﺘﺒﺎیﻨﺔ‬ ‫أآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫ﻋﺰﻟﺔ‬
‫ﻨﺲ‬‫ﺟ‬ ‫ﺸﺎر‬‫إﻥﺘ‬ ‫ﺎ‬
Streptomyces
‫أن‬ ‫اﻟﻨﺘﺎﺋﺞ‬ ‫أوﺿﺤﺖ‬ ‫آﻤﺎ‬
34
%
‫ﻥﺒﺎت‬ ‫أﻥﺴﺠﺔ‬ ‫ﻣﻦ‬ ‫آﺎن‬ ‫اﻟﻌﺰﻻت‬ ‫إﺟﻤﺎﻟﻰ‬ ‫ﻣﻦ‬
Mentha longifolia
.
‫ﺗﻢ‬ ‫وﻗﺪ‬
‫ﺸﺎط‬‫اﻟﻨ‬ ‫ﺗﻘﻴﻴﻢ‬ ‫ﺗﻢ‬
‫اﻟﺤ‬
‫ﻴ‬
‫اﻷآﺘﻴﻨﻮﻣﻴﺴ‬ ‫ﻟﻤﺰارع‬ ‫اﻟﻌﻀﻮیﺔ‬ ‫ﻟﻠﻤﺴﺘﺨﻠﺼﺎت‬ ‫ﻮى‬
‫ﻴ‬
‫ﺿﺪ‬ ‫اﻟﺨﻠﻮیﺔ‬ ‫اﻟﺴﻤﻴﺔ‬ ‫ﺡﻴﺖ‬ ‫ﻣﻦ‬ ‫وذﻟﻚ‬ ‫ﺘﺎت‬
Artemia salina
‫ﻰ‬‫اﻟﻤﻴﻜﺮوﺏ‬ ‫ﺸﺎط‬‫واﻟﻨ‬ ،
‫ﺿﺪ‬
10
‫إآﻠﻴﻨﻴﻜﻴﺔ‬ ‫ﻓﻄﺮ‬ ‫ﺱﻼﻟﺘﻰ‬ ‫إﻟﻰ‬ ‫إﺿﺎﻓﺔ‬ ‫وإآﻠﻴﻨﻴﻜﻴﺔ‬ ‫ﻣﺮﺟﻌﻴﺔ‬ ‫ﺏﻜﺘﻴﺮیﺔ‬ ‫ﺱﻼﻻت‬
.
‫ﻊ‬‫واﺱ‬ ‫ﻮى‬‫ﺡﻴ‬ ‫ﺸﺎط‬‫ﻥ‬ ‫ﻮد‬‫ﻟﻮﺟ‬ ‫ﺤﺔ‬‫ﻣﻮﺿ‬ ‫اﻟﻨﺘﺎﺋﺞ‬ ‫وﺟﺎءت‬
‫اﻟﻤﺨﺘﻠﻔﺔ‬ ‫اﻟﻨﺒﺎﺗﻴﺔ‬ ‫اﻟﻤﺼﺎدر‬ ‫ﻣﻦ‬ ‫اﻷآﻴﺒﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫ﻟﻌﺰﻻت‬ ‫وﻣﺘﺒﺎیﻦ‬
.
‫ﻼﻟﺔ‬‫ﺱ‬ ‫ﻊ‬‫ﻣ‬ ‫ﺰﻻت‬‫ﻟﻠﻌ‬ ‫ﻰ‬‫ﻣﻴﻜﺮوﺏ‬ ‫ﺪ‬‫ﺿ‬ ‫ﺸﺎط‬‫ﻥ‬ ‫ﻰ‬‫أﻋﻠ‬ ‫ﺎن‬‫وآ‬
Shigella
boydii ATCC 9207
‫واﻟﺴﻼﻟ‬ ‫اﻟﻤﺮﺟﻌﻴﺔ‬
‫ﺔ‬
Gardnerella vaginitis
‫ﺠﻠﺖ‬‫ﺱ‬ ‫ﺚ‬‫ﺡﻴ‬ ،‫اﻹآﻠﻴﻨﻴﻜﻴﺔ‬
29
%
‫ﺪ‬‫ﺿ‬ ً
‫ﺎ‬‫ﺸﺎﻃ‬‫ﻥ‬ ‫ﺴﺒﺘﺎت‬‫اﻷآﺘﻴﻨﻮﻣﻴ‬ ‫ﻦ‬‫ﻣ‬
‫اﻟﺴﻼﻟﺘﻴﻦ‬ ‫هﺎﺗﻴﻦ‬ ‫ﻣﻦ‬ ‫آﻞ‬
.
‫اﻹﺥﺘﻴﺎرى‬ ‫اﻟﻌﺰل‬ ‫إﻣﻜﺎﻥﻴﺔ‬ ‫ﻋﻦ‬ ‫اﻷوﻟﻰ‬ ‫هﻰ‬ ‫اﻟﺪراﺱﺔ‬ ‫ﺗﻠﻚ‬
‫ﺎآﻨ‬‫ﺱ‬ ‫ﻟﻸآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬
‫ﺎﺗﺮیﻦ‬‫آ‬ ‫ﺎﻥﺖ‬‫ﺱ‬ ‫ﺔ‬‫ﻣﻨﻄﻘ‬ ‫ﻰ‬‫ﻓ‬ ‫ﺎت‬‫اﻟﻨﺒﺎﺗ‬ ‫ﺔ‬
‫ﺔ‬‫وﻓﻌﺎﻟﻴ‬
‫اﻟﺤﻴﻮی‬ ‫ﻟﻠﻤﺮآﺒﺎت‬ ‫ﺟﺪیﺪ‬ ‫آﻤﺼﺪر‬ ‫اﻟﻌﺰﻻت‬ ‫هﺬﻩ‬ ‫ﻣﺜﻞ‬
‫اﻟﻨﺸﻄﺔ‬ ‫ﺔ‬
.

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Endophytic Actinomycetes of Some Medicinal Plants in Saint Katherine Area, Egypt

  • 1. CATRINA (2006), 1 (2): 91-96 © 2006 BY THE EGYPTIAN SOCIETY FOR ENVIRONMENTAL SCIENCES PROCEEDINGS OF THE FIRST INTERNATIONAL CONFERENCE ON CONSERVATION AND MANAGEMENT OF NATURAL RESOURCES, ISMAILIA, EGYPT, JUNE 18-19, 2006 ____________________________________________ * Corresponding Author: selshatoury@hotmail.com Endophytic Actinomycetes of Some Medicinal Plants in Saint Katherine Area, Egypt Sahar A. El-Shatoury*, Hesham M. Abdulla, Omnia A. El-Kraly, Waleed M. El-Kazzaz, and Ahmed Dewedar Botany Department, Faculty of Science, Suez Canal University, 41522 Ismailia, Egypt ABSTRACT A number of 41 morphologically different endophytic actinomycete isolates were recovered from surface-sterilized medicinal plants; Artemisia herba-alba, Echinops spinosus, Mentha longifolia and Ballota undulate. A simplified method for selective recovery of actinomycetes from internal plant tissues is described. Successful recovery was achieved on the media: starch-casein agar, 1/10 diluted starch casein agar, tap water-yeast extract agar and MG-plant extract agar. Preliminary description of the isolates, according to microscopic examination and amino acid composition of the cell wall hydrolystates, indicated prevalence of genus Streptomyces. The highest number of endophytic actinomycetes was recovered from Mentha longifolia and represented 34% of the isolates. Organic extracts of the growth metabolites from the cultures were tested for cytotoxicity against Artemia salina as well as for antimicrobial activities against 4 reference bacterial strains, 6 clinical bacterial cultures and two clinical fungal cultures. Variable activities were obtained with different actinomycete isolates; the highest activity could be detected against Gardnerella vaginitis and Shigella boydii strain ATCC 9207; being represented in 29% of the isolates in both cases. For the first time, the isolation of endophytic actinomycetes from Saint Katherine wild plants is reported and their potential use as novel source of bioactive compounds is discussed. Key words: Antimicrobial activity, cytotoxicity, endophytic actinomycetes, medicinal plants, Saint Katherine. INTRODUCTION Actinomycetes are a diverse group of filamentous Gram-positive bacteria well known for their production of an extensive array of chemically diverse and medically important secondary metabolites. Only a few recent studies have highlighted the bioactive importance of endophytic actinomycetes, including biocontrol of fungal plant pathogens (El-Tarabily, 2003; Coombs et al., 2004), production of antimalarial and antibimicrobial agents (Castillo et al., 2002), production of anticancer compounds (Caruso et al., 2000), production of plant growth regulators (Igarashi, 2004) and production of enzymes (Stamford et al., 2002). Plants growing in areas of great biodivesity usually have the prospect of harbouring endophytes with great biodiversity (Strobel and Daisy, 2003). The Saint Katherine World Heritage Site (WHS No. 954, UNESCO) of Sinai, Egypt, is one of the world's most biodiverse area; it is characterized by high percent of endimism in flora, fauna and related microbiota merging from the existing altitude gradient. However, to the best of our knowledge, microbiological studies on the endophytes residing in their plants were not considered yet. Thus, this habitat is one that deserves close examination for novel microbes that produce compounds with desired bioactivities. The present study describes the isolation of actinomycetes from internal tissues of some medicinal plants in the WHS of St. Katherine and evaluates metabolites for antimicrobial and cytotoxic activities. MATERIALS AND METHODS Site description and plant sampling Plants were collected during the early summer of 2005 from six sites with characteristic granitic country rocks, located in the ring dyke of the WHS. These were: Wadi Shreiag (29.8° 33' 57'' E, 51.9° 28' 33'' N and 1545.3 m Altitude), Wadi El Arba'een (18.2° 33' 59'' E, 24.7° 28' 40'' N and 1306.1 m Altitude), Al-Kharazen (53.2° 33' 57'' E, 12.2° 28' 43'' N and 1627.6 m Altitude), El-Talaa (55.8° 33' 55'' E, 00.6° 28' 34'' N and 1627.6 m Altitude), Wadi El-Sheikh (08° 33' 59'' E, 35.6° 28' 40'' N and 1281.7 m Altitude) and Wadi El- Raha (14.6° 33' 57'' E, 50.2° 28' 33'' N and 1544.7 m Altitude). Four plant species were collected for investigation: Artemisia herba-alba (Shieh) and Echinops spinosus (Khasheer), belonging to the family Compositae; Mentha longifolia (Habak) and Ballota undulate (Ghassa), belonging to the family Labiatae. These four plant species were selected for there medicinal value due to their high content of functional flavonoids, sesquiterpene, lactones and other essential oils (Rimbau et al., 1999). Healthy, green aerial parts were cut from these plants and placed in sealed plastic bags. Collected plant samples were dried at room temperature for 2 days and stored at 4o C for further processing. Isolation and characterization of endophytic actinomycetes Plant materials were processed with modification of the method described by Demain and Davies (1999) and plated into a battery of ten media: Tap Water Yeast Extract (TWYE) agar, Starch-Casein (SC) agar, 1/10 Starch-Casein agar, Tryptone Soya (TS) agar, MGA-PE medium, Potato-Dextrose (PD) agar, CYC agar, Arginine-Glycerol Salts (AGS) agar, Starch Nitrate (SN) agar and Actinomycetes Isolation (AI) agar (Basil et al., 2004; Demain and Davies 1999). Plants were aseptically cut into 1 g pieces (ca. 3-5 cm in length) and
  • 2. Endophytic actinomycetes of medicinal plants 92 immersed in 70% ethanol for 5 minutes for surface sterilization, followed by rinsing in sterile distillated water and air drying under a laminar flow hood. Surface sterility check and processing of plant material for endophytes isolation were performed as illustrated in flow chart (Fig. 1). All plants were processed in triplicates and all media were supplemented with 50µg/ml cycloheximide (antifungus) after sterilization. Plates were incubated at 28o C with regular monitoring; counts were recorded after 4 weeks. A total of 41 morphologically different actinomycete isolates were purified from plates using starch-casein agar medium and maintained as spore suspensions, in 20% glycerol, at 20o C (Hopwood et al., 1985). Isolates were characterized for their micromorphology and diaminopimelic acid (DAP) isomer in whole cell hydrolysate. Microscopic examinations of sporulated mycelia were performed on cultures grown on ISP4 medium (Shirling and Gottlieb, 1966). The determination of the DAP isomer in whole cell hydrolysate was performed as described by Schaal (1985) using dried mycelia obtained from cultures grown in TS broth in baffled flasks on shaking incubator (100 rpm), at 28 o C for 4-7 days. Enumeration of soil actinomycete Soil samples (10-15 cm depth) were collected aseptically from the target localities and kept in sterile polyethylene bags at 4 °C until isolation. Actinomycetes were isolated after serial dilution in phosphate buffer on SC agar supplemented with cycloheximide (50 µg/ml) using spread plate technique. Counts were recorded after incubation for 2-3 weeks at 28 °C. Fermentation procedure Preserved spore suspensions of actinomycete isolates were inoculated into 30 ml of SC liquid medium and incubated in a shaker incubator (100 rpm), for 5-7 days at 28 °C. Cultures were extracted using equal concentrations of ethyl acetate, for three successive times with vigorous shaking to thirty minutes. This allowed any organic molecules to be suspended in the less polar solvent. Ethyl acetate fractions were evaporated under vacuum into a preweighed vial, and then redissolved in ethyl acetate giving a final concentration of 10 mg/ml. Antimicrobial screening The raw extracts were tested by disk diffusion using 0.1 mg per disk against a representative panel of human pathogenic microorganisms as described by Castillo et al. (2002). This panel included 4 reference bacterial strains Salmonella typhimurium (NCMB 74), Escherichia coli (NCMB 11943), Shigella boydii ATCC 9207 and Pseudomonas aeruginosa NCMB 8295; 6 clinical bacterial cultures Vibrio sp., Proteus vulgaris, Klebsiella pneumonia, Gardnerella vaginitis, Staphylococcus aureus, and Streptococcus sp.; and two fungal cultures Candida albicans and Candida sp. Brine shrimp cytotoxicity assay The brine shrimp eggs were hatched in seawater (40 mg/l) as described by Svoboda and Hampson (1999). Hatched eggs were supplemented with 6 mg/l dried yeast and oxygenated for 48 hours at room temperature. Crude organic extracts (1 mg in ethyl acetate) were transferred into sterile microtubes and evaporated under Plant Sample Surface sterilization 70 % ethanol / 5min Aseptic Grinding Dilution in phosphate buffer plus 0.001% tween 80 Direct implanting Plating on SC, 1/10 SC, MGA-PE, AI, AGS, PD, CYC, TS, TWYE, SN Moist heat 55o C / 6min Washing in phosphate buffer Surface sterility check Plant pressing & washing buffer streaking on TSA plates Figure (1): Proposed strategy for selective isolation of endophytic actinomycetes from internal plant tissues.
  • 3. El-Shatoury et al. 93 vacuum,to which 10-20 A. salina larvae in 1 ml of seawater were added. Control blind sample was accompanied by adding ethyl acetate and duplication was performed. Cytotoxiciy was scored at intervals up to 84 hours compared to the blind samples. Statistical analysis Significant differences among different cultivation media and for different plant origins were studied by one-way analysis of variance. The means were separated at significant difference (p = 0.05). RESULTS Culturable endophytic actinomycetes The endophytic actinomycete populations from the investigated plants averaged between 102 -103 CFU/g dry plants. It was noticed that, recovery of actinomycetes was only successful using MGA-PE, SC, 1/10 SC and TWYE agar media, while the other media plates were over-grown with fungi and unicellular bacteria. Statistical analysis showed significant differences (p = 0.049) for actinomycete counts using the above mentioned four media. As shown in Figure (2), higher numbers were recovered on 1/10 SC and TWYE media compared to the other two media. The maximum population recorded was from Ballota undulata on TWYE agar. Direct implanting of grinded plant samples has recovered endophytic actinomycetes (data not shown); however, better separation of individual colonies was achieved by dilution and spread plate technique. In addition, the heat treatment did not enhance the recovery of actinomycetes from internal plant tissue as shown in Figure (3), and difference in actinomycetes counts between heat treated and untreated samples were not significant (p = 0.072). Tentative identification by microscopic examination and analysis of amino acid composition of the cell wall (Holt et al., 1994), indicated prevalence of genus Streptomyces, representing 41.5% of the isolated endophytic actinomycetes, in this study. Grouping of the isolates, based on DAP analysis of the whole cell hydrolysate revealed a number of 19 L-DAP containing isolates and 22 m-DAP containing isolates. As shown in Figure (4), high significant difference could be noticed between soil actinomycete population in the three investigated localities (p = 2.85 E-06). In contrast, endophytic actinomycete counts from Mentha longifolia collected from these localities were not significantly different (p > 0.05). Bioassay of antimicrobial and cytototoxic activities Results indicated bioactivity of 81% of the isolates (Fig. 5). Of the 41 endophytic actinomycete isolates, 30 exhibited microbial inhibitory effect. The highest activity was against Gardnerella vaginitis and Shigella boydii ATCC 9207, being represented in 29 % of the isolates in both cases. All the isolates failed to give activity against E. coli, P. Aeruginosa as well as the clinical cultures Proteus vulgaris and Candida sp. Cytotoxic effect against Artemia salina was positive for 27 endophytic actinomycetes. Of these, nine isolates exhibited high mortality rate reaching to 100% death after 12 hours. Those were mainly A. herba alba and E. spinosus isolates (Table 1). Figure (2):.Enumeration of endophytic actinomycetes on four selected media. Counts are average of triplicates with bars indicating Standard Error of the mean. Figure (3): Effect of heat treatment on recovery of endophytic actinomycetes from four plant species on 1/10 SC media. Counts are average of triplicates with bars indicating Standard Error of the mean. Figure (4): Actinomycetes population from Mentha longifolia and the surrounding bulk soil. Counts are average of three replications. 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 Count cfu / g of dry plant W. ElSheikh AlKharazen AlTalaa Sampling locality Bulk soil Mentha M. longifolia W. El-Sheikh Al-Kharazen El-Talaa 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Artemisia Echinops Mentha Ballota Count cfu / g of dry plant MGA-P E SC 1/10 SC TWYE A. herba alba E. spinosus M. longifolia B. undulata 0 500 1000 1500 2000 2500 3000 Artemisia Echinops Mentha Ballota Count cfu / g of dry plant heat treated untreated A. herba alba E. spinosus M. longifolia B. undulata
  • 4. Endophytic actinomycetes of medicinal plants 94 Figure (5): Total number of endophytic actinomycete isolates showing bioactivities. Isolates from the four investigated plant species are categorized into L-DAP and m-DAP containing groups. DISCUSSION It is recently apparent that plants can serve as a reserviore of endophytic microorganisms, meantime evidence thus far indicates that, metabolites from these sources hold pharmaceutical and agriculture promise (Castillo et al., 2002; Kunoh, 2002). In the present study, endophytic actinomycetes isolation was significantly higher on the low nutrient-content media 1/10 SC and TWYE, compared to the high nutrient- content media which allowed fast growing fungi and unicellular bacteria to dominate the isolation plates. These results were in agreement with Basil et al. (2004) for isolation of soil actinomycetes using TWYE and with May et al. (2003) for isolation of stone actinomycetes using 1/10 SC. McCarthy and Williams (1992) have described an autochthonous behaviour, i.e. sustained growth at low nutrient concentrations amongst actinomycetes, which may explain the successful selectivity of these low nutrient-content media for actinomycetes. Although average endophytic actinomycetes population was 102 to 103 cfu/g dry plants in the four plant species studied, the bioactivities of these endophytes varied widely. For example, 92% of M. longifolia endophytes showed broad antimicrobial activities and inhibited eight representatives of Gram positive, Gram negative bacteria and yeast strains. In contrast, only 31% of A. herba-alba endophytes exhibited antimicrobial activities against six of the tested microbial strains. Similarly, the cytotoxicity of M. longifolia isolates was 33% compared to 62% for A. herba-alba isolates. The high ratio of bioactive isolates obtained from this study (81%) compares well with other investigations, for example antimicrobial activity was 14% for actinomycetes endophytic in Taxus plant (Caruso et al., 2000) and 20% for endophytic actinomycetes isolated from a variety of Japanese wild plants (Igarashi et al., 2002). However, it should be emphasized that attempts to compare and contrast antimicrobial potential are susceptible to differences in protocols used. Although the available literature doesn't provide knowledge on the endophytes of the currently investigated plants, the four species are rich in metabolites of high medicinal value (Hanafy et al., 2000) and thus represent a promising source for bioactive microbial products. The recent discovery of different genera of actinomycetes and fungi that can produce quinoline alkaloids and diterpenoids identical to those originally characteristic of their host plants (Caruso et al., 2000; Puri et al., 2005) have arisen an interesting aspect that microbial endophytes may exhibit genetic recombination with their host plant to produce metabolites that facilitate the domination of its biological niche within the plant or even provide protection to the plant from harmful invading pathogens. While there was a significance difference in the culturable actinomycetes numbers in bulk soil of the studied localities, there were no qualitative differences in numbers and bioactivities of endophytic actinomycetes from M. longifolia collected from these localities. Bills et al. (2002) described a metabolic distinction between endophytes of the same plant origin in tropical regions and suggested the importance of the host plant in influencing the general metabolism of its endophytic microbes. Table (1): Source of actinomycetes and their bioactivities measured as diameter of the inhibition zone for antimicrobial activities and as percentage of dead larvae for cytotoxicty. Results were scored after 24 hours of incubation. Antimicrobial activity (avg ± SD) b Actinomycetes group (no. of isolates) a S. typhimurium Vibrio sp. S. boydii K. pneumonia Streptococcus sp. S. aureus G. vaginitis C. albicans Cytotoxicity (Death %) Artemisia herba-alba L-DAP group (2) 9 ± 0.7 9 ± 0.7 12 ± 0.5 16 ± 0.6 6 ± 0.7 17 ±.01 m-DAP group (5) 9 ± 0.6 7 ± 1.4 27± 0.7 100 ± 25 Echinops spinosus L-DAP group (3) 18 ± 0.3 7 ± 0.5 6 ± 0.6 100 ± 0.01 m-DAP group (1) 7 ± 0.3 100 ± 0.01 Mentha longifolia L-DAP group (7) 11 ± 0.7 10 ± 4.6 6 ± 0.7 15 ± 0.6 13 ± 0.6 7 ± 0.6 7 ± .05 33 ± 0.7 m-DAP group (4) 13 ± 7 12 ± 0.6 18 ± 3 12 ±0.7 8 ± 0.5 7 ± .01 67 ± 33 Ballota undulate L-DAP group (5) 7 ± 0.5 12 ± 0.5 7 ± 0.6 8 ± 0.7 9 ± 4 66 ± 40 m-DAP group (3) 15 ± 0.7 7 ± 0.6 0 a : Number of actinomycetes isolates in each group. b : Average (avg) of inhibition zone diameter ± Standard deviation (SD). 0 1 2 3 4 5 6 7 8 9 10 L-DAP m-DAP L-DAP m-DAP L-DAP m-DAP L-DAP m-DAP Artemisia Echinops Mentha Ballota Bioactive inactive A. herba alba E. spinosus M. longifolia B. undulata Number of isolates
  • 5. El-Shatoury et al. 95 Overall, it is concluded that, actinomycetes could be selectively isolated from the internal tissues of wild medicinal plants in the WHS using a simplified method. The bioactivities of these isolates varied with plant origin and the higher percentage of wide-spectrum antimicrobial strains were from M. longifolia. The reason for this requires further investigation. We hypothesize that actinomycetes which colonize internal tissues of these plants are highly adapted to this unique little-studied habitat and should be investigated for detailed identification and potential use as source of bioactive agents. ACKNOWLEDGMENTS We are grateful to Dr. Kamal Ghodeif, Geology Department, Faculty of Science, Suez Canal University for his invaluable guidance during the sampling trips. We also thank Dr. Raafat Abd El-Wahab, Botany Department, Faculty of Science, Suez Canal University for his effort to identify the plants. This study was funded by grant C/3801-1 from the International Foundation for Science (IFS), Sweden. REFERENCES BASIL, A.J., J.L. STRAP, H.M. KNOTEK-SMITH, AND D.L. CRAWFORD. 2004. Studies on the microbial populations of the rhizosphere of big sagebrush (Artemisia tridentata). Journal of Industrial Microbiology and Biotechnology 31: 278-288 BILLS, G., A. DOMBROWSKI, F. PELAEZ, J. POLISHOOK, AND Z. AN. 2002. Recent and future discoveries of pharmacologically active metabolites from tropical fungi. In R. Watling, J.C. Frankland, A.M. Ainsworth, S. Issac, and C.H. Robinson. (ed.), Tropical mycology: Micromycetes, volume 2. CABI Publishing, New York, New York 165-194. CARUSO, M., A.L. COLOMBO, L. FEDELI, A. PAVESI, S. QUARONI, M. SARACCHI, AND G. VENTRELLA. 2000. Isolation of endophytic fungi and actinomycetes taxane producers. Annals of Microbiology 50: 3-13. CASTILLO, U.F., G.A. STROBEL, E.J. FORD, W.M. HESS, H. PORTER, J.B. JENSEN, H. ALBERT, R. ROBISON, M.A. CONDRON, D.B. TEPLOW, D. 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  • 6. CATRINA (2006), 1 (2): 91-96 © 2006 BY THE EGYPTIAN SOCIETY FOR ENVIRONMENTAL SCIENCES PROCEEDINGS OF THE FIRST INTERNATIONAL CONFERENCE ON CONSERVATION AND MANAGEMENT OF NATURAL RESOURCES, ISMAILIA, EGYPT, JUNE 18-19, 2006 96 ‫ا‬ ‫اﻟﻄﺒﻴﺔ‬ ‫اﻟﻨﺒﺎﺗﺎت‬ ‫ﺑﻌﺾ‬ ‫ﻓﻰ‬ ‫اﻟﻤﻌﻴﺸﺔ‬ ‫داﺧﻠﻴﺔ‬ ‫ﻷآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫آﺎﺗﺮیﻦ‬ ‫ﺱﺎﻥﺖ‬ ‫ﺑﻤﻨﻄﻘﺔ‬ ، ‫ﻣﺼﺮ‬ ‫دویﺪار‬ ‫أﺣﻤﺪ‬ ،‫اﻟﻘﺰاز‬ ‫وﻟﻴﺪ‬ ،‫اﻟﻘﺮﻋﻠﻰ‬ ‫أﻣﻨﻴﺔ‬ ،‫ﻋﺒﺪاﷲ‬ ‫هﺸﺎم‬ ،‫اﻟﺸﻄﻮرى‬ ‫ﺱﺤﺮ‬ ‫ﻗﺴﻢ‬ ‫اﻟﻨﺒﺎت‬ ‫آﻠﻴ‬ ، ‫اﻹ‬ ،‫اﻟﺴﻮیﺲ‬ ‫ﻗﻨﺎة‬ ‫ﺟﺎﻣﻌﺔ‬ ،‫اﻟﻌﻠﻮم‬ ‫ﺔ‬ ‫ﺱﻤﺎﻋﻴﻠﻴﺔ‬ ‫ﻣﺼﺮ‬ ، ‫اﻟﻌﺮﺑـــﻰ‬ ‫اﻟﻤﻠﺨﺺ‬ ‫ا‬ ‫ﺏﺎﻟﻌﺰل‬ ‫اﻟﺪراﺱﺔ‬ ‫هﺬﻩ‬ ‫ﺗﺨﺘﺺ‬ ‫ﻟﻤﻴﻜﺮوﺏﻰ‬ ‫ﺔ‬‫ﻷرﺏﻌ‬ ‫ﺔ‬‫اﻟﺪاﺥﻠﻴ‬ ‫ﺴﺠﺔ‬‫اﻷﻥ‬ ‫ﻓﻰ‬ ‫اﻟﻤﺘﻮاﺟﺪة‬ ‫ﻟﻸآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫اﻟﺤﻴﻮى‬ ‫اﻟﻨﺸﺎط‬ ‫وﺗﻮﺹﻴﻒ‬ ‫ﻰ‬‫وه‬ ‫ﺔ‬‫ﻃﺒﻴ‬ ‫ﺎت‬‫ﻥﺒﺎﺗ‬ : Artemisia herba-alba ‫و‬ Echinops spinosus ‫و‬ Mentha longifolia ‫و‬ Ballota undulate ‫ﺪ‬‫ﺗﺤﺪی‬ ‫ﻦ‬‫أﻣﻜ‬ ‫ﺚ‬‫ﺡﻴ‬ ‫اﻟﻨﺒ‬ ‫ﺗﻠﻚ‬ ‫ﺱﺎآﻨﺔ‬ ‫ﻟﻸآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫اﻹﺥﺘﻴﺎرى‬ ‫ﻟﻠﻌﺰل‬ ‫ﻣﺒﺴﻄﺔ‬ ‫ﻃﺮیﻘﺔ‬ ‫اﻟﻐﺬاﺋﻴﺔ‬ ‫اﻷوﺱﺎط‬ ‫ﻋﻠﻰ‬ ‫اﻟﻌﺰل‬ ‫أﻓﻀﻠﻴﺔ‬ ‫اﻟﻨﺘﺎﺋﺞ‬ ‫وأوﺿﺤﺖ‬ ،‫ﺎﺗﺎت‬ : ‫ﺎر‬‫ﺁﺟ‬ ‫إﻟﻰ‬ ‫اﻟﻤﺨﻔﻒ‬ ‫واﻟﻜﺎزیﻦ‬ ‫اﻟﻨﺸﺎ‬ ‫وﺁﺟﺎر‬ ،‫واﻟﻜﺎزیﻦ‬ ‫اﻟﻨﺸﺎ‬ ‫اﻟﻨﺒﺎﺗﻰ‬ ‫اﻟﻤﺴﺘﺨﻠﺺ‬ ‫وﺁﺟﺎر‬ ،‫اﻟﺨﻤﻴﺮة‬ ‫ﻣﺴﺘﺨﻠﺺ‬ ‫وﺁﺟﺎر‬ ،‫اﻟﻌﺸﺮ‬ . ‫ﻋﺪد‬ ‫ﻋﻠﻰ‬ ‫اﻟﺤﺼﻮل‬ ‫ﺗﻢ‬ ‫وﻗﺪ‬ 41 ‫ﻟﻬ‬ ‫ﺪﺋﻰ‬‫اﻟﻤﺒ‬ ‫اﻟﺘﻌﺮیﻒ‬ ‫أﻇﻬﺮ‬ ،‫اﻟﻈﺎهﺮى‬ ‫اﻟﺸﻜﻞ‬ ‫ﻓﻰ‬ ‫ﻣﺘﺒﺎیﻨﺔ‬ ‫أآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫ﻋﺰﻟﺔ‬ ‫ﻨﺲ‬‫ﺟ‬ ‫ﺸﺎر‬‫إﻥﺘ‬ ‫ﺎ‬ Streptomyces ‫أن‬ ‫اﻟﻨﺘﺎﺋﺞ‬ ‫أوﺿﺤﺖ‬ ‫آﻤﺎ‬ 34 % ‫ﻥﺒﺎت‬ ‫أﻥﺴﺠﺔ‬ ‫ﻣﻦ‬ ‫آﺎن‬ ‫اﻟﻌﺰﻻت‬ ‫إﺟﻤﺎﻟﻰ‬ ‫ﻣﻦ‬ Mentha longifolia . ‫ﺗﻢ‬ ‫وﻗﺪ‬ ‫ﺸﺎط‬‫اﻟﻨ‬ ‫ﺗﻘﻴﻴﻢ‬ ‫ﺗﻢ‬ ‫اﻟﺤ‬ ‫ﻴ‬ ‫اﻷآﺘﻴﻨﻮﻣﻴﺴ‬ ‫ﻟﻤﺰارع‬ ‫اﻟﻌﻀﻮیﺔ‬ ‫ﻟﻠﻤﺴﺘﺨﻠﺼﺎت‬ ‫ﻮى‬ ‫ﻴ‬ ‫ﺿﺪ‬ ‫اﻟﺨﻠﻮیﺔ‬ ‫اﻟﺴﻤﻴﺔ‬ ‫ﺡﻴﺖ‬ ‫ﻣﻦ‬ ‫وذﻟﻚ‬ ‫ﺘﺎت‬ Artemia salina ‫ﻰ‬‫اﻟﻤﻴﻜﺮوﺏ‬ ‫ﺸﺎط‬‫واﻟﻨ‬ ، ‫ﺿﺪ‬ 10 ‫إآﻠﻴﻨﻴﻜﻴﺔ‬ ‫ﻓﻄﺮ‬ ‫ﺱﻼﻟﺘﻰ‬ ‫إﻟﻰ‬ ‫إﺿﺎﻓﺔ‬ ‫وإآﻠﻴﻨﻴﻜﻴﺔ‬ ‫ﻣﺮﺟﻌﻴﺔ‬ ‫ﺏﻜﺘﻴﺮیﺔ‬ ‫ﺱﻼﻻت‬ . ‫ﻊ‬‫واﺱ‬ ‫ﻮى‬‫ﺡﻴ‬ ‫ﺸﺎط‬‫ﻥ‬ ‫ﻮد‬‫ﻟﻮﺟ‬ ‫ﺤﺔ‬‫ﻣﻮﺿ‬ ‫اﻟﻨﺘﺎﺋﺞ‬ ‫وﺟﺎءت‬ ‫اﻟﻤﺨﺘﻠﻔﺔ‬ ‫اﻟﻨﺒﺎﺗﻴﺔ‬ ‫اﻟﻤﺼﺎدر‬ ‫ﻣﻦ‬ ‫اﻷآﻴﺒﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫ﻟﻌﺰﻻت‬ ‫وﻣﺘﺒﺎیﻦ‬ . ‫ﻼﻟﺔ‬‫ﺱ‬ ‫ﻊ‬‫ﻣ‬ ‫ﺰﻻت‬‫ﻟﻠﻌ‬ ‫ﻰ‬‫ﻣﻴﻜﺮوﺏ‬ ‫ﺪ‬‫ﺿ‬ ‫ﺸﺎط‬‫ﻥ‬ ‫ﻰ‬‫أﻋﻠ‬ ‫ﺎن‬‫وآ‬ Shigella boydii ATCC 9207 ‫واﻟﺴﻼﻟ‬ ‫اﻟﻤﺮﺟﻌﻴﺔ‬ ‫ﺔ‬ Gardnerella vaginitis ‫ﺠﻠﺖ‬‫ﺱ‬ ‫ﺚ‬‫ﺡﻴ‬ ،‫اﻹآﻠﻴﻨﻴﻜﻴﺔ‬ 29 % ‫ﺪ‬‫ﺿ‬ ً ‫ﺎ‬‫ﺸﺎﻃ‬‫ﻥ‬ ‫ﺴﺒﺘﺎت‬‫اﻷآﺘﻴﻨﻮﻣﻴ‬ ‫ﻦ‬‫ﻣ‬ ‫اﻟﺴﻼﻟﺘﻴﻦ‬ ‫هﺎﺗﻴﻦ‬ ‫ﻣﻦ‬ ‫آﻞ‬ . ‫اﻹﺥﺘﻴﺎرى‬ ‫اﻟﻌﺰل‬ ‫إﻣﻜﺎﻥﻴﺔ‬ ‫ﻋﻦ‬ ‫اﻷوﻟﻰ‬ ‫هﻰ‬ ‫اﻟﺪراﺱﺔ‬ ‫ﺗﻠﻚ‬ ‫ﺎآﻨ‬‫ﺱ‬ ‫ﻟﻸآﺘﻴﻨﻮﻣﻴﺴﻴﺘﺎت‬ ‫ﺎﺗﺮیﻦ‬‫آ‬ ‫ﺎﻥﺖ‬‫ﺱ‬ ‫ﺔ‬‫ﻣﻨﻄﻘ‬ ‫ﻰ‬‫ﻓ‬ ‫ﺎت‬‫اﻟﻨﺒﺎﺗ‬ ‫ﺔ‬ ‫ﺔ‬‫وﻓﻌﺎﻟﻴ‬ ‫اﻟﺤﻴﻮی‬ ‫ﻟﻠﻤﺮآﺒﺎت‬ ‫ﺟﺪیﺪ‬ ‫آﻤﺼﺪر‬ ‫اﻟﻌﺰﻻت‬ ‫هﺬﻩ‬ ‫ﻣﺜﻞ‬ ‫اﻟﻨﺸﻄﺔ‬ ‫ﺔ‬ .