BIOACTIVE MOLECULES

FROM ENDOPHYTIC

MICROORGANISMS

Prof. Vijeshwar Verma

Dean Faculty of Engineering &

Head, Department of Biotechnology

Shri Mata Vaishno Devi University

Katra (J&K state)

v.verma@smvdu.ac.in

Overview

What are plant endophytes?

Benefits of endophytes for plants

Only few endophytes are known

Result: endophyte diversity is completely

underestimated

Conclusions : Exiting future opportunities

ENDOPHYTE/PLANT RELATIONSHIP

– Endophyte gains

• shelter and nutrients

– Plant cost

• nutrients and resources – (e.g. more susceptible to pests and diseases: BCAs)

– Plant gains

• Growth promotion – (enhanced nutrient uptake)

• Increased tolerance to harsh environments – (e.g. drought tolerance)

• Induced Resistance to pests and diseases (BCAs)

Life cycle of Endophytes

Some endophytes

Some Known Endophytes

Pestalotiopsis microspora

Artemisia annua

Pestalotiopsis jesteri

Acremonium terricola

Monodictys castaneae

Penicillium glandicola

Phoma tropica

Tetraploa aristata etc.

Benefits Source of bioactive molecules.

Antibiotics

Alkaloids

Antioxidants

Immunosuppresive compounds.

Antiviral compounds

Insecticides and pesticides

Chemical structures of some bioactive compounds

produced by endophytic microorganisms.

FACTS

• Endophytes have proven to be rich sources of novel natural compounds.

• They have a wide-spectrum of biological activities and a high level of structural diversity.

• The use of endophytes as biocatalysts in the biotransformation process of natural products assumes greater importance.

• The application of microorganisms by the food and pharmaceutical industries to obtain compounds of interest is still modest.

• Endophytes are only example of a life form inhabiting plant species threatened with extinction.

Taxol

Biotechnological applications of endophytic microorganisms

Why Plants let them in?

Nutrient acquisition

Nitrogen fixation, phosphate, etc.

Pathogen resistance

Antimicrobial compounds

Stress tolerance

Heat, salt, drought

Growth promotion

Phytohormone production

Overall, endophytes provide the means to

rapidly adapt to new environmental conditions

Taxus

Scientific classification

Kingdom: Plantae

Division: Pinophyta

Class: Pinopsida

Order: Pinales

Family: Taxaceae

Genus: Taxus

L.

Species

Taxus baccata - European Yew

Taxus brevifolia - Pacific Yew, Western Yew

Taxus canadensis - Canadian Yew

Taxus chinensis - Chinese Yew

Taxus cuspidata - Japanese Yew

Taxus floridana - Florida Yew

Taxus globosa - Mexican Yew

Taxus sumatrana - Sumatran Yew

Taxus wallichiana - Himalayan Yew

Taxus is a genus of yews, small coniferous trees or shrubs in the yew

family Taxaceae. They are relatively slow-growing and can be very long-lived,

and reach heights of 1-40 m, with trunk diameters of up to 4 m.

Taxus baccata (European Yew)

shoot with mature and

immature cones

Taxol

Potent antifungal agent

Prevents cell division

Same effect on human

cancer cells

Endophytes of Yew but

also of other plants

(non-Taxol producer;

medicinal plants)

Signals from plant activate

fungus to make

Taxol

Taxol Facts

Very effective treatment against ovarian

cancer, breast cancer, melanoma, and colon

cancer

Stops cell division, thus blocking cancer. It

does this by interfering with microtubule

function. Microtubules are responsible for

pulling apart the sets of chromosomes in

mitosis.

Taxol Needs

It is estimated that 250 kg of pure Taxol are

needed to treat cancer in the USA. This would

require the bark of about 360,000 trees per

year!

Obviously Taxol woud be very expensive by this

method (approximately $200,000 to $300,000

per kg).

Taxol is a very good target for

biotechnology

• a) tissue culture of bark cells

• b) fungus produces taxol

• c) alternative species

• d) genetic engineering

• e) chemical synthesis

The Search for Taxoid-Producing Microorganisms –

Stierle et al. (1993); an endophytic microorganism

colonizing yew tree

Within two years, Stierle groups isolated more than 300

fungi from the bark and needles of Taxus brevifolia

Taxomyces andreanae; the first described endophytic

fungus of pacific yew -Strobel et al. (1996)

A second endophytic fungus; Pestalotiopsis microspora

from Taxus wallachiane, a Himalayan yew growing on

steep, moist mountain slopes(1500-3000 m)

Advantages of microbial fermentation

large biomass production(microorganisms typically

respond favorably to routine culture conditions)

productivity amplification and stability

Availability of less sophisticated and time consuming

methods for genetic manipulation of microorganisms

Isolating Endophytes

Surface sterilized

Plant Microbes

grow out of the cut

tissue when put on agar

plate

Surface sterilized plant

Microbes grow out of the cut tissue when put on agar

Sliced stems on plates and endophytes

Endophyte cultures result on different media

WORK in PROGRESS

Isolation of endophytic fungi from different plants of medicinal

importance collected from different regions of J&K state.

Purifying the fungal/bacterial endophytes by further subculturing

Morphological features of isolated endophytic fungi

Screening of the isolates for the presence of enzymes like Amylases,

Proteases, Cellulases, Lipases, laccases & unique bioactive molecules

Screening of the isolates for antimicrobial activity against several

pathogenic strains

Identification of selected fungal endophytes based on amplification and

sequencing of the ITS region of the fungal rRNA using PCR with

universal primers (ITS1 and ITS4)

Collection of Sample

RESEARCH METHODOLOGY

Isolation/ Characterization of Endophytes (Fungi)

DNA Isolation Optimization of Media/

Culture Condition

Endophytes

Enzymatic/Anti-microbial Screening

Photographs showing Endophytes

isolated from different Plants

RESULTS OBTAINED SO FAR • 23 isolates of endophytes have been isolated and

purified from different medicinal plants.

• All the 23 isolates have been screened for important

enzymes like Amylases,Cellulases,Lipases,Laccases

and Proteases.

• Most of the isolates showed good enzymatic potential.

• All these 23 isolates have also been screened for

antimicrobial activity against different test organisms

and some of the endophytes showed good antimicrobial

activity against some of the test organisms.

• Some of the endophytes have been identied based on

morphological and ITS sequencing.

• Analysis of sequencing results of the six endophytes from W.somnifera and M.koengii revealed significant similarity :

• sample1,WLU1-(1)(b): Alternaria alternata(98%)

• Sample2,WLU1-B2: Alternaria sp.(98%)

• Sample3,WLU2-(1)(a): Alternaria tenuissima(96%)

• Sample4,WLU2-(1)(b): Alternaria compacta(99%)

• Sample5, MKLU2-B2: Glomerella cingulata(94%)

• Sample6,MKLU4-2: Alternaria brassicae(83%)

1. Puri, S.C., Verma, V., Amna, T., Qazi, G.N. and Spiteller, M. (2006) An Endophytic Fungus from

Nothapodytes foetida that Produces Camptothecin. J. Nat. Prod. (Published on line) NP0502802.

2. Amna, T., Puri, S.C., Verma, V., Sharma, J.P., Khajuria, R. K., Musarrat, J., Spiteller, M. and Qazi, G.N.

(2006) Bioreactor Studies on the Endophytic Fungus Entrophospora infrequens for the Production of an

anticancer alkaloid Camptothecin. Canadian Journal of Microbiology, 52(3): 189-196.

3. Puri, S.C., Nazir, A., Chawla, R., Arora, R., Riyaz-ul-Hasan, S., Amna, T., Ahmed, B., Verma, V., Singh, S.,

Sagar, R., Sharma, A., Kumar, R., Sharma, R. K. and Qazi, G.N. (2006) The endophytic fungus Trametes

hirsuta as a novel alternative source of Podophyllotoxin and related aryl tetralin lignans. J. Biotechnology

122(4): 494–510.

4. Amna, T., Khajuria, R. K., Puri, S.C., Verma, V. and Qazi, G.N. (2006) Determination and quantification of

camptothecin in an endophytic fungus by liquid chromatography-positive mode electrospray ionization

tandem mass spectrometry (LC-MS/MS). Current Science 91(2):208-212 .

5. Verma, V., Sudan, P. and Kour, A. (2008) Endophytes: A Novel Source for Bioactive Molecules. Proc. of

Indian Natn. Sci. Acad. 74(2): 73-86

6. Kour, A., Shawl, A.S., Rehman, S., Sultan, P., Qazi, P.H., Suden, P., Khajuria, r.K. and Verma, V. (2008)

Isolation and identification of an endophytic starin of Fusarium oxysporum producing podophyllotoxin from

Juniperus recurva. World J. Microbiol. & Biotechnol. 24(7): 1115-1121.

7. Kour, A., Shawl, A.S., Rehman, S., Qazi, P.H., Suden, P., Sultan, P., Verma, V. and Qazi, G. N. (2010) An

endophytic fungus from Juniperus recurva producing podophyllotoxin with other biological activity. Annals

of Microbiology. Volume 59, Number 1, Pages 157-161.

PUBLICATIONS IN ENDOPHYTE RESEARCH

Patents

• Puri, S.C., Verma,V., Amin, T., Qazi, G.N. and Spiteller, M. Camptothecin and camptothecinoids from endophytic fungi of Nothapodytes foetida. 469NF/2002 application filed in Indian & PCT.

• Puri, S.C., Nazir, A., Handa, G., Dhar, K.L., Parshad, R., Verma, N., Khajuria, R.., Verma, V., and Qazi, G.N. Podophylotoxin and related cytotoxic aryl-tetrahydronaphthalene lignans from endophytic fungi. N/F 0474/2004.

• Puri, S.C. and Verma, V. Camptothacin and Camptothacinoids from endophytic fungi of Nothapodytes Foetida. US Patent No. 7378268, Patent NFNO 0469NF2002/US, Patent granted: 27/05/2008.

• Puri, S.C., Verma, V., Amin, T., Handa, G., Gupta, V., Verma, N., Khajuria, R.K. and Saxena. A Novel endophytic Camptothecin and Camptothecinoid producing fungi and process of producing the same. India Patent No. 238011, Patent NFNO 0469NF2002/IN. Patent granted 18/01/2010.

• Puri, S.C., Verma, V., Amin, T., Handa, G., Gupta, V., Verma, N., Khajuria, R.K. and Saxena. A Novel endophytic Camptothecin and Camptothecinoid producing fungi and process of producing the same. GB Patent No.: 1828374, Patent NFNO 0469NF2002/GB, Patent granted 17/11/2010.

Collected samples of metagenomic water, and soil samples from :

• Apharwat Glacier,Gulmarg

•Panamick,Ladakh

•Walna, Ladakh

•Reen, Anantag

Collection of Samples

Microscopic view of endophytic fungal

mycelia from N.foetida

si_okt_30_02 # 2587-2679 RT: 22.54-23.27 AV: 15 NL: 4.04E5 F: + c APCI SRM ms2 349.00@-40.00 [ 49.98-349.99]

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z

0

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tive

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247.5

248.3 304.8

219.6

249.1

56.9 245.7 218.7

220.2

167.6

221.1 205.3 305.3 276.6

180.8 274.7

258.7 203.8 235.0 348.7

291.2 193.7 319.3

166.5 331.1 142.7 94.9 122.6 57.4 108.5 80.9

CPT aus

Pflanzen

Mass spectrum of Camptothecin

si_okt_30_05 # 1746-2249 RT: 15.18-19.50 AV: 84 NL: 1.00E4 F: + c APCI SRM ms2 349.00@-40.00 [ 49.98-349.99]

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z

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305.0 219.8

249.1

56.8 218.8

220.7

167.7

247.1

277.0

205.2 274.8

180.6 204.0 348.8 290.8 258.9 245.5

181.3 320.3 57.4 81.0 193.6 319.6 109.1 91.0 143.2 166.6 117.0

331.2

Probe 1 CPT neu

Mass spectra of fungal camptothecin

IR Spectrum of camptothecin

IR Spectrum of fungal camptothecin

1H-NMR Spectrum of fungal camptothecin

13C-NMR Spectrum of fungal camptothecin

N

N

O

O

O

HO

H3C

12 3

4

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67

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18 19 2021

1316a

13C-NMR Spectrum of fungal camptothecin

Identification, isolation and bioactive properties of

endophytic fungi from medicinal plants “Withania

somnifera” and “Murraya koengii”

INTRODUCTION

Endophytes are bacteria and fungi which live within

plant tissues for all or part of their life cycle and cause

no apparent infection.

Mutualism interaction between endophytes and host

plants may result in fitness benefits for both partners.

• Poorly biotechnologically explored.

• Diverse biological activity (antibacterial, antifungal,

antitumoral, antiviral, antioxidant, immunosupressor,

inseticide and control of diabetes and malaria).

WHY TO STUDY ENDOPHYTIC

MICROORGANISMS?

• Nutrient acquisition

• Nitrogen fixation, phosphate, etc.

• Pathogen resistance

• Antimicrobial compounds

• Stress tolerance

• Heat, salt, drought

• Growth promotion

• Phytohormone production

• Overall, endophytes provide the means to adopt to rapidly environmental conditions

Why let them in?

S.No

ENDOPHYTE PLANT PROPERTIES

1 Cryptosporiopsis quercina Tripterigeum wilfordii

Against pests pyricularia oryzae and other pathogenic fungi.

2

phomopsissp Erythrina crista-galli

Anti-inflammatory,antifungally and antibacterially active polyketide lactone.

3 Taxomyces andreanae

Taxus brevifolia anti-cancer

Endophytes from different plants:

• Medicinal plants have been used by mankind for its

curative quality since the starting of human

civilization.

• Plants produce huge amount of medicinal agents and

we can get maltitude of medicines from these agents.

• Various plants have been studied and reported.

MEDICINAL PLANTS

S.NO Common name botanical name

Plant part use Medicinal use

1 Amla Emblica officinnalis

Fruit VitaminC,Cough,Diabetes,Cold,Lxative,hyper acidity

2 Aswagandha Withania somnifera

Roots,leaves Restorative Tonic,Stress,Nerves disorder,anticarcinogenic,anti-inflammatory

3 Brahmi Bacopa Whole plant Aenimic,jaundice,dropsy

4 Sandal wood Santalum album

Heart wood,oil Skin disorder,burning sensation,jaundice,cough

5 Tulsi Ocimum sanctum

Leaves/seed Cough,cold,bronchitis.

6 Curry patta Murraya koengii

Leaves,stem,roots

Anti-diabetic,anti-inflammatory,cholestrol control,antifungal.

Selected plants for this project:

Murraya koengii Withania somnifera

Murraya koengii

Withania somnifera

CLASSIFICATION:

Kingdom: Plantae

Order: Sapindales

Family: Rutaceae

Genus: Murraya

Species: Koengii

Kingdom:Plantae

Order:Solanales

Genus:Withania

Species:Somnifera

Family:Solanaceae

Aims and Objectives

Isolation of endophytic fungi from leaves and stem of medicinal plants Withania somnifera and Murraya koenigii

Purifying the fungal endophytes by further subculturing

Morphological features of isolated endophytic fungi

Screening of the isolates for the presence of enzymes like Amylases, Proteases, Cellulases, Lipases and laccases

Screening of the isolates for antimicrobial activity against several pathogenic strains

Identification of selected fungal endophytes based on amplification and sequencing of the ITS region of the fungal rRNA using PCR with universal

primers (ITS1 and ITS4)

Collection of Sample

RESEARCH METHODOLOGY

Isolation/ Characterization of Endophytes (Fungi)

DNA Isolation Optimization of Media/

Culture Condition

Endophytes

Enzymatic/Anti-microbial Screening

• Collection of Explants

• Transfer of explants to Potato –Dextrose agar plates

and incubation at 250C .

• After 5-6 days endophytes grow out of explants.

• Endophytes transferred to fresh PDA Plates.

Isolation of Endophytes

Endophytes isolates from Murraya

koengii and Withania somnifera

(a) W.somnifera(leaf) (b) M.koengii(leaf)

Leaves were inoculated on Potato dextrose agar showing initial growth

of endophytic fungi.

Photographs showing mixed cultures of

Endophytes:

(a) MKLU1-2, (b) MKLU1-2(+), (c)MKSU4-A2, (d)WLU1-3(b)

• Repeated subculturing was done in order to purify the endophytic cultures:

Purification of fungal endophytes

(a)MKLU1(2), (b)MKLH2-B1, (c)MKSU2A1-(1), (d)WLU2-(1)(b)

Identification of Endophytic Fungi on the basis of

their macroscopic and microscopic characters

WLU1(2)(a) WLU1(2)(a)

WLU2(3)(b) WLU2(3)(b)

Screening:Enzymatic activity

Endophytes Showing Amylase Activity on 1% Starch PDA

Plates.

Endophytes showing Laccase activity

Laccase activity with 1-napthol on PDA Plates and zone of clearance

Protease activity.

Endophytes showing Protease Activity On Skimmed Milk PDA Plates .

Lipase Activity.

Endophytes Showing Lipase Activity with tributyrin on PDA Plates .

Cellulase Activity

Endophytes showing Cellulase Activity on 1%CMC PDA Plates.

S No. Plant part

used

Isolates Amylase Protease Laccase Lipases Cellulase

1 Leaf WLU1-

(1)(b)

+++ + - - -

2 Leaf WLU2-

(3)(b)

+++ + - - -

3 Leaf WLU1-

(3)(b)

++ ++ - - +++

4 Leaf WLU2-

(1)(a)

++ + - - -

5 Leaf WLU1-

(2)(a)

+++ + ++ - -

6 Leaf WLU1B2 - - ++ - -

7 Leaf WLU2(1)(b) +++ + - + -

Table showing enzymatic activity of endophytes isolates from W.somnifera

:

S No. Plant part

used

Isolates Amylase Protease Laccase Lipase Cellulase

1 Stem MKSU1A1(+

)

+++ + - + -

2 Leaf MKLU4A2 - - - + -

3 Leaf MKLU1(2) +++ - - + -

4 Leaf MKLU4-A3 _ - - - -

5 Leaf MKLH2-B1 ++ - - ++ -

6 Stem MKSU2A1(1) - - - + -

7 Leaf MKLU4-(2) - ++ ++ + +

8 Leaf MKLU4-A1 - - - - -

9 Leaf MKLU4-B1 ++ + ++ - +

10 Stem MKSU4-A2 ++ - - - +

11 Leaf MKLU1-2(+) _ - + - -

12 Leaf MKLU4-2(+) _ - - ++ +

13 Leaf MKLU2-A ++ ++ - - +

14 Leaf MKLU2-B2 ++ +++ - ++ ++

15 Leaf MKLU2-2(+) ++ +++ - +++ ++

Table showing enzymatic activity of endophytes isolate from M.koengii

Antimicrobial Activity

TEST ORGANISMS USED FOR ANTI MICROBIAL ACTIVITY

TEST ORGANISMS MEDIA FOR TEST ORGANISMS

Bacillus subtilis(MTCC-10619) Luria-Bertani

Staphylococcus aureus (MTCC-3160) Luria-Bertani

E.coli (MTCC-1652) Luria-Bertani

Klebsiella pneumonia (MTCC- 39) Luria-Bertani

Enterobacter aerogenes(MTCC -111) Luria-Bertani

Mycobacterium smegmatis (MTCC-994) Nutrient broth

Proteus vulgaris (MTCC-426) Nutrient broth

Enterobacter sps. (MTCC-7104) Nutrient broth

Pseudomonas aeruginosa (MTCC-3542) Luria-Bertani

Candida albicans(MTCC-227) Malt Yeast Extract

Aspergillus fumigates (MTCC-8673) Czapek Yeast Extract Agar

Aspergillus Niger(MTCC-1344) Czapek Yeast Extract Agar

Overnight grown test organisms were spread on LB Agar plates.

Supernatant were loaded in wells.

Zones of clearance were observed after incubation at 30ºC for 24 hours.

Screening of isolates for antimicrobial activity

Agar well diffusion method

Plates showing anti-microbial activity with different strains

WLU2-(1)(a)

WLU2-(1)(a)

WLU2-(1)(a)

MKSU2-

(A1)(1)

S.NO. Supernatant A.fumigatus A.niger C.albican P.vulgaris k.pneumoniae

1 WLU1-B2 ++ ++ - - -

2 WLU1-(2)(a) ++ - ++ - -

3 WLU2-(3)(b) + - - ++ -

4 WLU1-(3)(b) - - - - -

5 WLU1-(1)(b) - - - - -

6 WLU2-(1)(b) - - - - -

7 WLU2-(1)(a) ++ ++ ++ +++ -

Table showing antimicrobial activity of endophytes isolated from

W.somnifera with different test organisms:

S.NO

.

Supernatant E.aerogenes P.aerogenosa S.aureus M.smigmatis B.subtilis

1 WLU1-B2 - - - - -

2 WLU1-(2)(a) + - - - +

3 WLU2-(3)(b) - - - - -

4 WLU1-(3)(b) - - - - -

5 WLU1-(1)(b) - - - - -

6 WLU2-(1)(b) + - - - -

7 WLU2-(1)(a) +++ ++ - - +++

Table showing antimicrobial activity of endophytes isolated from W.somnifera with

different test organisms:

S.NO. Supernatant E.aerogenes P.aerogenosa S.aureus M.smigmatis B.subtilis

1 MKLU4-A(3) - - - - -

2 MKLU1(2)(+) - - - - -

3 MKLH2-B + - - - -

4 MKLU1(2) - - - - -

5 MKLU4-2 - - - - -

6 MKLU2-B2 - - - - -

7 MKLU4A1 + - - - -

Table showing antimicrobial activity of endophytes isolated from M.koengii with

different test organisms:

S.NO. Supernatant A.fumigatus A.niger C.albican P.vulgaris k.pneumoniae

1 MKSU1A1(+) - - - - -

2 MKLU4-B1 - - - - -

3 MKSU2A1(1) - - - - -

4 MKLU2-A 4/10 - - - - -

5 MKLU2(+) - - - - -

6 MKSU4-A2 - - - - -

7 MKLU4-A2 - - - - -

8 MKLU2-(A) - - - - -

Table showing antimicrobial activity of endophytes isolated

from M.koengii with different test organisms

• DNA from endophyte was isolated

DNA isolation

• Amplification of ITS sequence from endophytes

PCR

• Amplified fragments were purified and given for sequencing.

SEQUENCING

0.8% Agarose gel electrophoresis of endophytes(withania somnifera and murraya koengii) DNA Lanes;

Lane 1 uncut λDNA, Lane 2WLU13B, Lane 3MKLU4A3, Lane 4MKLU4-2(+) Lane5MKSU4A2, Lane 6

MKLU2B2, Lane7WLU2-1A

Isolation of DNA from fungal endophytes

1% Agarose gel electrophoresis of endophytes (Withania somnifera and Murraya koengii)

DNA. Lanes:Lane1.1.1kb Ladder, Lane 2.WLU1-1B,Lane 3.WLU1-2A,Lane 4.WLU2

3B,Lane4 MKLU4-A3,Lane5.MKLU4A3,Lane6MKLU42(+).

PCR of DNA isolated from fungal

endophytes using ITS primers

• Six isolated endophytes (four of W.somnifera and two

of M.koengii) were identified by sequencing.

• Sequencing of six endophytes and their BLAST

results are shown as

Sequencing

• Analysis of sequencing results of the six endophytes from W.somnifera and M.koengii revealed significant similarity :

• sample1,WLU1-(1)(b): Alternaria alternata(98%)

• Sample2,WLU1-B2: Alternaria sp.(98%)

• Sample3,WLU2-(1)(a): Alternaria tenuissima(96%)

• Sample4,WLU2-(1)(b): Alternaria compacta(99%)

• Sample5, MKLU2-B2: Glomerella cingulata(94%)

• Sample6,MKLU4-2: Alternaria brassicae(83%)

CONCLUSION • 23 isolates were isolated from the two medicinal

plants(W.somnifera and M.koengii)out of which seven are

from W.somnifera and fifteen from M.koengii.

• All the 23 isolates were screened for important enzymes

like Amylase,Cellulase,Lipase,Laccase and Protease.

• Most of the isolates from both the medicinal plants showed

good enzymatic potential.

• All these 23 isolates were also screened for antimicrobial

activity against different test organisms and most of the

endophytes isolated from W.somnifera showed good

antimicrobial activity against most of the test organisms.

Thank you.