In Vitro Microbial Competence of the Aerial roots of Rhaphidophora aurea enlaced over four diverse host trees and their Metabolite Impact

 

P. Arulpriya and P. Lalitha*

Department of Chemistry, Avinashilingam Institute for Home Science and Higher Education for Women University, Coimbatore-641043

*Corresponding Author E-mail: goldenlalitha@gmail.com

 

 

ABSTRACT:

Antimicrobial competence of various solvent extracts of the aerial roots of Rhaphidophora aurea entwined over four different host trees were examined against   Salmonella sp, Pseudomonas fluorescens, Klebsiella sp and  Aspergillus fumigates, Candida tropicalis by disk diffusion and streak plate method. Overall results bring out significant antimicrobial activity, which might due to the impact of secondary metabolites presences. Also, activity results were comparable to that of standard medicate gentamycin and fluconozole.  These finding suggest that the aerial roots of Rhaphidophora aurea can be utilize as potent antimicrobial inhibitors

 

KEYWORDS: Rhaphidophora aurea, Aspergillus fumigates, Salmonella sp, Klebsiella sp, Pseudomonas fluorescens, Candida tropicalis

 


 

INTRODUCTION:

Naturally plant living system has a huge tendency to generate natural products and secondary metabolite; these constituent have shown great latent in treating multiple diseases (human, animals and birds). A vast finding has been grown up seeking antimicrobial drugs from plant. In addition, plant based synthetic drugs are harmless and reliable as compared to purely synthesized.  Most often plant have rich source of phytoconstituent like alkaloids, phenols, flavonoids, terpenoids, tannins, steroids and etc, which have an importance in the field of bio and pharma exploit.

 

Rhaphidophora aurea (Linden ex Andre) is a popular decorative foliage plant. It grows rapidly over the trunks of huge trees by attaches its aerial roots to their surfaces.  The root and leaf extracts of Epipremnum aureum (Araceae) has been reported to have significant   antitermites, antioxidant and antibacterial properties1.

 

The present antimicrobial study was carried out with the aerial roots of Rhaphidophora aurea intertwined over host trees like Areca catechu (betel nut palm), Lawsonia inermis (Mehandhi), Cocos nucifera (coconut tree) and Azadirachta indica (Neem tree) against selective human pathogens.

 

MATERIALS AND METHOD:

Plant collection and extraction:

Aerial roots of Rhaphidophora aurea intertwined over Lawsonia inermis (MM) and Azadirachta indica (MN) were collected from Coimbatore District and also Areca catechu (MB) and Cocos nucifera (MC) were collected from Palakkad District. The aerial roots of plant were extracted with solvents like petroleum ether, ethyl acetate, ethanol and aqueous by refluxing with suitable volume of solvent for 12 hours. The procedure was repeated till complete extraction as noted from the colour of the solvents. The solvents were then filtered and distilled to get the extracts.

 

Phytochemical screening:

The phytochemical screening of solvents and aqueous extract of MM, MB, MC and MN were carried out by using standard procedures2-4, to identify the constituent like alkaloid, flavonoid, tannin, saponins, steroids, terpenoids, glycosides, anthraquinons, phnoles, carbohydrates, quinines and reducing sugars.

 

Antimicrobial assay:

The plant extracts were tested for their antibacterial and antifungal activity. Three different bacteria and two fungi, reported to cause human disease were taken up in the study (Table 1).

 

 

Table 1

Micro organism

Effects

Salmonella sp

A etiologic agents of enteric fever and food poisoning5

Pseudomonas fluorescens

Affect the immune system of cancer patients6

Klebsiella sp

Urinary tract infections7

Candida tropicalis

Nosocomial candidaemia8

Aspergillus fumigates

Infections in human (respiratory, lung, etc.,)9

 

Preparation of culture media

Muller-Hinton Medium (Bacteria)

Contents

g/liter

Acid Casein Peptone (H)

17.50g

Starch

1.50g

Beef Infusion

2g

Bacteriological Agar

17g

 

Suspended 38g of the medium in one liter of purified water. Heated with frequent agitation and boiled for one minute to completely dissolve the medium. Autoclaved at 121°C for 15 minutes and cooled to room temperature10.

 

SDA medium (Sabouard Dextrose Ager) for fungi

Contents

g/liter

SDA

65g

Distilled water

100ml

Suspended 65.0g of SDA in 100ml distilled water and boiled to dissolve the medium

 

Antimicrobial Testing:

Disk Diffusion Method (Antibacterial):                         

Paper discs of 4 mm diameter and glass Petri plates of 90 mm diameter were used throughout the test. Paper discs were untainted in an autoclave and dried at 100oC in an oven. Then the discs were soaked with test chemicals at the rate of 50 µg (dry weight) per disc for antibacterial analysis. One drop of bacterial suspension was taken in a sterile Petri dish and then approximately 20 ml of sterilized and melted nutrient agar (~45oC) was poured into the plate and then mixed thoroughly10.

 

The paper discs after soaking with test chemicals were placed at the center of the inoculated pour plate. A control plate was also sustained in each case with alcohol. First, the plates were maintained at low temperature (4oC) for 4 hours. The plates were then incubated at 35oC for growth of test organisms and were observed at 24 hour intervals for two days. The activity was expressed in terms of zone of inhibition in mm. each experiment test was repeated thrice. The standard antibiotics gentamycin were used as a positive control and compared with test chemicals under identical conditions10.

 

Streak Plate Isolation Method (Antifungal):

The required amount of SDA medium was taken in a conical flask separately and was sterilized in autoclave (at 121oC and 15 Psi) for 15 min. A tube of SDA was liquefied and dispensed into the petri dish. The plate was rotated gently for uniform distribution of the medium. The inoculating loop was held at a 60oC angle in the hottest part of the Bunsen burner flame. The entire tube was heated to redness. The loop was allowed to cool for 15 to 20 seconds before it touches the culture. A small amount of the culture was taken from the tube with a sterilized inoculating loop and the microorganisms were streaked in a plate following quadrant. The stock solutions were equipped following the quadrant by dissolving the compounds in ethanol. The process of inoculation was done under aseptic condition and the spores were vaccinated in the medium and warmed for five days. A clear zone or ring on the SDA plate develops, the diameter of which is measured as the zone of inhibition. The antimicrobial activities of the compounds were recorded by photographing the Petri dishes10.

 

Sterilization:

The Petri dishes were sterilized by means of hot air oven at 160oC for 1 hour. Melted Mueller Hinton agar for bacteria and SDA for fungi, were poured into separate dishes to a uniform depth of 5 mm and refrigerated for solidification. The plates were then kept in an incubator at 37oC for 10 to 20 minutes to dry off the moisture that develop on the agar surface.

 

Activity index:

Measured zone of inhibition (ZOI) were compared with standard antibiotics. Activity index11 of each extract was calculated by using the formula given below.

 

Activity index (AI) = ZOI of sample / ZOI of standard

 

RESULTS AND DISCUSSION:

The phytochemical screening results are shown in table 2a, 2b, 2c and 2d, its revealed the presence of most important secondary metabolite like alkaloids, flavonoids, tannins, saponins, steroids, terpenoids, phenols, glycosides anthraquinone and other constituents.

 

Alkaloids are alkaline in nature, nitrogenous substances and reveal pharmacological activities. Several alkaloids are used to cure cardiac, respiratory and cancer disorders12.  Flavonoids are hydroxlated phenolic substances and distributed in higher plants. Also act as antioxidant, support entire cardiovascular system, including arterial walls and effective antimicrobial substances12.

 

Phenolic compounds are effective antifungal agents and several uncomplicated bioactive phytochemical consist of single substituted phenolic ring16. Tannins are astringent, aromatic acidic glycosides. These occur in all parts of plants including roots, stem, bark, leaves, fruits and even hairs. Tannins promote healing of wounds. These are effective in diarrhoea, colitis and peptic ulcers. Glycosides are active and complex substances containing carbon, hydrogen and oxygen. They have characteristic actions on contractile forces of cardiac muscle. Cardiac glycosides are known as cardiotonic glycosides and are characterized by their simulative effects on heart12.

 

 


Table 2a: Phytochemical constituents of the petroleum ether extract of aerial roots of Rhaphidophora aurea twined over four diverse host trees

Solvents

MM

MB

MC

MN

Petroleum

ether

Alkaloids

Flavonoids

Tannins

Terpenoids

Phenols

Anthraquinone13*  Anthocyanin

Alkaloids

Flavonoids

Tannins

Terpenoids

Steroids

Anthraquinone 13*

Anthocyanin

Alkaloids

Steroids

Phenols

Anthraquinone

Anthocyanin

Terpenoids  Anthraquinone Anthocyanin

13* Lalitha et al., 2010

 

Table 2b: Phytochemical constituents of the ethyl acetate extract of aerial roots of Rhaphidophora aurea twined over four diverse host trees

Solvents

MM

MB

MC

MN

Ethyl acetate

Alkaloids

Flavonoids

Phenols

Anthraquinone Anthocyanin

Alkaloids

Flavonoids

Phenols

Tannins

Anthraquinone14*

Flavonoids

Phenols

Anthraquinone Anthocyanin

Alkaloids

Flavonoids Anthraquinone Anthocyanin

14* Arulpriya et al., 2011

 

Table 2c: Phytochemical constituents of the ethanol extract of aerial roots of Rhaphidophora aurea twined over four diverse host trees

Solvents

MM

MB

MC

MN

Ethanol

Alkaloids

Flavonoids

Tannin

Saponins

Glycosides13*

 phenols

Anthraquinone Anthocyanin

Alkaloids

Flavonoids 

Taninin

Glycosides

Phenols

Anthraquinone13*

Anthocyanin

Alkaloids

Flavonoids

Steroids

Glycosides

Phenols

Carbohydrates

Reducing Sugar Anthraquinone

Anthocyanin

Alkaloids 

Flavonoids Anthraquinone Anthocyanin

13* Lalitha et al., 2010

 

Table 2d: Phytochemical constituents of the aqueous extract of aerial roots of Rhaphidophora aurea twined over four diverse host trees

Solvents

MM

MB

MC

MN

Aqueous

Alkaloids

Flavonoids

Saponins

Phenols

Glycosides

Anthraquinone

Anthocyanin

Alkaloids

Flavonoids

Saponins

Phenols

Anthraquinone 

Anthocyanin

Alkaloids

Phenols

Carbohydrates

Reducing sugar

Anthraquinone

Anthocyanin

Alkaloids

Flavonoids Carbohydrates

Reducing sugar Anthraquinone Anthocyanin

 

 


Table 3: Zone of inhibition and type of microbe

Zone of Inhibition (mm)

Type of antimicrobe

> 13

Highly sensitive or susceptible

8-13

Moderately sensitive or intermediate

< 8

Resistant

 

The saponins form strong insoluble complexes with body cholesterol and thus cause its depletion by preventing its reabsorption, due to increasing its excretion, in the same way as other cholesterol lowering drugs do. Saponins also show anti-fungal, antibacterial and anti-protozoal effects12. Terpenoids, which are generally deemed safe, restrain the growth of malignant cells, diminish tumor size, cholesterol level and micro-organism concentration17. Anthraquinones are chemically known as 9, 10 dione derivatives of anthracene. Anthraquinone, glycisodes are present in madder, a climbing plant and used as dye stuff (Alizarin). They possess astringent, purgative anti-inflammatory, fair anti-tumor and bactericidal belongings12

 

The presence of flavonoids, triterpenes and steroids in crude extract showed significant antimicrobial activity against microbes18. Steroids are potential therapeutic agents for infectious diseases.  In this context, several steroid-antibiotics have been developed to mimic the antibacterial behavior of endogenous peptide antibiotic. In addition, several studies suggest that functional groups of steroid-derivative are involved in the bacterial activity. Steroidal saponins were reported for antimicrobial activities15.

 

The chemical constituent and bioactive compounds occurring in several plant materials are known to have fungicidal and bactericidal property. Higher plants produce organic compounds with high structure diversity of all research and academic faculty.  So this knowledge is the basic of finding herbal medicine and the recognition of medicinal plants in indigenous pharmacopoeias.

 

Antimicrobial assay:

Plants have immeasurable capability to synthesize aromatic substances of different functional groups, most of which are oxygen substituted derivatives and the majority are secondary metabolites. In numerous cases, these substances provide as plant defense mechanisms against predation by germs and insects.  Some plants used for their odour (terpenoids), pigment, (quinones and tannins) and flavour (terpenoid capsaicin from chili peppers) were found to be endowed with medicinal properties. Some of the herbs and spices used by humans as season food yield useful medicinal compounds15. Zone of inhibition and type of microbe indications are given in Table 3. The antimicrobial activities of MM, MB, MC and MN extracts were tested against selected human pathogens (Table 4 and 5).  The ethyl acetate and ethanol extract of all the four species had significant potential against test microbes.

 

From table 4, it is obvious that M3 exhibited highly significant activity when compared to all the other extracts tested.  This could be an indication of the potential of this extract as an effective source of antimicrobial agent and this might be due to the presence of saponins.  The M2, B2, C2, N2 and N3 showed moderate activity against tested pathogens, and the activity of these extracts were comparable to that of standard. B3 and C3 did not show any activity against the microbes except Salmonella sp, Pseudomonas fluoresces and Aspergillus fumigates where it showed moderate activity. M4 and C4 showed highly significant activity against Candida tropicalis, whose activity was almost equal to that of standard which might be due to the synergistic effect of active antifungal agent’s present in the extract (Table 2a, 2b, 2c and 2d). Extracts code and its indications are given below.

 

M1, B1, C1 and N1 – Petroleum ether extract of MM, MB, MC and MN

M2, B2, C3 and N2 – Ethyl acetate extract of MM, MB, MC and MN

M3, B3, C3 and N3 – Ethanol extract of MM, MB, MC and MN

M4, B4, C4 and N4 – Aqueous extract of MM, MB, MC and MN

 

Activity index of a substance more than 0.5 is considered to have significant activity20. The activity index of M3 and B3 reveals significant activity against Pseudomonas fluorescens; M2, B1, B2, N2 and N3 showed significant activity against Pseudomonas fluorescens and Klebsiella sp. Antifungal activity of M3, M4 and C4 were more significant against Candida tropicalis; M1, M2, M3, B2, C2 and C3 were significant against Aspergillus fumigates. 

 

Phytomedicines are major components of traditional system of healing in emerging countries, which have a vital part of their history and culture. Plants are great source of thousands new useful phytochemicals of great diversity, which have inhibitory effects on all types of microorganisms in vitro15. The antimicrobial activity of aerial roots of Rhaphidophora aurea twined over four different host trees reveals that the activity against selected human pathogens is in the order: Lawsonia inermis > Areca catechu > Cocos nucifera > Azadirachta indica

 

The antimicrobial activity of ethanol and ethyl acetate extract of the aerial roots of Rhaphidophora aurea twined over four different host trees possesses significant antibacterial and antifungal activity, because of synergistic effect of secondary metabolites in the extracts. In future isolation of antibacterial and antifungal agents from aerial roots of Rhaphidophora aurea from different host trees may provide good herbal remedy in the field of medicine

 

ACKNOWLEDGEMENT:

The authors thank the authorities of the Avinashilingam Institute for Home Science and Higher Education for Women University (Etd. u/s 3 of UGC Act 1956), Coimbatore-43 for having providing the facilities to carry out this research work.

 

 

 


 

Table 4: Zone inhibition of inhibition (mm) of the aerial roots of Rhaphidophora aurea twined over four diverse host trees against human pathogens

Extract code

Salmonella sp

Pseudomonas fluorescens

Klebsiella sp

Candida tropicalis

Aspergillus fumigates

M1

R

5

R

519*

1019*

M2

10

5

15

5 19*

10 19*

M3

10

20

10

15 19*

15 19*

M4

R

R

R

20 19*

5 19*

B1

5

10

R

R

R

B2

5

10

10

10

15

B3

10

20

5

R

5

B4

R

R

R

10

R

C1

10

R

5

1019*

R 19*

C2

5

5

10

1019*

1019*

C3

10

5

5

519*

2019*

C4

R

5

R

2419*

R19*

N1

R

R

10

10

5

N2

10

10

20

5

5

N3

10

10

15

3

5

N4

R

R

R

5

R

Standard

25

30

20

30

20

19*Arulpriya et al., 2012



 

Table 5: Activity index (AI) of the aerial roots of Rhaphidophora aurea twined over four diverse host trees against human pathogens

Extract code

Salmonella sp

Pseudomonas fluorescens

Klebsiella sp

Candida tropicalis

Aspergillus fumigates

M1

 

0.25

-

0.16

0.5

M2

0.33

0.25

0.6

0.16

0.5

M3

0.33

1

0.4

0.5

0.75

M4

-

-

-

0.66

0.25

B1

0.16

0.5

-

-

-

B2

0.16

0.5

0.4

0.33

0.75

B3

0.33

1

0.2

-

0.25

B4

-

-

-

0.33

-

C1

0.33

-

0.2

0.33

-

C2

0.16

0.25

0.4

0.33

0.5

C3

0.33

0.25

0.2

0.16

1

C4

-

0.25

-

0.8

-

N1

-

-

0.4

0.33

0.25

N2

0.33

0.5

0.8

0.16

0.25

N3

0.33

0.5

0.6

0.1

0.25

N4

-

-

-

0.16

-

 

 


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Received on 19.01.2013       Modified on 02.02.2013

Accepted on 10.02.2013      © RJPT All right reserved

Research J. Pharm. and Tech. 6(3): March 2013; Page 255-259