Antibacterial Activity Of Roots Biology Essay

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The present study was undertaken to investigate the antibacterial activity of roots, stem bark leaves of various solvent (Hexane, Benzene, Isopropanol, Ethyl acetate, Methanol & Water) extracts of Alstonia scholaris using well diffusion techniques on gram positive (Staphylococcus aureus, Bacillus cereus & Lactococcus lactis) as well as gram negative bacteria (Aeromonas sp., Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa & Proteus mirabilis). Among the various extracts tested,Methanol extract of root was found to be most potent against E.coli with a zone of inhibition of 18 mm followed by Methanol extract of leaf against Lactococcus lactis (13.5 mm) and Isopropanol extract of stem bark against Lactococcus lactis(13.3mm). Phytochemical analysis revealed the presence of Alkaloids, Carbohydrates, Terpenoids, Steroids& Saponins in major amount in various extracts of roots. Leaf & Stem bark extracts revealed the presence of Alkaloids, Terpenoids, Steroids& Saponins in major amount.

Keywords:- Alstonia scholaris, antibacterial activity, phytochemical analysis

Introduction

Phytochemistry is a branch of science that deals with the chemicals obtained from plants with desirable biological activities (K. Karthishwaran, 2010). Several plant species endowed with these phytochemicals have been documented to serve as potent antimicrobial agents against several pathogenic microoraganisms (Anonymous, 1976; Ray and Majumdar, 1976; Farnsworth, 1988; Rastogi and Mehrotra, 1991, 1993; Asolkar et al., 1992; Cox, 1994; Rastogi, 1998; Perry and Metzer, 1998; Khan et al.,2002).Although several conventional chemical based drugs are quite popular but herbal and plant based medicinal drugs, also known as herbal medicine, traditional medicine or complementary medicine are quite popular in developing countries like India. Plants have been a major source of therapeutic agents since time immemorial and traditional herbal systems of medicine, like ayurveda, resulted in the revival of ancient traditions of medicine(Kurian and Sankar, 2007 ). Therefore indispensable scientific authentication of these medicinal values of plants will pave the way for future herbal drugs (K. Karthishwaran, 2010; Trivedi, 2006 ). These herbal and natural products of folk medicine have been used by men since the advent of human race for the major purpose of curing human ailments (Baliga et.al., 2004). Hence it is the urgent need of the hour to study the various pharmaceutical applications of medicinal plants and harvest the important medicinal potential of these plants in various human pathologies.

. Alstonia scholaris (family:Apocynaceae) also known as Saptaparna or Dita Bark tree is an evergreen tree commonly found in South Asia and Africa grows to a height of hundred meters and is used in the Indian system of medicine to treat various ailments (Satyavati et al., 1987).Almost all parts of plant are used in medicine. The bark is anthelminthic, astringent, and antiperiodic. It is used against chronic diarrhoea, dysentery, and bowel movements (Nadkarni 1976). Alkaloids present can cause a rapid fall in the blood pressures and are hypotensives. Leaves are used against beri-beri, congestion of liver, Dropsy and ulcers. The latex is useful as an application for ulcers, sores, tumours, and in rheumatoid pain(Daniel, 2006). Methanolic extracts of leaf stem & root bark extracts have been reported as potent antimicrobial agents (Khan et.al.,2003)

In the present study, various solvent extracts of root were found to be more potent as antimicrobial agent as compared to extracts of leaf and stem bark.Therefore the present study focuses on drawing a comparison of potency of root extracts with those of leaf and stem bark extracts in terms of phytochemical analysis and antimicrobial activities.

Materials and Methods:-

Collection of plant samples:

The fresh and fully grown plant of Alstonia scholaris was collected from VIT University campus, Vellore Tamilnadu. The plant was authenticated by plant Biotechnology division, VIT, Vellore.

The fresh weight of the plant samples were taken and then separated into Leaves, Stem, Stem Bark and root.

The plant material was thoroughly washed under running water to remove dirt particles and were dried to remove water and subjected to shade drying for about a week. Stem bark and roots were crushed into small pieces to ease the pulverization after drying.

Processing of plant materials:-

The dried plant material (Leaves, Stem Bark and Roots) was taken separately and grinded using a sterile electric blender to obtain a fine powder.

Solvent Extraction:-

Each powdered plant material were subjected to successive solvent extraction in Soxhlet apparatus using solvents in increasing order of polarity like hexane, benzene, isopropanol, ethyl acetate, methanol and water respectively. 25g each of leaves and roots and 40g of stem bark was taken and subjected to extraction with 175ml of suitable solvent for 12hrs. The extracts were concentrated using vacuum distillation to obtain semi-solid or solid extracts and weight of each extract was obtained. The concentrated extracts were stored at 200 C for further analysis.

Phytochemical analysis of plant extracts:-

All the extracts were suspended in dimethyl sulfoxide(DMSO) except water extract and it was dissolved in distil water prior to use(Beyer and Walter, 1997).

The plant extracts of all solvents were used for the phytochemical analysis (Khyade and Vaikos, 2009) for the identification of various classes of chemical compounds using the standard protocol (Harborne, 1984; Kumar et al, 2009; Gothandam et al, 2010).

Screening procedure:

Test for alkaloids: [Wagner's Test]

200 µl of extract was added in test tube. Few drops of Wagner's reagent added to sides of test tube. A reddish brown precipitate produced immediately indicates the presence of alkaloids.

Test for amino acids:[Ninhydrin test]

200 µl of the extract was treated with few drops of Ninhydrin reagent. Appearance of

purple colour shows the presence of amino acids.

Test for carbohydrates:[Fehling's test]

200 µl of the extract is boiled over water bath at 60˚C in test tube.200 ul of Fehling solution A and B was added in test tube. A red precipitate formation indicates the presence of carbohydrate and glycosides.

Test for phenolic compounds:[Ferric chloride test]

200 µl of the extract was added in test tube.2 ml of distill water was then added.Few drops of 5% ferric chloride was added by sides of test tubes.A dark green colour shows the presence of phenolic compounds.

Test for terpenoids:[Salkowski's test]

200 µl of the extract was dissolved in 1ml of chloroform and then few drops of concentrated sulfuric acid was added. A reddish brown precipitate produced immediately indicates the presence of terpenoids.

Test for cardiac glycosides:[Kellar kiliani test]

200 µl of the extract was dissolved in 100 µl of glacial acetic acid. Few drops of 5% ferric chloride was added by sides of test tubes and then few drops of concentrated sulfuric acid was added. A greenish blue colour shows the presence of glycosides.

Test for fixed oils and fats:[Spot test]

A small quantity of extract is pressed between two filter papers. Oil stain on the paper indicates the presence of fixed oil and fats.

Test for Steroids:

200 µl of extract is added with 2 ml of chloroform and equal volume of conc. Sulphuric acid was added by the sides of the test tubes.the upper layer turns red and sulphuric acid layer showing yellow with green fluorescence confirms the presence of steroids.

Test for Saponins:

The extract was diluted with 5ml of distil water and was agitated in a graduated cylinder for 10 minutes. The formation of foam shows the presence of saponins.

Tested Microorganisms

Various culture of human pathogenic bacteria were used which were maintained in VIT under fully sterile condition. These are Staphylococcus aureus, Bacillus cereus, Lactococcus lactis Aeromonas sp., Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis. Bacterial cultures were regularly sub-cultured in order to maintain pure isolates (Khyade,2009).The loop full of tested organism was inoculated in peptone water(HIMEDIA) for 1hour before being tested for antibacterial property.

Screening of antibacterial properties

Plant extracts were tested for their antibacterial activity against gram positive and gram negative strains by well diffusion method (Kavanagh, 1972;Khyade ,2009). The culture plates were prepared by pouring 20ml of nutrient agar (HIMEDIA) in petriplates. The sterile well borer (8 mm) was used to make wells in each plate for extracts. Sterile swab was then used to take the inoculum suspension to spread uniformly on agar plates to get uniform distribution of bacteria. These plates were properly labelled and 100µl of each extract was added in wells (Ayfer and Turgay(2003); Sai Ramesh et al (2010)) and incubated over night at 370 C. The concentration of plant extract is presented in Table 1. The antibacterial activity was observed next day as evidenced by the zone of inhibition surrounding the well.

Table 1: Concentration of plant extract of Alstonia scholaris in different solvent

Solvents

Concentration(mg/100 µl)

Leaves

Stem bark

Roots

Hexane

20.74

9.52

10.16

Benzene

13.3

7.22

1.8

Isopropanol

14.78

20.28

26.74

Ethyl acetate

3.86

2.62

1.58

Methanol

31.82

83.2

11.51

Water

32.63

30.01

82.07

Table 2: Phytochemical screening of extracts from various plant parts of Alstonia scholaris*

Compounds

Leaves

H B I E M W

Stem bark

H B I E M W

Roots

H B I E M W

Alkaloids

++++

++++

++++

++++

++++

++++

--

++++

++++

--

++++

++

--

--

+++

++

+++

++++

Carbohydrates

--

--

+ ++

++

+

--

--

--

+++

+++

++++

--

++

+++

+++

++

++++

++++

Amino acids

--

--

--

--

--

--

--

--

--

--

--

++

--

--

--

--

--

--

Fixed oils and fats

--

--

--

--

--

--

--

--

--

+++

--

--

+++

++

---

++

+

--

Phenolic compounds

--

++++

--

--

++++

--

--

--

--

--

--

--

+++

++++

++

--

++

--

Terpenoids

--

--

+++

+++

--

++

++++

--

++++

++++

+++

++

++

++

++++

+

+++

++++

Cardiac glycosides

--

--

--

--

--

--

--

+++

--

--

--

--

--

--

--

--

--

--

Steroids

++++

++++

++++

+

++

++

--

++++

++++

--

++

++

--

+++

++

+

+

++

Saponins

+

+++

++

--

++++

++++

--

--

+++

--

++++

++++

--

--

++++

+++

++++

++++

*Weak (+), moderate (++) strong (+++) very strong (++++), absent (--)

H,Hexane extract ;B, Benzene extract; I, Isopropanol extract ;E, Ethyl acetate extract;M, Methanol extract;W, Water extract

Table 3: Antibacterial activity of extractives from various plant parts of Alstonia scholaris*

Organisms

Gram stain +/-

Leaves

H B I E M W

Stem bark

H B I E M W

Roots

H B I E M W

DMSO

Chl

Staphyloccocus aureus

+

0

0

8.0

5.5

6.3

0

0

3

9.3

6

0

7

4.5

14.8

15.5

14

14.8

1.8

0

25

Bacillus cereus

+

0

0

6.5

6.3

0

0

8.5

9

12.3

13.2

0

11.8

0

9.3

10.8

9.8

8.5

0

0

18

Lactococcus lactis

+

0

0

7.8

7.8

13.5

0

0

5.3

13.3

4.5

4.5

0

0

3.8

10.3

2.5

14.8

5.8

0

22

Aeromonas sp.

-

0

0

0

0

4.8

5.3

3.8

0

0

4

0

0

4.0

4.3

0

4.8

5.3

0

0

21

Enterobacter aerogens

-

0

3.8

0

0

6.0

10.8

4.3

2.8

5.8

3.5

0

0

4.0

6.0

11.3

6.3

9.5

2.0

0

24

Escherichia coli

-

0

0

4.0

4.8

5.8

5.0

4.0

0

4.8

4.0

4.3

0

6.3

4.5

4.8

6.3

18

9

0

23

Pseudomonas aeruginosa

-

5.0

4.0

4.0

5.0

8.8

11.8

6.3

5.0

6.3

4.5

0

0

6.5

6.0

5.0

6.3

8.0

0

0

22

Proteus mirabilis

-

2.0

2.0

4.3

4.0

8.0

2.0

2.0

2.0

4.3

4.0

8.0

2.0

6.0

3.8

5.3

4.8

7.0

1.0

0

19

*Values are inhibition zone (mm) and average of replicate.

H, Hexane extract; B, Benzene extract; I, Isopropanol extract; E, Ethylacetate extract; M, Methanol extract; W, Water extract

Result and Discussion:-

Results obtained for qualitative screening of phytochemical components in leaves, stem bark and roots performed on different solvent extracts of A. scholaris are presented (Table 1).

In case of roots, hexane extracts showed the presence of fats & oils in major amounts, phenolics & terpenoids in moderate amounts whereas carbohydrates in low amounts. A higher proportion of phenolic compounds and steroids followed by carbohydrates , fixed oils and fats and terpenoids in benzene extract. In case of isopropanol extract, terpenoids & saponins were the major components followed by steroids , phenolics carbohydrates & alkaloids. Ethylacetate extract showed fats & oils in moderate amounts whereas alkaloids, carbohydrates, terpenoids, steroids & saponins in low amounts. Methanol extracts showed the presence of carbohydrates and saponins in major amounts, alkaloids & terpenoids in moderate amounts whereas fats & oils, phenolics & steroids in low amounts. Alkaloids, carbohydrates, terpenoids & saponins were found to be the major components in water extract. As shown in Table 1, amino acids & cardiac glycosides were found to be absent in all the extracts.

Hexane extracts of stem bark exhibited a higher amount of terpenoids and an absence of all other components unlike roots. In case of benzene extract alkaloids, cardiac glycosides & steroids were found to be present which were not found to be present in case of root extracts .Steroids as present in roots was also found to be a major component in isopropanol extracts of stem bark along with alkaloids, terpenoids ,carbohydrates & saponins (in moderate amounts). As in case of ethyl acetate extract of roots, fats & oils was found to be present in stem bark extract followed by terpenoids & carbohydrates (moderate amount). Methanol extracts of stem bark showed the presence of carbohydrates & saponins as major components as seen in case of root extracts whereas a higher amount of Alkaloids was noted in this case as compared to former case. Unlike roots, water extract of stem bark showed the presence of amino acids. As seen in case of water extracts of roots, alkaloids, terpenoids & saponins were found to be the major components in water extracts of Stem bark.

In case of leaves of Alstonia scholaris, hexane extracts showed the presence of alkaloids & steroids as major components unlike root & stem bark extracts. Benzene extract showed the presence of alkaloids (similar to that of Stem bark extract) & phenolics (as seen in Root extract) as well as saponins in moderate amount. Amount of alkaloids & carbohydrates was found to be more in isopropanol extracts of leaf as compared to that of Root & Stem bark extract whereas terpenoids were found in high amounts in isopropanol extracts of both Roots & Leaves as compared to that of Stem bark. Steroids were found to be a major component in Isopropanol extracts of both Leaves & Stem bark as compared to Root extract. In case of Ethyl acetate extract, alkaloids was found to be present in higher amount as compared to the extract of Roots whereas it was found to be absent in Stem bark extract. Along with Alkaloids, Carbohydrates,Terpenoids & Steroids were found to be present in moderate amounts. Methanol extract of leaves exhibited the presence of higher amounts of Alkaloids & Saponins similar to that of Stem bark extract as compared to Root extract. Amount of Carbohydrates was found to be more in Methanol extract of leaves(Khyade & Vaikos 2009) similar to that of Root & Stem bark extract whereas Steroids was found to be present in moderate amounts in present case when compared to Root & Stem bark extract. In case of water extract, Alkaloids, Terpenoids & Saponins was found to be the major components in Leaf extract similar to that of Root extract & Stem bark extracts.

Results for antibacterial activity as obtained with different solvent extracts of various plant parts of A.scholaris are presented in Table 2. Roots extracted with Benzene, Isopropanol, Ethyl acetate & Methanol exhibited maximum antimicrobial activity against Staphylococcus aureus. Methanol & Water extracts showed major activity against Lactococcus lactis whereas hexane extracts exhibited a major activity against Pseudomonas aeruginosa. Stem bark extracted with Hexane, Benzene, Ethyl acetate, & Water showed maximum activity against Bacillus cereus whereas Isopropanol & Methanol extract showed maximum activity against Lactococcus lactis & Proteus mirabilis respectively. In case of leaves, Hexane extract showed a major activity against Pseudomonas aeruginosa similar to that of Root extract. Benzene & Water extract of leaves were also found to exhibit a major antimicrobial activity against Pseudomonas aeruginosa. As seen in case of Root extract, leaves extracted with Isopropanol & Methanol showed maximum antimicrobial activities against Staphylococcus aureus & Lactococcus lactis. Ethyl acetate extract was also found to show maximum antimicrobial activity against Lactococcus lactis.

Conclusion:-

Phtytochemial screening of six solvent extracts viz., Hexane, Benzene, Isopropanol, Ethylacetate, Methanol & Water of different plant parts viz., leaf, stem bark and root revealed that Alkaloids, Carbohydrates ,Terpenoids, Steroids, Saponins were the major phytochemical constituents in various solvent extracts of several plant parts of Alstonia scholaris.

Solvent extracts of plant parts viz., leaf , stem bark & roots were used to test the antimicrobial activities against three Gram positive and five Gram negative bacteria. Methanol extract of root was found to exhibit maximum antimicrobial activity against E.coli. With respect to the zone of inhibition, it can be concluded that the maximum range of zone of inhibition was exhibited by root extract followed that of Stem bark and the least by leaf extracts.With respect to the results obtained, the Gram-positive bacterial strains were found to be more susceptible to the extracts of various plant parts tested as compared to Gram-negative bacteria. From the present study it can thus be concluded that medicinal properties of Alstonia scholaris is by virtue of the presence of several Phytochemical constituents and the antimicrobial activities is exhibited against several pathogenic microorganisms. However further research is needed in this aspect to investigate the potentialities of a wide range of extractants to cure several ailments.

Activities of the various extracts were comparable to those of standard antibacterial agent chloramphenicol and DMSO as control. (Khyade and Vaikos,2009). A varying degree of solubility of the active constituents in the six solvents used may be considered as the major cause behind the differences observed in the activities of the various extracts against different bacterial strains tested.

Demonstration of antibacterial activity of A. scholaris against the various bacterial strains tested is an indication that the possibility of sourcing alternative antibiotic substances in this plant for the development of newer antibacterial agents(Khyade and Vaikos,2009).

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