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Investigation of phytochemical constituents in Azolla microphylla for antibacterial activity.

INTRODUCTION

Azolla is a pteridophyte having agronomic significance in developing as well as developed countries. [1-6] It produces maximum biomass in a relatively shorter period of time. [3] Azolla acts as a nitrogen biofertilizer, and it increases the productivity of rice. [4] The oceanic plant Azolla turned out to be progressively prominent as bioenergy feedstock on account of its high development rate, generation of biomass with elevated amounts of biofuel-creating capacity. Besides, Azolla has been appeared to be fit for hyper aggregating an incredible assortment of overwhelming metal toxins and additionally purifying ammonium and phosphorous in wastewater. [5] Azolla is utilized as a feed for animals, food for humans, water purifier, green fertilizer, hydrogen fuel, biogas, and weed and bug controller. [7] Azolla enhances the water quality by expelling nitrates and phosphorous. [8] In the present study, we have encapsulated about Azolla's attributes such as antimicrobial and phytochemical activities, but still, there are many fields in which Azolla can act as fern hero in many more areas which can be future enhanced. [9]

Objectives

* Current study was done to investigate phytochemical and antibacterial action in Azolla microphylla.

* This study was conducted to assess whether Azolla extract found to possess better antimicrobial action on bacteria.

MATERIALS AND METHODS

Phytochemical Activity

Leaves of diverse extracts of the A. mycrophylla were analyzed for the presence of alkaloids, saponins, tannins, phenols, amino acids, and monosaccharides.

Screening for Alkaloids

Alcoholic extract was heated with 5 ml of 2N HCL. To the filtrate, Mayer's reagent was added and observed for cream color precipitate which indicated the nearness of alkaloids.

Screening of Tannins

To 5 ml of concentrate, a couple of drops of 1% lead acetate were included. Yellow color shows the nearness of tannins.

Screening of Phenols

Around 2 ml of the concentrate was added to 2 ml of ferric chloride ([Fecl.sub.3]), a deep bluish green solution is formed which indicated the nearness of phenols.

Screening of Steroids

One ml of the concentrate was disintegrated in 10 ml of chloroform, and an equivalent volume of concentrated sulfuric acid ([H.sub.2]S[O.sub.4]) was included in the test tube. The upper layer turns red, and [H.sub.2]S[O.sub.4] layer showed yellow with green fluorescence. This demonstrated the nearness of steroids.

Screening of Cardiac Glycosides

To the concentrate of glacial acetic acid, few drops of [Fecl.sub.3] and concentrated [H.sub.2]S[O.sub.4] were included and watched for the reddish-brown coloration at the intersection of two layers, and the bluish green color in the upper layer demonstrates the nearness of cardiac glycosides.

Screening for Anthraquinones

To 5 ml of the concentrate, 10 ml of [H.sub.2]S[O.sub.4] was added and filtered. The filtrate was shaken with 5 ml of chloroform. The chloroform layer was pipette out, and 1 ml of dilute ammonia was included. The solution was observed for color changes.

Screening for Flavonoids

To 1 ml of the concentrate, a couple of dilute sodium hydroxide was included. Yellow color was developed in the plant extract, which became colorless when few drops of dilute acid were added. This indicated the nearness of flavonoids.

Screening for Terpenoids

The concentrate was dispersed in 1 ml of chloroform; 1 ml of acidic anhydride was included after the inclusion of 2 ml of conc. [H.sub.2]S[O.sub.4]. Formation of reddish-violet color demonstrated the nearness of triterpenoids. 1 ml of the concentrate was treated with few drops of Ninhydrin reagent. Appearance of purple color demonstrates the nearness of amino acids.

Screening for Reducing Sugars

To 1 ml of the concentrate, few drops of Fehling's solution were included and observed for the brick red precipitate.

Screening for Monosaccharides

To 1 ml of the concentrate, 1 ml of Barfoed's reagent was included and heated which lead to the development of red cupric oxide indicated the nearness of monosaccharide.

Test Organisms

The test organisms used in this study include Bacillus sp., Staphylococcus sp, Escherichia coli, Klebsiella sp., and Proteus sp.

Antibacterial Assay

The crude extracts (methanol, ethanol, chloroform, petroleum ether, and hot water) obtained from the leaves of Azolla microphylla were studied for its antibacterial activity using agar well diffusion and filter paper disc diffusion methods.

Antifungal Assay

Ethanol and methanol extracts of the leaves alone were tested for its antifungal activity. The media used were Potato Dextrose Agar (HiMedia).

RESULTS

The current study on A. microphylla explains the presence of medically active components. The phytochemical constituents of the Azolla were investigated, and the results were tabulated.

Phytochemical Activity

Table 1 showed phytochemical constituents of various extracts of the leaves. Aqueous extracts of leaves showed the incidence of alkaloids and the ethanolic extract contained cardiac glycosides. All the extracts showed the absence of anthraquinones, Monosaccharide's, and cardiac glycosides. Among the various extracts, aqueous extracts showed the presence of significant amount of phytochemicals followed by methanol, ethanol, water, chloroform, and petroleum ether extract and this may contribute to the better antibacterial activity. Hence, in our study, leaves extracts were investigated for its antimicrobial activity.

Antibacterial Activity

Table 2 showed the antibacterial action of leaves extracts of A. microphylla against the test organisms using agar well diffusion method. Among the test organisms used in the study, Gram-positive bacteria showed more inhibitory effect than Gram-negative bacteria, and especially, Bacillus species showed a higher zone of inhibition than Staphylococcus species. Among the Gram-negative bacteria, E.coli showed better activity than Klebsiella species. All the extracts did not exhibit any inhibitory effect on Proteus species [Figures 1-4].

Antifungal Activity

Methanol and ethanol extracts of the leaves alone were tested for its antifungal activity. The media used were Potato Dextrose Agar. Various concentration of extract was mixed into 20 ml molten PDA media and the content was allowed to solidify in plates [Figures 5 and 6].

DISCUSSION

The summary of this work revealed that the Azolla sp. contained bioactive agents such as alkaloids, saponins, tannins, flavonoids, steroids, and cardiac glycosides and these agents could be responsible for its antimicrobial activity. In many cases, alkaloids, flavonoids, and terpenoids prove to showed good antimicrobial action. [10]

Prasad et al. investigated the phytochemicals present in Azolla. [11] Temmink et al. discussed that saponins are a special class of glycosides which have a soapy characteristic and facilitate the absorption of foods and medicine. [12] Thagela et al. discussed that saponins and steroids are main source for normal activities of the CNS (central nervous system). [13] Vannini et al. observed that tannins act as an antimicrobial agent by inhibiting extracellular enzymes, deprecating the substrates required for microbial growth or by inhibiting oxidative phosphorylation of microbial metabolism. [14] From the above result, the Azolla extract found to possess good antimicrobial action on bacteria. Similar reports were shown by some other investigators. [15]

Strengths and Limitations of the Study

Strength of this study includes it is easy to find and establish the vegetative material which helps in quick recovery. Azolla requires no extra fertilization beyond P applied to crop and aids in nitrogen fixation which, in turn, increases crop yields. Limitations of this study include it can be used only in wet fields. It may require a nursery pond and difficult to establish in winter.

CONCLUSION

The organic solvent extracts of A. mycrophylla leaves were analyzed separately for its phytochemical constituents. The species was found to contain alkaloid, tannin, saponin, steroid, terpenoid, flavonoid, and phenols. Almost all the phytochemical components were present in ethanol, methanol, and water. All the extracts of the leaves were screened for its antimicrobial activity against bacterial species such as Bacillus species, Staphylococcus species, Klebsiella species, E. coli, and Proteus species and the maximum antibacterial activity was shown against like Bacillus species followed by Staphylococcus species. The result of the current study suggested that the extract of A. mycrophylla possesses phytochemical compounds with significant antimicrobial activity.

REFERENCES

[1.] Acharya P, Mohanty GP, Pradhan CR, Mishra SK, Beura NC, Moharana B. Exploring the effects of inclusion of dietary fresh Azolla on the performance of white pekin broiler ducks. Vet World 2015;8:1293-9.

[2.] Bharali A, Baruah KK, Baruah SG, Bhattacharyya P. Impacts of integrated nutrient management on methane emission, global warming potential and carbon storage capacity in rice grown in a northeast India soil. Environ Sci Pollut Res Int 2018;25:5889-901.

[3.] Brouwer P, Brautigam A, Buijs VA, Tazelaar AO, van der Werf A, Schluter U, et al. Metabolic adaptation, a specialized leaf organ structure and vascular responses to diurnal N2 fixation by nostoc Azollae sustain the astonishing productivity of Azolla ferns without nitrogen fertilizer. Front Plant Sci 2017;8:442.

[4.] Carlozzi P, Padovani G. The aquatic fern Azolla as a natural plant-factory for ammonia removal from fish-breeding fresh wastewater. Environ Sci Pollut Res Int 2016;23:8749-55.

[5.] De AK, Dey N, Adak MK. Bio indices for 2,4-D sensitivity between two plant species: Azollapinnata R.Br. and Vernonia cinerea L. with their cellular responses. Physiol Mol Biol Plants 2016;22:371-80.

[6.] Dijkhuizen LW, Brouwer P, Bolhuis H, Reichart GJ, Koppers N, Huettel B, et al. Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify. New Phytol 2018;217:453-66.

[7.] Gomes MP, de Brito JC, Carneiro MM, da Cunha MR, Garcia QS, Figueredo CC. Responses of the nitrogen-fixing aquatic fern Azolla to water contaminated with ciprofloxacin: Impacts on biofertilization. Environ Pollut 2018;232:293-9.

[8.] Liu J, Xu H, Jiang Y, Zhang K, Hu Y, Zeng Z. Methane Emissions and microbial communities as influenced by dual cropping of Azolla along with early rice. Sci Rep 2017;7:40635.

[9.] Mishra DB, Roy D, Kumar V, Bhattacharyya A, Kumar M, Kushwaha R, et al. Effect of feeding different levels of Azolla pinnata on blood biochemicals, hematology and immunocompetence traits of Chabro chicken. Vet World 2016;9:192-8.

[10.] Park H, Song U. Microcosm investigation of growth and phytoremediation potential of Azolla japonica along nitrogen gradients. Int J Phytoremediation 2017;19:863-9.

[11.] Prasad SM, Kumar S, Parihar P, Singh A, Singh R. Evaluating the combined effects of pretilachlor and UV-B on two Azolla species. Pestic Biochem Physiol 2016;128:45-56.

[12.] Temmink RJ, Harpenslager SF, Smolders AJ, van DG, Peters RC, Lamers LP, et al. Azolla along a phosphorus gradient: Biphasic growth response linked to diazotroph traits and phosphorus-induced iron chlorosis. Sci Rep 2018;8:4451.

[13.] Thagela P, Yadav RK, Mishra V, Dahuja A, Ahmad A, Singh PK, et al. Salinity-induced inhibition of growth in the aquatic pteridophyte Azolla microphylla primarily involves inhibition of photosynthetic components and signaling molecules as revealed by proteome analysis. Protoplasma 2017;254:303-13.

[14.] Vannini A, Paoli L, Vichi M, Backor M, Backorova M, Loppi S. Toxicity of diclofenac in the fern Azolla filiculoides and the lichen Xanthoria parietina. Bull Environ Contam Toxicol 2018;100:430-7.

[15.] Yadav RK, Tripathi K, Ramteke PW, Varghese E, Abraham G. Salinity induced physiological and biochemical changes in the freshly separated cyanobionts of Azolla microphylla and Azolla caroliniana. Plant Physiol Biochem 2016;106:39-45.

Sathammaipriya N, Thamilmaraiselvi B, Steffi P F, Sangeetha K

Department of Microbiology, Cauvery College for Women, Trichy, Tamil Nadu, India

Correspondence to: Steffi P F, E-mail: steffi.titu@gmail.com

Received: March 25, 2018; Accepted: July 30, 2018

DOI: 10.5455/njppp.2018.8.0310430072018

How to cite this article: Sathammaipriya N, Thamilmaraiselvi B, Steffi PF, Sangeetha K.Natl J Physiol Pharm Pharmacol 2018;8(11):1500-1504.

Source of Support: Nil, Conflict of Interest: None declared.
Table 1: Phytochemical screening of leaf extract of A. microphyla

Phytochemicals      Methanol  Ethanol  Chloroform  Petroleum ether

Tannins             +++       ++       +           -
Phenols             +++       +++      -           -
Saponins            ++        ++       +           -
Alkaloids           +         +        +           -
Flavonoids          ++        +++      ++          -
Anthraquinones      -         -        -           -
Amino acids         ++        ++       +++         -
Monosaccarides      +         -        -           -
Reducing sugars     +         +        +           ++
Terpenoids          ++        ++       +++         -
Cardiac glycosides  -         +        -           -
Steroids            +++       +++      ++          +

Phytochemicals      Aqueous

Tannins             -
Phenols             -
Saponins            -
Alkaloids           -
Flavonoids          -
Anthraquinones      -
Amino acids         -
Monosaccarides      -
Reducing sugars     +
Terpenoids          -
Cardiac glycosides  -
Steroids            -

+++: Highly present, +: Weakly present, ++: Fairly present, -:
Nil. A. microphyla: Azolla microphyla

Table 2: Antibacterial action of leaf extracts of A. microphylla

Extracts    Conc. ([micro]l)  Bacillus  Staphylococcus  Klebsiella

Methanol    10                1.0       1.0             1.0
            20                1.5       1.5             1.4
            30                2.0       2.0             1.6
            40                2.5       2.3             2.4
Ethanol     10                1.0       0.5             1.6
            20                1.4       0.9             1.8
            30                1.6       1.2             2.0
            40                2.0       1.5             2.5
Water       10                0.4       -               -
            20                0.6       -               -
            30                1.3       -               -
            40                1.7       0.5             0.3
Chloroform  10                1.0       0.5             0.5
            20                1.6       1.0             1.2
            30                2.0       1.2             2.0
            40                2.5       1.6             2.5
Pet Ether   10                -         -               -
            20                -         -               -
            30                1.0       -               1.0
            40                2.0       0.5             1.4

Extracts    E. coli  Proteus

Methanol    1.3      0.2
            1.8      0.5
            2.4      1.5
            2.7      1.7
Ethanol     0.7      -
            1.9      -
            2.0      -
            2.7      -
Water       -        -
            -        -
            1.0      -
            1.5      -
Chloroform  0.5      -
            1.8      -
            2.3      -
            2.7      -
Pet Ether   0.5      -
            0.7      -
            1.5      -
            2.0      -

The values on the table are[+ or -]mean value. A. microphyla:
Azolla microphyla
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Title Annotation:RESEARCH ARTICLE
Author:Sathammaipriya, N; Thamilmaraiselvi, B; Steffi, P F; Sangeetha, K
Publication:National Journal of Physiology, Pharmacy and Pharmacology
Article Type:Report
Date:Nov 1, 2018
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