IN-VITRO ANTIFUSARIAL ACTIVITY OF MEDICINAL PLANTS

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International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 3(2): March-April 2014

INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES

IMPACT FACTOR 1.89*** ICV 5.13*** Research Article ……!!!

Bio Sciences

IN-VITRO ANTIFUSARIAL ACTIVITY OF MEDICINAL PLANTS Uma Maheswari N and Kanimozhi C P.G and Research Department of Microbiology, Sengamala Thayaar Educational Trust Women”s College, Sundarakkottai, Mannargudi-610016, Thiruvarur District, Tamil Nadu, India.

KEYWORDS: Fusarium spp , Momordica charantia, Ricinus communis, Datura metel, Well diffusion method. For Correspondence: Uma Maheswari N * Address: P.G and Research Department of Microbiology, Sengamala Thayaar Educational Trust

ABSTRACT In the present study, Fusarial species were isolated and identified from the paddy field soil and tested for antifusarial activity of medicinal plants. Isolated Fusarium species was susceptible to Momordica charantia, Ricinus communis and Datura metel. Phytochemical compounds

such as

Tannin,

Phlobatannin,

Saponin,

Steroid,

Triterpenoid, Cardiacglycoside were analyzed in the above plants. Tannin, Phlobatannin, Saponin Triterpenoid, Cardiacglycoside were present in all tested plants. Steroids was absent in all the three plants. The Phytochemical compounds such as Alkaloid, Phenols, Tannin, Saponin were quantitatively analyzed in three test plants. Antifusarial

Women”s College,

activity of aqueous and methanol solvent extract of three plants were

Sundarakkottai, Mannargudi-

analyzed against pathogenic Fusarium spp by well diffusion method.

610016, Thiruvarur District, Tamil Nadu, India.

Momordica charantia had exhibit the highest antifungal activity against Fusarium oxysporum compared to other test plants Ricinus communis and Datura metel. The Momordica charantia, Datura metel was found to be significantly recommended for controlling F. oxysporum, F.solani and F. roseum pathogens. Comparison with standard antibiotic namely amphotericin B was showed better controlled effect on Fusarium solani.

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International Standard Serial Number (ISSN): 2319-8141

INTRODUCTION: The use of medicinal plants as a source for relief from illness can be traced back over five millennia to written documents of the early civilization in china, India and the near east, but it doubtless an art as old as mankind. Neanderthals living 60,000 years ago in present day ,Iran used plants such as holly back, these plant are still widely used in ethnomedicine around the world (11). The potential of higher plants as source for new drugs is still largely unexplored. Among the estimated 250,000-500,000 plant species, only a small percentage has been investigated phytochemicals and the fraction submitted to biological or pharmacological screening is even smaller. Thus, any phytochemical investigation of a given plant will reveal only a very narrow spectrum of its constituents. Historically pharmacological screening of compounds of natural or synthetic origin has been the source of innumerable therapeutic agents. Random screening as tool in discovering new biologically active molecules has been most productive in the area of antibiotics (1). MEDICINAL PLANTS Medicinal plants represent a rich source of antimicrobial agents. Plants are a source of many potent and powerful drugs (10). A wide range of medicinal plant part is used for extract as raw drugs and they possess varied medicinal properties. The different parts used include root, stem, flower, fruit, twigs exudates and modified plant organs. While some of these raw drugs are collected in small quantities by the local communities and folk healers for local used, many other raw drugs are collected in larger quantities and traded in the market as the raw material for many herbal industries (12). Momordica charantia (Bitter gourd) This herbaceous, tendril- bearing vine grows to 5 m .It bears simple, alternate leaves 4-12cm across, with three to seven deeply separated lobes. Each plant bears separate yellow male and female flowers. In the Northern hemisphere, flowering occurs during June to July and fruiting during September to November. The fruit has a distinct warty a relatively thin layer flash surrounding a central seed cavity filled with large, flat seeds and pith. The fruit is most often eaten green, or as it is beginning to turn yellow (7). Ricinus communis (Castor oil plant) The castor oil plant can vary greatly in its growth habit and appearance. The variability has been increased by breeders who have selected a range of cultivars for leaf and flowers, colours, and for oil production. Its seed is the castor bean, which, despite its name, is not a true bear. Castor is indigenous to the south eastern Mediterranean basin, eastern Africa, and India, but is widespread throughout tropical regions .Castor seed is the source of castor oil, which has a wide variety of uses (7).

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International Standard Serial Number (ISSN): 2319-8141

Datura metel (Thorn apple) Datura metel is a shrub –like perennial herb, commonly known as devil’s trumpet and metel grows in the wild in all the warmer parts of the world, and is cultivated worldwide for its chemical and ornamental properties. Datura is 30 to 150cm tall, with erect, forking and purple stems. The leaves are large, 7 to 20 cm long and have irregular teeth. The flowers are one of the most distinctive characteristics of Datura (5). Fusarium Fusarium species are grown rapidly colonies are initially white and cottony. Often becoming pigment rose or violet with age and developing mucoid are as when sporulating. Two types of conida are produced, curved, multicellular macroconida and unicellular microconida. Some species produce thick walled chlamydospores, wich are solitary or in aggregates conida are formed is slimly maruses. Fusarium species are emerging pathogens, causing superficial and invasive infections particularly in the neutropenic patient. Fusarium is a facultative parasite occurring commonly in the soil as saprophyte. Some of the species are “root rot” of various economically important vascular plants are due to different species of ‘Fusarium’. Fusariosis induced by Fusarium oxysporum is one of the most difficult to control and is a severe disease of several crops, Green house plant and trees. It causes significant losses in crop production and has been reported in of least 32 countries (4). MATERIALS AND METHODS The soil samples were collected from paddy field at Paravakkottai, Thiruvarur, District, Tamilnadu, South India. The collected soil sample kept in sterile polythene bags and stored in refrigerator at 4oc for further studies. The soil sample was used isolation of Fusarial spp. Qualitative determination (9) Test for Tannin 10ml of each extract was evaporated and the residue was extracted by 10 ml of hot 0.9% Nacl solution filtered and derided into 3 equal portions. Nacl solution was added to one portion of the extract, 1% solution to a second portion and gelatine. Salt reagent to the 3 rd portion precipitation with the latter reagent or to both the 2nd and 3rd reagent is indicative of the presence of tannin. Positive test are confirmed by the addition of Fecl3 solution to the extract and should result in a characteristic blue, blue-black, green or blue-green colour or precipitate. Test for Phlobatannin The 1% Hcl was powered into the plant extract. Then the sample was mixed thoroughly and boiled at 450c to 15 minutes. After that to observe the tube for red colour precipitate it indicates the presence of phlobatannin. Full Text Available On www.ijupbs.com

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Test for Saponin About 2.5g of the dried powdered sample were extracted to boiling water. After cooling, the extract was shaken rigorously to forth and was then allowed to stand for 15-20 minutes and classified for saponin contents as no froth it indicates the absence of saponin. Test for Steroids Dried powdered sample was extracted to chloroform 2ml of acetic anhydride was added to 0.5ml chloroform extract. Then 1 ml of concentrated sulphuric acid was added from the sides of the test tubes. A reddish brown ring at the function of two layers indicates positive test for steroids. Test for Triterpenoid Dried powdered sample was extracted to chloroform 2ml of acetic anhydride was added to 0.5ml chloroform extract. Then 1 ml of concentrated sulphuric acid was added from the sides of the test tubes. A red ring at the function of two layers indicates positive test for Triterpenoid. Test for Cardiacglycoside 5ml of each extract was treated to 2ml of glacial acetic acid containing one drop of ferric chloride solution. This was underplayed to 1ml of concentrated sulphuric acid. A brown ring to the interface indicates a deoxy sugar characteristic of cardenolids. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may from just gradually throughout this layer. Quantitative determination (13) Determination of total phenols by Spectrophotometric method The fat free sample was boiled with 50ml of either for extraction of the phenolic component for 15minutes then 5 ml of extract was pipetted into a 50ml volumetric flask then 10 ml distilled water was added to it. 2ml of ammonium hydroxide solution and 5ml concentrated amyl alcohol were also added the samples were made up to mark and test to react for 30 minutes for colour development. The absorbance of the sample was measured at 505nm using spectrophotometer. Alkaloid determination 5g of the sample was weighed into a 250ml beaker and 200 ml of 10% acetic acid in ethanol was added to it and allowed to stand for 4 hours. Then it filtered and the extract was placed in water bath for concentration. Then to add concentrated ammonium hydroxide solution until the precipitate was complete the whole solution was allowed to settle and the precipitate was collected and washed with dilute ammonium hydroxide solution and then filtered the final residue s the alkaloid, which was dried and weighed.

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Tannin determination 500mg of the sample was weighed into an 50ml plastic bottle. Then 50ml of distilled water was added and shaken for 1 hour in a mechanical shaker. This was filtered and the filtrate was made up to 50ml using volumetric flask then 5ml of the filtrate was pipetted out into a test tube and mixed to 2ml of 0.1ml Fecl3 in 0.1 N Hcl and 0.008m potassium Ferro cyanide. Then the absorbance was measured at 120nm within 10 minutes using spectrophotometer. Saponin determination 10gm of the plant sample was extracted repeatedly with 100 ml of 80% aqueous, methanol at room temperature. The whole solution was filtered through whatmann filter paper. The filtrate was placed in the hot air oven at 550c for 45 minutes. After that the constant weight of the sample was measured. Antifusarial activity by well diffusion method (6) Potato – Dextrose Agar medium was prepared and transferred to sterile petri dishes and allowed to solidify. A suspension of identified two Fusarial spp was added to media and swabs the entire surface of the agar medium separately. The inoculums were equally distributed surface of the media by rotating the plate. The well was made in agar plates by using cork borer. Then poured the different concentration of various solvent methanols and aqueous plant extracts of different plant. Then the standard (Amphotericin B) and control were placed on the surface of agar plates. The plates were left for 1 hour at room temperature as a period of pre incubation diffusion to minimize the effects of variation in time between the applications of the different solutions. Then the plates were incubated at 240c for 2-3 days and observed for antifusarial activity. The diameters of the zone of inhibition was observed and measured. The average area of zone of inhibition was calculated and compared with the standard. RESULTS AND DISCUSSION Isolation and identification of fungi Fusarium spp was isolated and identified from paddy field soil. Identified Fusarium spp namely F.oxysporum, F.solani, F.roseum. Momordica charantia, Ricinus communis, Datura metel medicinal plants were collected from paravakkottai, Thiruvarur Dt. Phytochemical compounds were analyzed by both quantitatively and qualitatively. Antifusarial activity against the isolated pathogen was done by well diffusion method. Characteristics of Fusarium spp After incubation, the PDA plate contains numerous type of fungal spp. Such as white, black, green and grey colour colonies were observed. In that the Fusarial spp were identified by microscopic observation using staining technique. In lacto phenol colon blue mounting, sickle shaped cells were Full Text Available On www.ijupbs.com

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observed. Hence, the identified Fusarial spp, confirmed as Fusarium oxysporum, Fusarium solani and Fusarium roseum. Qualitative determination of phytochemical analysis The Tridex procumbens contain Tannin, Saponin, Triterpenoid and Cardiacglycoside, and also analyze the phytochemical compound such as Tannin, Gallic acid etc in both qualitative and quantitative method (8). Phytochemical compound such as Tannin, Phlobatannin, Saponin, Triterpenoid, Cardiacglycoside were present in all the medicinal plants. Steroids are absent in all the tested plants . Quantitative determination Phytochemical screening can help to reveal the chemical constituents of the plant extract and one that predominates over the other. Phytochemical screening of the M.oleifera plant seed showed the presence of Saponins, Steroids and flavonoids, Saponin have been implicated as possible bioactive agents responsible for the aphrodisiac effect in Tribules terrestris extract (3). In quantitative analysis the Alkaloid, Phenols, Tannin and Saponin were analyzed. The plant extract of the Momordica charantia have contained Alkaloid (3.59±0.16), Phenols (9.15+0.10), Tannin (12±1.27), Saponin (4.38±0.10), respectively Ricinus communis contain Alkaloid (4.40.±0.12), Phenols (8.11+0.10), Tannin (10±1.99), Saponin (7±0.58) respectively. Datura metel contained Alkaloid (2.03±19), Phenols (6.13+0.09), Tannin (9±2.49), Saponin (2±0.55). All the medicinal plants have contained maximum amount of tannin compound. Antifusarial activity The aqueous extract of Tridex procumbens plant unable to kill all the Fusarial spp. The ethanolic extract to show low suppression activity against Fusarium oxysporum and Fusarium solani (06±0.02 and 04 ± 0.09) also reported that the methanolic and ethanolic extract of some Indian medicinal plant effectively to kill the fungal pathogen (2). Antifusarial activity of Momordica charantia, Ricinus communis, and

Datura metel were tested

against Fusarium oxysporum, Fusarium solani and Fusarium roseum. The antifusarial activity of aqueous extract (100%) of Fusarium

oxysporum

Momordica charantia had showed high inhibitory effect against

(13±0.03)

and

Ricinus

communis

had

high

inhibitory effect

Fusarium solani (10±0.05) and Datura metel had high inhibitory effect of

of

Fusarium roseum

(10±0.06). Maximum growth suppression were observed in aqueous extract (100%) of Momordica charantia, against Fusarium oxysporum, Fusarium solani and Fusarium roseum. (13±0.03, 12±0.04 and 10±0.59) respectively. At the same time moderate activity was observed (100%) methanolic extract of Full Text Available On www.ijupbs.com

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Momordica charantia against Fusarium oxysporum, Fusarium solani and

Fusarium roseum.

(13±0.42, 12±0.03 and 11± 0.01). Aqueous extract (100%) of Ricinus communis was showed inhibition against Fusarium oxysporum, Fusarium solani and Fusarium roseum (11+0.04,10±0.05 and 11±0.05) and the (100%) methanolic extract of Ricinus communis was showed modetate antifusarial activity of against Fusarium oxysporum, Fusarium solani and Fusarium roseum (10+0.63,11±0.61 and 11+0.59). At the same time antifusarial activity were noted in aqueous extract (100%) of Datura metel against Fusarium oxysporum, Fusarium solani and Fusarium roseum (11+0.01,10±0.07 and 10±0.41) respectively and (100%) methanolic extract of Datura metel was found to moderate antifusarial activity of against Fusarium oxysporum, Fusarium solani and Fusarium roseum.(9±0.85, 10±0.38 and 9±0.06) . Maximum antifusarial activity noted was in Momordica charantia, followed by Ricinus communis and Datura metel. And Fusarium oxysporum was the best pathogen controlled by Momordica charantia. Comparison with standard antibiotic namely amphotericin B was showed better controlled effect on F.solani (11±0.02). In the aqueous extract of Momordica charantia was showed better antifusarial activity of tested pathogen Table-1: Qualitative Analysis of Plant Extracts.

S. No

Plant Extracts

Phytochemical Compounds

Momardica

Ricinus

Datura

charantia

communis

metel

1

Tannin

Positive

Positive

Negative

2

Phlobatannin

Negative

Negative

Positive

3

Saponin

Positive

Negative

Negative

4

Steroids

Negative

Negative

Negative

5

Triterpeonid

Positive

Positive

Positive

6

Cardiacglycoside

Positive

Positive

positive

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Table-2: Quantitative Analysis of Plant Extracts.

Plant extracts S.No

Phytochemical Compounds

Momardica charantia

Ricinus communis

Datura metel

1

Alkaloid

3.59±0.16

4.40±0.12

2.03 ± 0.19

2 3 4

Phenols Tannin Saponin

9.15±0.10 12±1.27 4.38±0.10

8.11±0.10 10±1.99 7±0.58

6.13±0.09 09±2.49 02±0.55

Values are expressed as Mean ± Standard deviation Table 3: Antifusarial Activity Of Aqueous Plant Extracts Against Fusarium oxysporum.

S. No 1 2 3

Plant extracts Momordica charantia Ricinus communis Datura metel

Zone of inhibition (mm) 25% 50% 07± 0.03 07±0.01 03±0.04

09± 0.4 08±0.06 06±0.01

75%

100%

10±0.05 09±0.04 10±0.05

13±0.03 11±0.04 11±0.01

Values are expressed as Mean ± Standard deviation Table 4: Antifusarial Activity Of Aqueous Plant Extracts Against Fusarium solani . Zone of inhibition (mm) S. No

1 2 3

Plant extracts

Momordica charantia Ricinus communis Datura metel

25%

50%

75%

100%

09± 0.02 06±0.05 05±0.02

10± 0.01 03±0.05 05±0.02

11±0.04 09±0.06 08±0.05

12±0.04 10±0.05 10±0.07

Values are expressed as Mean ± Standard deviation

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Table 5: Antifusarial Activity Of Aqueous Plant Extracts Against Fusarium roseum.

Zone of inhibition (mm) S. No 1 2 3

Plant extracts Momordica charantia Ricinus communis Datura metel

25%

50%

75%

100%

05± 0.18 03±0.09 06±0.13

07± 0.13 08±0.09 08±0.01

09±0.21 10±0.30 09±0.01

12±0.30 11±0.21 10±0.41

Values are expressed as Mean ± Standard deviation Table 6: Antifusarial Activity Of Methanolic Plant Extracts Against Fusarium oxysporum.

Zone of inhibition (mm) S. No

Plant extracts

25%

50%

75%

100%

1

Momordica charantia

08± 0.01

10± 0.06

11±0.09

13±0.42

2

Ricinus communis

05±0.02

06±0.07

09±0.23

10±0.63

3

Datura metel

04±0.03

06±0.079

08±0.39

09±0.85

Values are expressed as Mean ± Standard deviation Table 7: Antifusarial Activity Of Methanolic Plant Extract Against Fusarium solani Zone of inhibition S. No 1 2 3

Plant extracts Momordica charantia Ricinus communis Datura metel

25%

50%

75%

100%

07± 0.71 06±0.80 05±0.71

09± 0.81 08±0.90 07±0.063

11±0.03 09±0.81 08±0.46

12±0.71 11±0.61 10±0.38

Values are expressed as Mean ± Standard deviation

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Table 8: Antifusarial Activity Of Methanolic Plant Extracts Against Fusarium roseum. Zone of inhibition (mm) S. No 1 2 3

Plant extracts Momordica charantia Ricinus communis Datura metel

25%

50%

75%

100%

06± 0.71 04±0.35 05±0.60

08± 0.86 09±0.71 07±0.036

10±0.49 09±0.40 08±0.45

11±0.59 11±0.90 09±0.06

Values are expressed as Mean ± Standard deviation Table 9: Antifusarial Activity Of Amphotericin B Against Fusarial spp Zone of inhibition (mm) S. No 1

Microorganism F. oxysporum

Amphotericin B 09±0.02

2

F. solani

11±0.02

3

F.roseum

06±0.02

Values are expressed as Mean ± Standard deviation CONCLUSION: In the present study, Fusarial species were isolated and identified from the paddy field soil. Isolated Fusarium species was susceptibilie to Momordica charantia,

Ricinus communis and Datura metal.

Phytochemical compounds such as Tannin, Phytobatannin, Saponin, Steroid, Triterpenoid, Cardiacglycoside were analyzed in the above plants, steroids was absent in all the three plants. Antifusarial activity of aqueous and methanol solvent extract of three plants were analyzed against pathogenic Fusarium spp. Momordica charantia has highest antifungal activity against

Fusarium

oxysporum, compared to other test plants Ricinus communis, Datura metal. Finally concluded that the Momordica charantia, Datura metal was formed to be significantly recommended for controlling F.solani F. oxysporum, F. roseum pathogens. REFERENCES: 1. Gerhartz, W, Y.S. Yamamota, F.T. Campbell, R. Pfefferkorn and J.F. Rounsavile, (1985). Ullmann, Encyclopedia of chemical industrial.84: 250-261. 2. Goun, E,Cunningham, G, Chu, D., Ngucin,C. and Miles,D., (2003). Antibacterial and antifungal activity of Indonesian ethanomedical plants. Fitoterapia, 76:592-596. 3. Gouthaman, K, Adaikan PG, Prasad RN, (2002). Aphorodisic properties of tribules terrestris extract in normal and castrated rats. Life sci. 71:592-596. Full Text Available On www.ijupbs.com

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