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Antibacterial Activity of Mussa acuminata Colla- medium (Interest to Wild Edible Plants of Tribal People of Tripura)

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About Authors:
Jyotirmoy Das Choudhury, Dr. Biplab De,
Department of Pharmaceutical Chemistry.
Regional Institute of Pharmaceutical Science and Technology. Abhoynagar, Agartala,
Tripura(W)

INTRODUCTION
Nature always stands as a golden mark to exemplify the outstanding phenomenon of symbiosis. The biotic & abiotic elements of nature are all interdependent. The plants are indispensable to man for his life. The knowledge of drug has accumulated over  thousands of years as a result of man’s inquisitive nature so that today we posses many effective means of ensuring health care.
In the part, almost all the medicines used were from the plants, the plant being men’s only chemist for ages. The history of medical plants dates back to RIG VEDA, perhaps the oldest repository of human knowledge, which was written in about 4500-1600 B.C. then there is the Ayurveda which given us detail accounts of many herbal drugs. Most of the medicinally active substance are identified in the 19th & 20th centuries was used in crude form. But now in modern days we use to take drug in the form of dosage form.

Reference Id: PHARMATUTOR-ART-1226

The North-Eastern part of India including Tripura is very rich in plants & herbs because of plenty of rainfalls & availability of deep forest. More than 800 thousand tribals of Tripura belonging to 19 communities once in successive waves settled down on the hill tract that are generally covered with thick vegetation and dense jungles.
The deep forest & jungles of Tripura in and around which they once concentrated were rich in flora and fauna. This bountiful nature serves as an allurement to the early for food gathering(1). The average annual income of the tribal jhumias on the whole is very low which hardly can provide even a meager subsistence to the extent of keeping body & soul together(2). Perhaps low economic return has been compelling the large number of tribal people since the early times to use forest products as their food. It is necessary to inform that the neglected or the little known species(3) are used by the tribal population as food.
The roots, tubers, & leaves discovered and used by the traditional societies are now appearing as the effective sources of food for growing population and also proving to be an important source of potentially therapeutic drugs(4). The ‘ethnobotanical approach’ strongly suggest to study the relationship between plants & people(5). As such the interest in the present study of forest resources used as food bythe tribal people of Tripura results on this emerging appreciation.
In Tripura, forest occupies 3552 sq.kms of the total area of 10,491 sq.kms.
In continuation of our interest to tribal life & culture, the present research project is to highlight the medicinal importance of the edible plants by the tribal people of Tripura, especially the food value of the vegetables marketed by tribal farmers in local market Agartala. Tripura has also been evaluated in some of the cases. Looking  forward about our  research on those  vegetables,  I have  taken  one  plant  for  detailed  studies on pharmaceutical application- Mussa acuminata Colla. 

PLANT PROFILE:

o   Bengali name:

Ram Kala.

o   Kokborok name:

Bugili, Chupui.

o   Source:

Champaknagar, Agartala.

o   Family:

Musaceae.

o   Genus:

Musa.

o   Species:

Acuminata colla.

DESCRIPTION OF PLANT:
It is a large perennial rhizomatous herb. Stem is unbranched pseudostem formed of convolute. Leaf sheath possessing brown black mark on it. Leaves are simple, very large & arranged spirally to form a compact crown. Lamina, glaucous, entire, oblong, truncate with distinct midrib, lateral nerves parallel. The adaxial surface of the lamina is green but the abaxial surface is waxy & green in colour with a tinge of purple. Midrib is greenish yellow above & underneath it is tinged with red. Inflorescence paniculate i.e. mixed spadix. Each group of flowers is covered by a large spathe like bract. The colour of the bract is yellowish with a tinge of purple. Bisexual flowers grow in rows with about twenty flowers per bract. Compound tapae yellowish.  Stamens as long as perianth. Ovary yellowish green, glabrous. Fruits are elegant, elongated berry. Seeds with hard testa, black, irregularly angular.
The hill ranges of Baramura, Atharamura & Jampui are the common places where this plant can be found growing widely.

EDIBLE PARTS USED BY THE TRIBAL PEOPLE:
Musa acuminata Colla: The flowers of this plant are eaten as vegetable & are used to prepare ‘Gudhak’; fruits are also eaten when ripe. The tribals take both young and mature stem. They peel the outer layer of the stem of the young plant to take the inner immature soft portion which they call “Laifung”. After giving off the fruits the mature stem of the plantain which they take as vegetable is called as “Bugili” or “Chupui”. It is the inner comparatively soft portion. Tribals prepare “chakhal, gudhak, & mesideng” by these Laifung & Bugili. Even the tribals use the plantain stem & leaf ash to prepare ‘alkali water’.

LITERATURE SURVEY
Musa species (Musacaea) a tropical plant have been consumed since many years by mankind for its nutritious and delicious fruit. In addition to this musa species have been reported such as antiulcergenic, antidiarrhoel, antidiabetic, antiatherogenic, antitumoral, antimutagenic, & have also found to be effective in treatment of migraine, hypertension, cholesterol & hyperoxalury,

Introduction: Musa sp. (Musacaea) called banana in English, are one of the interesting tropical plantwhich have been consumed since centuries by humans & animals as a nutritious food. Yet ethnobotanist Dr. not know exactly where the plant has been originated.

DescriptionMusa acuminata is a perennial herb but the leaf sheath produce several trunk like structure called pseudostems.

Blooming time: Bananas may flower any time of the year.

Culture: Musa acuminata do best in well drained soils high in organic matter with a pH 5.5- 7.0. The development of the plant in first 3-4 month determines the weight of the bunch similar formulation with 2 – 3% Magnesium applied every 2 months unit fruiting, 10 – 18 months later.

Propagation: Musa acuminatais most commonly propagated by removal of suckers of piece of rhizomes from the original plant.

ØEffect on GI System: In a study by Best et al. various preparation of dried unripe plantain banana were used in aspirin induced ulceration in rats. Although ripe fruits of bananas were inactive, dried unripe bananas showed antiulcerogenic activity & is effective both as a prophylactic treatment and in healing ulcers already induced by aspirin. They found that the active fraction was water soluble & the antiulcerogenic action of banana is due to its stimulating action to grow the gastric mucosa.

Ref: - Best et al. “The Antiulcerogenic Activity of the Unripe Banana (Musa sp.)”; J Pharmacol; Year: 1984; Page no. 82, 107 – 116.

ØEffect on Blood Glucose & Cholesterol: Banana is a tasty fruit which is often restricted in the diet for diabetes owing to its high free sugar content. In ripe banana 80 – 90% of its starch contents changes into free sugar. In other study on banana(Musa sapientum), it is found that oral administration of chloroform extract of the banana flowers in alloxan induced diabetes mallitus in rats for 30 days resulted in a significant reduction in blood glucose, glycosylated haemoglobin & an increase in total haemoglobin.

Ref: - Pari, L. Maheswari; “Hypoglychemic Effect of Musa spienetum in Alloxan induced Diabetic Rats”; J Pharmacol; Year: 1999; 8th Edition; Page: 321 – 325.

ØEffect on Diarrhea: Diarrhea is among the foremost disorder responsible high mortality & morbidity in children of 3rd world countries. The patients with diarrhea & receiving enteral feeding were randomized to receive either banana flakes & medical treatment for diarrhea. 

Ref: - Emery et al; “Banana Flakes Controls diarrhea in Enterally Fed patients”; Year: 1997, 12th Edition; Page: 72 – 75.    

ØEffect on Urinary System: Influence of the stem extract of banana was studied on glycolic acid oxidase (GAO) & lactate dehydrogenase enzymes in liver tissue of sodium glycolate induced hiperonaturic rats. Activity of GAO was significantly lowered in thye extract treated rats compared to that of the glycolate fed rats. LHD increases significantly in glycolate administered rats when compared to to the extract treated rats.

Ref: - Kailash P. Varalakhshmi P; “Effect of Banana Stem Juice on Biochemical Changes in Liver of Normal & Hypernaluric Rats”; Year: 1992;Vol. 30; Page: 440 – 442.

ØEffect on Muscular System: An experiment with banana trunk juice as a neuromuscular blocker was considered out by Lee et al. They found that the juice of banana trunk produces a nondepolarising neuromuscular block & oxygenation of the extract enhances its potency(24, 25).

ØEffect against Cancer & mutagenecity: A case control study was conducted in Thailand with 279 incident cases against cancer. Each subject was interviewed with regard to bowel pattern information, family history, past history of illness & dietary information. The major finding indicated that there was a protective effect provided by banana & papaya for colorectal cancer(29).

Ø  Effect on Hypertension: The effect of banana on cold stress induced hypertension, peak expiratory flow rate & plasma ACE activity in healthy human volunteers was tested. Systolic blood pressure, diastolic blood pressure & mean blood pressure were significantly decreased during cold stress  after banana treatment(34).

Ø  Effect Against Bacteria Growth: Extract prepared from & pulp of banana in increasing stages of ripening were evaluated for their ability to modulate the growth of non-pathogenic & pathogenic bacteria. Extracts increased the growth of Gm –ve bacterial strain E.coli , Shigella flexneri. The growth of Gm +ve was altered by any of the extract.

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PRESENT WORK
The Bugili of Musa accuminata Colla were taken for present investigation. The plants were collected from Agartala, Champak nagar, of Tripura in the month of March 2011 & were shed dried for 10 days, plants are macerated in methanol, keeping material in methanol for 7 days. The liquid portion was collected through filtration & subjected for further studies.
The physico-chemical, phyto-chemical & anti-microbial studies were carried out for the extracts.
Physico-chemical parameters include determination of density, colour, pH etc.
Phyto-chemical parameters include Rf value, Qualitative chemical studies & antimicrobial study.

PHYSICO – CHEMICAL STUDIES
1.     
Ph determination: The pH of the extract was determined by using Digital pH meter ((ECPC Tutor, Eutech Instruments, Serial No. 242510).) where the pH meter is calibrated by using buffer tablets of pH 4, pH 7 & pH 9.
2.      Colour determination:Colour of the extract was visualised by naked eye at sunlight.
3.      Density determination: Spacific gravity bottles are used to determine the density of the extract at room temperature by using an electronic balance (Anamed Instrument Pvt. Ltd. Serial No. V300DR).

The results are depicted in table 1.

TABLE – 1. Characterization of the Extract

Parts of the Plant

Colour

pH

 

Density(gm/cm3)

Musa accuminata Colla

Dark Brown

5.77

 

1

* Density of Water at 25.5oC is 0.99707 g/cm3

PHYTOCHEMICAL STUDIES

1.      Rf Value determination: The Rf value of the components present in the extract was determined by Thin Layer Chromatography (TLC). Taking Silica Gel G as stationary phase & the mobile phase are:
i) Butanol : Water : Dioxan = 4 : 2 : 1
ii) Butanol : Acetic acid : Water = 4 : 1 : 1.

The spots were visualized under Iodine vapour chamber & marked accordingly. The Rf values were calculated by following formula:

Distance travelled by the solute / Distance travelled by the solvent font.

The values are represented in Table. 2.

2. Chemical Analysis: The chemical tests were carried out for the extract as described in Table No. 3

The results obtained are tabulated in Table No. 4

Table No. 2; Rf values

 

Sample

 

Solvent system

 

Spot No.

 

Distance travelled by the components (Cm)

 

Distance travelled by the solvent (Cm)

 

Rf value

 

 

 

Musa accuminata Colla

 

BAW

1

5.5

5.8

0.94

2

4.9

5.8

0.84

3

4.3

5.8

0.74

4

3

5.8

0.51

 

BWD

1

5.4

6

0.9

2

5.5

5.9

0.93

3

5.3

5.9

0.89

4

4.5

5.9

0.76

5

3.3

5.9

0.55

*BAW = Butanol : Acetic acid : Water (4 : 1 : 1).

*BWD = Butanol : Water : Dioxan (4 : 2 : 1).

Table No. 3; Chemical test

 

Test

Observation

Inference

 

 

a. Mayer’s test:2ml of extract treated with 0.2ml of HCl, filtered & treated with 0.1ml of Mayer’s reagent (Pot. Mercuric Iodide solution).

Cream coloured ppt. does not formed.

Alkaloid absent.

 

b. Dragendroff’s test:2ml of extract treated with 0.2ml of HCl, filtered & 0.1ml of Dragendroff’s reagent (Pot. Bismuth Iodide solution) was added.

Reddish brown ppt. does not appear.

Alkaloid absent.

 

C.Wagner’s test: 2ml of extract treated with 0.2ml of HCl, filtered & 0.1ml of Wagner’s reagent (Iodine-Pot. Iodide solution) was added.

Reddish brown ppt. does not appear.

Alkaloid absent.

 

Saponin Tests

a. 1ml extract diluted with 20ml distilled water & shaken in graduated cylinder for 15 minute

1cm layer foam is not formed.

Saponin absent.

 


Carbohydrate Tests

a.Molish’s test:1ml of extract was diluted with 5ml distilled water, then Molish’s reagent was added.

Purple violet ring appeared at the interface of two layers.

Carbohydrate present.

 

b. Benedict’s test: 5ml of extracted was mixed with 1ml Benedict’s qualitative reagent & heated.

Brick red ppt appeared.

Reducing sugar present.

 

Fixed Oil Tests

Few drops of 0.5 N alcoholic KOH was added to the small quantity of extract, phenolphthalein was added & heated for 1-2hr on water bath.

Soap formation on partial neutralization of alkali occurs.

Fixed oil present.

 

 

Test

Observation

Inference

Starch Tests

 

2ml extract treated with 5drops of iodine solution.

 

 

Blue colorization appears.

 

Starch present

Amino Acid Tests

 Ninhydrin test:To 1ml of extract few drops of distilled water was added & treated with 2ml of Ninhydrin solution & warmed.

Violet color is produced.

Amino acid present.


Tannin Tests

a.2ml extract treated with 1ml FeCl3 solution.

Bluish black ppt appeared.

Tannins present.

b.2ml extract was treated with 1ml of 10% Lead acetate solution.

Yellow ppt appeared.

 Tannin present.


Steroid Tests

a.Salkowski test: 5ml of extract was treated with 1ml of CHCl3 & conc. H2SO4.

Red coloritation appears.

Steroid present

b. 10 gm of dried extract was dissolved in 1ml CHCl3 & 1ml of acetic anhydride & 2ml of conc. H2SO4 was added.

Reddish purple colorization appeared.

Steroid present

c. 10gm of dried extract was dissolved in 5ml glacial acetic acid & 1ml conc. H2SO4 was added

Reddish ring appeared.

Steroid present

Table No. 4; Results of Chemical test.

Plant

Constituents present.

Musa accuminata Colla

Carbohydrate, Fixed oil, Starch, Amino acid, Tannin, Steroids.

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ANTIMICROBIAL STUDIES
The antimicrobial study was carried out for the extract by adopting paper disc method. The MIC (Minimal Inhibitory Concentration) 51 of all the extracts was also determined by observing optical density at 600nm by following serial dilution technique

* Procedure for Antimicrobial Studies.
The antibacterial studies carried out for the extract were done systematically against 3 strains of bacteria by Agar diffusion method using paper disc.
The nutrient Agar media was first prepared. The prepared media was sterilized by autoclaving at 1210c for 15 min at 15 psi pressure. The slants were prepared & the test organisms were subcultured in the fresh media, which were incubated for 24 hr. at 37 ± 10C.
Then they were stored in refrigerator & considered as stock culture. Bacterial inoculums were prepared by transferring a loop full of stock culture to sterilized nutrient broth taken in the test tube; they are incubated at 37 ± 10C for 8 hr, before the experiment was carried out.
All the glasswares were sterilized in hot air oven for 1hr. Fresh sterilized nutrient media for bacteria was again prepared & taken in the petridishes & left at room temp. to become solidified. 2ml of inoculated broth was then transferred in each of the petridish aseptically. The broth was then spread uniformly throughout the entire media.
In each of the plates paper disc of 6mm diameter were soaked with standard drug solution (Tetracyclin), extract is placed aseptically & accordingly. The plates were kept undisturbed atleast 2hr at room temperature. Then after incubation the zone of inhibition was measured. The value for the solvent was deducted from the zone of inhibition occurred due to extracts. All the experiments were carried out in triplicate.

Results of antimicrobial study.

 

 

 

Sample used

 

Zone of inhibition (mm).

 

Cornybacterium diptheriae.

 

Enterobactor acrogens.

 

Pyrogens

 

Musa accuminata Colla

 

13

 

10

 

10

RESULTS & DISSCUSION
The stems of Musa accuminata Colla mostly available in Tripura were taken for present investigation Methanolic extract of the plant is weakly acidic. Density of the extract is also determined. Different components were also determined by TLC.
The qualitative tests were also done to determine the different chemical groups present in the extract. It is found that the extract contain carbohydrate, fixed oil, amino acid, tannins & steroids.  
The extract is also tested against Gram ‘+ve’ & Gram ‘-ve’ bacteria. Extract is also able to inhibit the growth of C. diphtheria, B. pyrogens & E. acroginosa.

FUTURE SCOPE OF THE STUDY
Active components of Musa accuminata Colla – further claims the necessity for structural elucidation by various spectral analysis such as IR, NMR, Mass Spectroscopy, which will be helpful for further determination of more active constituents with minimal side effects. Some other pharmacological activities & LD50 should also be determined.

REFERENCES
1.    J.Ganchaudhury,The  Rianges of Tripura,Govt of Tripura,1983,p-3
2.    S.L.Doshi Tribal: an assinilationist Society and National Integration as contributed in Tribal Situation in India,edicted by K.S Singh   p-466
3.    N.K Chakroborty,Plant Resources as contributed in an anthology of Tripura, edicted by J.Ganchaudhury,Inter India Publication
4.    M.Debnath,B.Bhaumick,T.K Majumder & B.Dc,TVI(xi-2)
5.    P.Dc,S.Dutta,S.Paul,P.Bhattacharjee & B.Dc TVI,(xi-3)
6.    R.Chaudhury,M.De,M.Debnath &B.De TVI(xi-3)
7.    B.S Vahl .Essentials of Physical Chemistry,24th Edition ,2001.S.Chand & Co Ltd,New Delhi
8.    C.K.Kokate,,A.P Purohit & S.B Gokhle,”Pharmacognosy”20th  Edition 2002,Nirali Prakashan,Pune 114-119
9.    J.B Harborne,”Phytochemical Methods, 2nd  Edition,1984,Champman & Hall Ltd, London.P-60,84,181
10.    Dr. C.K. Kokate,”Practical Pharmacognosy” 1st edition 1986,Vallabh Prakashan,New Delhi 110052
11.    M.J.Pelzar, E.C.S.Chan & I.V.R,Krieg,Microbiology,5th edition 1993. Tata McGraw Hill publishing, Co, New Delhi 536-539
12.    Cappuccino, J.G & Sharman,N, “Microbiology- A  Labrotary  Mannual, Addison,Wesky, Delhi,199,263
13.    Lee, SK, Ng, LL, Lee, S.I.”Experiment with banana trunk juice as neuromuscular  blocker” MED J. Malaysia, 34, 423-425(1980)
14.    Lohsoonthorn,P,Danvivat, D”colorectal cancer risk factor,a case control study in Bangkok”Asia pac,J.public health,8,118-122
15.    Sarkar.C,Bairy,K.L Rao, N.M Udopa,E.G “Effect of banana on cold stress test & peak expiratory flow rates in health volunteers” Indian  J.Med, Res 110,27-29
16.    Lyte,M, “Induction of gm –ve bacterial growth by neuro chemical containing banana extracts FEMS Microbial Lett, 154,245-250

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