Drug Invention Today ISSN: 0975-7619 Research Article

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Corresponding Author:

Premnath D, School Of Biotechnology And Health Science , Karunya University, Coimbatore, India; e-mail<prems.bioinfo@gmail.com>

Received 10-08-2012; Accepted 03-11-2012 November, 2012 Drug Invention Today, 2012, 4(11), 564-568

Antifungal and Anti Bacterial Activities of Chemical Constituents from Heliotropium indicum Linn. Plant

Premnath D*, Priya J.V, Ebilin Shabthika E, Patric Gomez M School of Biotechnology And Health Science, Karunya University, Coimbatore, India.

INTRODUCTION Herbal medicine is a major component in all indigenous peoples”. Herbal medicine sometimes referred to as Herbalism or Botanical medicine is the use of herbs for their therapeutic or medicinal value. Substances derived from the plants remain the basis for a large proportion of the commercial medications used today for the treatment of heart disease, high blood pressure pain, asthma, and other problem. Nature products were also recognized as a continuous source of newer compounds with higher biological activity when compared to the present synthetic drugs.Finding healing power in plants is a ancient idea. People of all continents and civilizations used plant in one form or other like poultice or decoction. Due to problem like adverse effect, limited lifespan and misuse of traditional antibiotics, effects are currently underway to look for products of natural origin. Presently there is an increasing interest in use of plant microbicides because of the necessity of finding safer microbicides and the need for preventing environmental degradation. Heliotropium indicum1is an annual plant which grows in all parts of India and is given different names by the local communities. The leaves of this widely distributed plant are help in esteem in various part of the world as an external application to ulcer, wounds, and local inflammation2. Their action is probably only that of an emollient. Diuretic properties are also assigned to the plant. In patna, the leaves are employed in case of fever; the dose given being form half a drachma to three drachms3. The leaf is considered by the native of Porto-Rieo to be an excellent remedy for all kinds of ulcer. But it has a far greater reputation for the cure of sore throat. This study investigated the isolation of the various components from Heliotropium indicum4.

MATERIALS AND METHODS The Whole parts of the plant were collected from Tamilnadu State and in the place place on Coimbatore in August 2010. It was identified by School of biotechnology and Health science Karunya University INDIA .The plant material was air-dried,

pulverized by the use of a wooden mortar and pestle and stored in bags. Extraction Procedures The whole parts of Heliotropium indicum were dried in shade and powerdred to get a coarse powder. About 200 gms of dry powder was extracted with chloform by continuous hot percolation using soxlet apparatus. The extraction was continuous for 72 hours. The chloroform extract was concentrated and a light brown residue was obtained. The marc left after chloroform extraction was dried and extracted with ethanol, continued upto 72 hours. The ethanolic extract was filtered and concentrated by vacuum distillation. A greenish brown residue was obtained. The marc left after ethanolic extraction was dried and extracted with chloroform water by simple maceration. The maceration was filtered and concentration by Vaccum distillation. A deep brownish black residue was obtained. Phytochemical evaluations The crude plant was subjected to phytochemical screening using standard techniques of plant secondary metabolites [Sofowora, 1993], [Harborne and Turner, 1984], [Trease and Evans, 2002]. It was tested for alkaloids, saponins, glycosides, phlobotamins, tannins, steroids,flavonoids, cardiac glycosides and coumarin. Bioassay analyses of the various crude extracts4,5,6: In order to confirm scientifically and correlate with the ethno-medicinal uses of Heliotropiumindicum, antimicrobial sensitivity tests were conducted with the extract of the aerial parts against some clinical pathogens. The meat extract , peptone and sodium chloride were taken in the above proportion and dissolved in 500 ml of water by boiling and cooling. Sodium hydroxide was added to neutralize the solution to pH-7. The pH was tested using universal indicator paper,which showed green colour at pH-7.Then the solution was heated to boil and a weighed quantity of agar to form 2% of the medium was added and mixed well in the beaker containing the

The whole plant ethanol and chloroform aqueous extract of Heliotropium indicum Linn. showed promising antibacterial, antifungal activities. (Family: Boraginaceae) were separately evaluated for their antifungal and antimicrobial activity using micro organism. The extracts were obtained by concentration in vacuo at 40oC using rotary evaporator. Phytochemical screening of the extracts showed the presence of Alkaloids, saponins, tannins, glycosides, carbohydrates, phytosterols, and flavonoids while phlobotamins, steroids, coumarin and cardiac glycosides are absent in all the crude extracts. The antimicrobial screening of the extracts showed significant zones of inhibition against 70% of the test organisms. The Minimum Inhibition Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC) against various organisms were conducted. The antimicrobial analysis of the isolated components was also conducted. These prove the folkloric usage of the plant and also indicated a broad spectrum activity of the plant extracts. The methanolic extract afforded white crystalline solids which were characterized using Infra-red Spectroscopy. Key words: Heliotropium indicum, Antifungal, Antibacterial.

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Premnath et al. : Antifungal and Anti Bacterial Activities of Chemical Constituents from Heliotropium indicum Linn. Plant

November, 2012 Drug Invention Today, 2012, 4(11), 564-568

solution.Then it was transferred to boiling tubes in hot condition and plugged with cotton and kept in the autoclave and sterilized at 121�c at 151 lbs pressure/square inch for 20 minutes. Antimicrobial Sensitivity Test7: Antimicrobial sensitivity test was carried out in vitro to determine the extent to which an extract can inhibit the growth of pathogenic microorganism. The sensitivity test was carried out using paper disc agar diffusion technique8,9. Isolation of active components Chromatography was the major technique used in the separation of the extracts’ constituents. 1gm of plant extract is mixed with calcium hydroxide .A smooth slurry is prepared &kept for 5 min and extracted with chloroform.Chloroform layer is repeated &concentrated. Thin Layer Chromatography (TLC)10: This was used to determine the number of chemical components in the petroleum spirit and methanol extract and the most suitable mobile phase for good resolution. The plant extract was dissolved in minimal amount of methanol, spotted at the base of the plate and developed using petroleum spirit, ethylacetate, methanol and chloroform in different ratios. The resulting chromatogram, after air-drying was viewed under the short wavelength (254nm) and long wavelength (366nm) ultraviolet light. Suitable solvent system is prepared in a TLC tank and tightly closed with glass lid.The chamber is allowed to saturate with vapour and baseline is marked under lower end of the plate spots of both sample and saturated solution are applied above baseline ,using capillary tube ,then the spots are allowed to dry .It is placed inside the chamber.Chromatogram is allowed to devolpe by ascending technique.The solvent system reaches the 90%distance of the plate .It is taken out and solvent front is marked and it is sprayed with the suitable reagent. Bioassay analyses of the compound isolated Antimicrobial screening of the compound isolated: The meat extract, peptone and sodium chloride were taken in the above proportion and dissolved in 500 ml of water by boiling and cooling. Sodium hydroxide was added to neutralize the solution to pH-7. The pH was tested using universal indicator paper, which showed green colour at pH-7.Then the solution was heated to boil and a weighed quantity of agar to form 2% of the medium was added and mixed well in the beaker containing the solution. Then it was transferred to boiling tubes in hot condition and plugged with cotton and kept in the autoclave and sterilized at 121�c at 151 lbs pressure/square inch for 20 minutes. The sterilized nutrient agar medium was heated on a water bath to meet the media. When the media was lukewarm, the organisms, Staphylococcus aurous, E.coli , Candida albicans , Pseudomonas aeruginosa , Salmonella typhi were inoculated separately in separate nutrient agar and poured aseptically into sterile petridish and allowed to solidity.The filter paper discs impregnated with chloroform , alcoholic and aqueous and were placed on the surface and the standard drug amikacin (5 µg) and Griseofulvin impregnated in the flter paper disc placed on the surface of the media . It was kept in the incubator for a period of 15 hours at 37°C and the zone of inhibition was measured.

RESULTS AND DISCUSSION Physio Chemical Evaluation Air dried coarsely poedered material of the plant Heliotropium indicum was subjected to the following analysis. Determination of Moisture Content (Loss on Drying): Glass stoppered weighing bottle was weighed and dried for 30 min under the same condition which was employed for the determination. The drug sample (5gms) was placed in the bottle and weighed .The sample was gently shaken side wise to produce even distribution .Then the bottle was placed in the drying chamber(Hot air oven)after removing the stopper.Then it was heated to 105�c for the half an hour.After that the oven was opened and the bottle was closed with the stopper and removed from the oven and was allowed to cool to room temperature .The weight was taken and again the sample was dried in hot air oven ,placing in the weighing bottle.This procedure was repeated until three constant weights were obtained.The difference in weight was determined and percentage of the weight loss was calculated (vide table1) Table 1: Physio chemical evaluation

S.NO

PARAMETERS

VALUES(%)W/W

1 LOSS ON DRYING 8 2. ASH VALUES

A.TOTAL ASH B.ACID INSOLUBLE ASH C.WATER SOLUBLE ASH D.SULPHATED ASH

16.00 16.72 6.37 9.1 8.1

3 EXTRACTED VALUES A.WATER SOLUBLE EXTRACTIVE B.ALCOHOL SOLUBLE EXTRACTIVE

- 8.1 6

4 CRUDE FIBRE 4.5 5 SWELLING INDEX 1

Determination of Ash Value: Ash value includes total ash ,acid insoluble ash,water soluble and sulphate ah values.residues was allowed to cool in a dessicator for 30 min and the weight was taken .The content of the acid insoluble ash in mgs/gms of air dried material was calculated (vide table1) Water Soluble Ash: Water soluble ash is calculated difference in weight between the total ash and the residue remaining after treatment of the total ash with water. To the crucible containing the total ash ,25ml of the distilled water was added ad boiled in 5 min.The insoluble residue was collected in ash less filter paper. The residue was ashed hot and ignited for 15 min at a temperature not exceeding 450�c. The weight of the residue obtained in mgs was subtracted from the weight of the total ash.The content of the water soluble ash in mgs/gms of the air dried material was calculated (vide table1) Sulphated Ash: 2gms of the accurately weighed powdered drug was moistened with the concentrated suphuric acid ,ignited gently in a crucible and again moistened with concentrated sulphuric ,reignited ,cooled and weighed.The content of the sulphated ash in mgs/gms of air dried material was calculated(vide table1)

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Premnath et al. : Antifungal and Anti Bacterial Activities of Chemical Constituents from Heliotropium indicum Linn. Plant

November, 2012 Drug Invention Today, 2012, 4(11), 564-568

Extracted Values Water Soluble Extractives: 10gms of the powdered material was taken and macerated with 100ml of chloroform water in a stoppered flask for 24 hours,shaking frequently during the first 6 hours.filtered and evaporated 20ml of the filtrate to dryness in a china dish .Dried to constant weight at 105�c and the percentage of the water soluble extractive was calculated in reference to the air dried powder (vide table1) Alcohol Soluble Extractives: 10gms of powdered material was taken and macerated with 100 ml of 90%ethanol in a stopped flask for 24 hour,shaking frequently during the first 6 hours.Filterd and 20 ml of the filtrate was evaporated to dryness in a china dish.Dried to constant weight at 105�c and the percentage of the alcohol soluble extractive was calculated in reference to the air dried powder (vide table1) Determination of the Crude Fibre About 2gm of the drug sample ,weighed is extracted with either .Then 200 ml of 1.25%sulphuric is added to the extracted drug and the whole mixture boiled for 30 min..Under reflux in a 500 ml flask .The mixture is the filtered through a haredened filter and the residue washed with the boiling water until free of acid. all of the residue is rinsed back into the flask with 200ml of the boiling 1.25%NaOH solution &again boiled under reflux for 30 min.The liquid is the quickly filtered through a tared filter &residue on the filter is washed with boiling water until neutral ,dried at 110�c to constant weight &incinerated .The difference between the weight of the dried residue and that of the incinerated residue represents the weight of the crude fibre. Determination of Swelling Index

1gm of the lant is taken in a glass stoppered measuring cylinder and add 25ml water.the mixture is shaken thoroughly every 10 min for 1 hour.Allow to stand for 3 hour at room temperature and measuer the volume of the material. Phytochemical Screening Phytochemical screening of the extracts showed the presence of Alkaloids, saponins, tannins, Glycosides and flavonoids while phlobotamins, steroids, carbohydrates and cardiac glycosides are Absent in all the crude extracts. Chemical Evaluation: The solvent system reaches the 90%distance of the plate .It is taken out and solvent front is marked and it is sprayed with the suitable reagent.The Rf value of both test and standard are Rf= Distance traveled by solute Distance traveled by solvent front Distance traveled by solute =7.5cm Distance traveled by solvent front =9cm Rf= 7.8cm =0.866 cm 9cm

Qualitative Analysis Table 2: .Test for lead ion

S.NO EXPERIMENT OBSERVATION INFERENCE 1. The ash is treated with equal volume ofdil.HCL No white precipitate Absence of lead 2. The above solution was treated with hydrogen

sulphide(H�S) gas No black precipitate was observed Absence of lead

3. The ash is treated with dil.Sulphuric acid(H�So�) No white precipitate was observed Absence of lead 4. The ash is treated with Potassium Iodide(KI) No yellow precipitate was observed Absence of lead

Table 3: Test for mercury

S.No EXPERIMENTS Observation Inference 1. The ash is treated with dil.Hydrochloric acid No white precipitate Absence of mercury 2. The ash is treated with Sodium hydroxide No black precipitate Absence of mercury 3. The ash is treated with Potassium Iodide solution No red crystalline precipitate Absence of mercury 4. The ash is treated with Sodium Carbonate No yellow precipitate Absence of mercury

Table 4: Test for arsening S.No EXPERIMENTS OBSERVATION INFERENCE 1. The ash is treated with Silver Nitrate(AgNo�) No brownish red precipitate Absence of Arsenic 2. The ash is treated with Potassium Iodide solution in

the presene of Con.Hydrochloric acid No Iodine was black precipitate out Absence of Arsenic

Phytochemical Analysis: All extract of whole parts of Heliotropium indicum were subjected to qualitative tests for the identification of various plant constituents and reported.(Table No . 3) Pharmacological Screening Antibacterial and Anti Fungal Activities13,14: The meat extract , peptone and sodium chloride were taken in the

above proportion and dissolved in 500 ml of water by boiling and cooling. Sodium hydroxide was added to neutralize the solution to pH-7. The pH was tested using universal indicator paper,which showed green colour at pH-7.Then the solution was heated to boil and a weighed quantity of agar to form 2% of the medium was added and mixed well in the beaker containing the solution.Then it was transferred to boiling tubes in hot condition and plugged with cotton and kept in the autoclave and sterilized at 121�c at 151 lbs pressure/square inch for 20 minutes.

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Premnath et al. : Antifungal and Anti Bacterial Activities of Chemical Constituents from Heliotropium indicum Linn. Plant

November, 2012 Drug Invention Today, 2012, 4(11), 564-568

Table 5: Test for arsening

Positive: [+] Negative : [-]

The sterilized nutrient agar medium was heated on a water bath to meet the media. When the media was lukewarm, the organisms, Staphylococcus aurous, E.coli , Candida albicans, Pseudomonas aeruginosa , Salmonella typhi were inoculated separately in separate nutrient agar and poured aseptically into sterile petridish and allowed to solidity.The

filter paper discs impregnated with chloroform , alcoholic and aqueous and were placed on the surface and the standard drug amikacin (5 µg) and Griseofulvin impregnated in the flter paper disc placed on the surface of the media . It was kept in the incubator for a period of 15 hours at 37°C and the zone of inhibition was measured.

S.No Plant constituents-Test Reagent used Chloroform Extract Ethanolic Extract Aqueous Extract 1. Test for carbohydrates

a.Molisch’s Test b.Fehling’s Test c.Benedict’s Test d.Barfoed’s Test Test for starch

- - - -

+ + + + +

+ + + + -

2. Test for gum and mucilage Alcoholic precipitate and Molosch’s test

-

+

+

3. Test for protein and amino acid a. Ninhydrin Test b. Biuret Test c. Million Test or Coles’s mercuric Nitrites test d. Xanthoproteic Test e. Tannic Test

- - - - -

- - - - -

+ + + + +

4. Test for fixed oils and fats a. Spot Test b. Saponification Test

- -

- -

- -

5. Test for alkaloids a.Mayer’s Test b.Dragendorff’s Test c.Wagner’s Test d.Hager’s Test

+ + + +

+ + + +

+ + + +

6. Test for Glycosides a.Legal’s Test b.Balijet’s Test c.Borntragers’s Test d.Keller-Killiani’s Test e.Cyanogentic glycosides

- - - - -

- - - - -

- - - - -

7. Test for Phytosterols a.Libermann’s Test b.Libermann-Burchard’s Test c.Salkowski Test

+ + +

+ + +

- - -

8. Test for Flavonoids a.Ferric chloride Test b.Shinoda’s Test c.Fiuoresence Test d.Alkaline reagent Test f.Lead Acetate solution Test g.Minerals Acid reaction Test h.Boric acid Test

- - - - - - -

+ + + + + + +

+ + + + + + +

9. Test for Tannins and phenolic compounds a.5% Ferric Chloride solution test b.Reaction with copper sulphate c.Rection with potassium dichromate Reaction with lead acetate. e.Gelatin Test f.Potassium ferric cyanide test

- - - - -

+ + + + + +

+ + + + + +

10. Test for Saponins a.Fothing Test b.Haemolysis Test

- -

- -

- -

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Premnath et al. : Antifungal and Anti Bacterial Activities of Chemical Constituents from Heliotropium indicum Linn. Plant

November, 2012 Drug Invention Today, 2012, 4(11), 564-568

Table 6: Spectroscopical analysis FREQUENCY (cm¯¹) GROUPS ASSIGNED

835-862 Isolated hydrogen 927-939 OH-bending (out of plane) 1018-1040 C-O-C stretching 1332-1360 O-H bending 1470-1477 C=C stretching ( aromatic) 1521-1685 C=O carbonyl compounds 3310-3350 N-H strtching

Stephelococus aureus Salmonella thyphe Pseudonomas aeruginosa

The effect of a known concentration of amikacin(5gm/disc) was compared with effect of various extract of the plant Heliotropium indicum using Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi. It was observed that the zone of inhibition shown by chloroform extract (12mm) against Aeruginosa and same using (10mm) against Salmonella typhi were maximum among other extract where as the zone of inhibition of standard drug(amikacin) has shown (19mm) against Saphylococcus aureus. Antifungal Effect

ORGANISM

ZONE OF INHITION(in mm)

standard queous Extract

CHCL� Extract

Ethanoic Extract

Candida albicans

16

6mm

12

5mm

CONCLUSION Heliotropium indicum is a prostrate herb.It grows on water places or road sides .The gnuinity of the plant was tested using several parameters .It was done by studying the plant for its microscopial examination and the diagnostic characters were identified .The plants having high medicinal values . The medicinal component which provides value to fight the microbs.Using different extraction solvent the phytochemicals are isolated and identified the physiochemical properties. Almost all extracts were tested for their pharmological efficacy like antifungal and antibacterial activities. Fungal strain candidaalbicans and bacterial strain staphylococcus aureus, salmonella typhi and pseudomonas aeruginosa used to for pharmaceutical analysis amoung the different solvent extraction analysis Chloroform extract has maximum antifungal and antibacterial activity againt candida albicans and staphylococcus aureus,pseudomonas aeruginosa respectively. IR also reported. The study can be further extended so as to fix the extracted phytoconstituent responsible for this activity.

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printing press, New York, 2010.

Source of support: Nil, Conflict of interest: None Declared

ORGANISM

ZONE OF INHIBITION (mm)

Standard Aqueous

Extract CHCl3

Extract Ethanolic

Extract Staphylococcus aureus gram(+)

19 10 12 8

Pseudomonas aeruginosa gram(+)

16 - 12 -

Salmonellatyphi gram (-)

17 8

10

6

Candida albicans

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