Phytochemical Screening and Antimicrobial Activity of ...

Phytochemical Screening and Antimicrobial Activity of Secondary Metabolite Excreted by Ervatamia species

 

Submitted By KADAM NITIN SURYAKANT M.Sc. Sem II Application number- LFS1845 Indian Academy of Science Branch of Biochemistry Department of Chemistry University of Pune, Maharashtra, India

Supervisor Prof. Ashwani Kumar Rai Department of Botany BANARAS HINDU UNIVERSITY

Varanasi-221005, India

  BOTANY DEPARTMENT BANARAS HINDU UNIVERSITY Varanasi-221005, India

P Phone : 0542-230 07147(Ext.330) : 0542-6701098 M Mobile : 0954086 67006 E E-mail :akrai.bhu@ @gmail.com

   

Dr. Ashwani K. Rai  FNA AAS, FNASc, FBRSI Professor in Botaany Deppartment of Bootany

 

 

 

CER RTIFICA ATE Thiss  is  to  cerrtify  that  the  t projecct  work  entitled  e “Phytochem P mical screeening

andd Antimicrrobial actiivity of Seecondary metabolite m e excretedd by Ervataamia speccies’’ was  carried  out  o in  my  laboratorry  by  Mr..  KADAM  NIITIN  SURYAKANT  (Sum mmer Research Felllow, Indian Academ my of Scien nces, Banggalore).

                         

 

      

ACKNOWLEDGEMENT

I am grateful to Indian Academy of Science to provide me fellowship perform research work at Banaras Hindu University, Varanasi 221 005. I owe a great thank to many people who helped and supported me during my summer fellowship programme. I would like to express my deepest appreciation to my supervisor Prof. Ashwani K Rai, who has the substance of a genius, for guiding and correcting various works of mine with attention and care. I would like to thank Prof. Gopal Nath, who gave me opportunity to work in his lab for antimicrobial activity. I would like to thank with gratitude to research scholar, Mr. Abhishek Kumar, Mayank and Dharmendra (Dept. of Micro. IMS, BHU), Ms. Neha Sharma (Dept. of Chem., BHU) for their advise and assistance during the tenure of my work. In addition, I would also like to thank all the research scholars, Mrs. Meenakshi Singh, Mr. Umesh Pravin Dhuldhaj, Ms. Apurva Gupta, Ms. Sweta Singh, Mr. Pradip Kumar Rai for their constant suggestions and support during conduction of various experiments associated with my work. I thank the non- teaching staff, which had helped me in various ways. Last but not the least I would like to thank almighty God for his blessing and mercy, without which the completion of this project would not have been successful.

KADAM N. S. (LFS1845, IAS, Bangalore)

 

CONTENTS

1. Introduction 2. Materials and Methods a) Plant source b) Grinding of solid sample c) Gradient elution chromatography d) Phytochemical examinations e) Spectrophotogram f) Thin layer chromatography g) Extraction of lipids h) Estimation of carbohydrates i) Antimicrobial activity

# Microbial samples # Culture media # screening for antibacterial activity # screening for antifungal activity

3. Results 4. Discussion 5. References

Abstract: The extract obtained with various solvents showed the presence of cumarins, flavonoids, triterpenes, phenols, glycoproteins. All these compounds have a valuable role in disease control. Petroleum Ether extract exhibited higher activity to that of other extracts against the microbial strains tried. Compared to that of antibacterial, the extracts were more active against fungal activity. Thus, it can be used as broad spectrum antibiotic for intestinal infection. Candida tropocalis is a fungus, which grows in chocolates and spoils it. The petroleum ether extract is highly active against Candida tropocalis, so may be used as preservative in chocolate industry. The acetone extract showed moderate antibacterial and antifungal activity. It may also be used. The water extract contains high percentage of saponins and may be of use to soap industries as a foaming agent. Thus, the secondary metabolite secreted by Ervatamia species contains various bioactive molecules active against bacteria and fungi and of be much use in pharma and other industries.

Introduction:According to World Health Organisation (WHO) about 80% of world population relies on traditional medicine for primary health care needs. Plants are natural source of medicines. Nature has blessed us with the cures of all diseases. We have just to find it out and use it. However, it is not so simple task. From an ancient time, plants have been in medicinal use. Knowledge about their use is based on trial and error. Plants are rich in a wide variety of secondary metabolite such as tannins, terpenoids, alkaloids, and flavanoids, and in vitro show antimicrobial properties. The use of plant extracts and phytochemicals with known antimicrobial properties can be of great significance in therapeutic treatments (Gislene et al. 2000). India has a rich history of using plants for medicinal purposes. Turmeric (Curcuma longa L.) is a medicinal plant extensively used in Ayurvedha, Unani and Siddha medicine as home remedy for various diseases. Turmeric rhizome is used as a food additive (spice), preservative and colouring agent (Lotempio et al. 2005) in Asian countries, including China and South East Asia. In India, it is considered auspicious, and is a part of religious rituals. In old Hindu medicine, it is extensively used for the treatment of sprains and swelling caused by injury. In recent times, traditional Indian medicine uses turmeric powder for the treatment of biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic disorders, rheumatism and sinusitis. Various sesquiterpenes and curcuminoids have been isolated from the rhizome of C. longa, attributing a wide array of biological activities such as antioxidant (Majumdar, et al. 2000) anti-inflammatory (Manimegalai et al. 2011), wound healing (Menon and Sudheer, 2007), anticancer (Nair et al. 2005) and antibacterial activity (Pathak et al. 2010). My project is aimed to study secondary metabolites excreted by Ervatamia species as gum including separation of active biomolecules, its identification and antimicrobial activities. Materials and methods:Plant source: The plant Ervatamia species used in the present investigation is selected from Ayurvedic garden of University of Pune, Pune-411007, Maharashtra. The secretion is yellow colored, which later solidifies. The solid secretion was collected.

Methodology: Grinding of solid sample: Yellow colored solid secretion was powdered using mortal and pestle and stored in glass bottle until use. Gradient elution chromatography: The crude extract powder was mixed with silica gel and loaded on silica gel column chromatogram. The compounds were eluted serially with solvents of increasing polarity from non polar to polar such as petroleum ether, chloroform, ethyl acetate, acetone, ethyl alcohol and water. Ten fractions each, of 10 ml eluants was collected in separate tubes. Absorption spectra of each fraction was recorded at 800 -200 nm with corresponding solvent as blank. Phytochemical examinations: Phytochemical examinations of solvent fractions were carried out using standard methods. 1. Detection of alkaloids: A known amount of extract fractions (0.5 ml) was dissolved in diluted HCl and added two drops of Mayer’s reagent. Yellow colored precipitate indicated the presence of alkaloids. 2. Detection of flavonoids:

To the 0.5 ml extract fraction added 3 drops of 0.1 N

NaOH, which gave intense yellow colour. After the addition of dilute HCl, it turned colorless. This indicated the presence of flavonoids. 3. Detection of phytosterols:

To the 0.5 ml extract fraction added equal volume of

chloroform and 5-6 drops of concentrated sulphuric acid and shaken well. Appearance of golden yellow colour indicated the presence of triterpenes. 4. Detection of phenols: 20 microliters of extract fraction was loaded on silica gel chromatography plates, ran in selected solvent. Plates was visualised by spraying with FCR reagent gave a blue colour spot represented presence of phenol. 5.

Detection of saponins:

To the 0.5 ml of extract fraction added 2 ml of water and

shacked well. Foam came at the surface persisted for 10 min, indicated presence of saponins. 6.

Detection of Cumarins: To the 0.5 ml extract fraction added 3 drops of 0.1 N NaOH, which gave yellow colour, indicated the presence of coumarin.

7. Detection of proteins: To the 0.5 ml extract fraction added 2-3 drops of concentrated HNO3. Formation of yellow colour indicated presence of proteins. 8. Detection of glycoside: Benedict test and Fehling’s did not give response, while tests were positive for hydrolysed extract with concentrate H2SO4. Spectrophotogram: Spectrophotogram of each fraction was taken at 800-200 nm in a spectrophotometer. The spectrophotogram of the fractions in different solvents differed from each other. However, all the fraction of same solvent showed identical curves, indicating the presence of the same compound. Thin Layer Chromatography: The measure compounds were present in chloroform fraction so it’s the best mobile phase was detected. The stationary phase was silica gel. The best mobile phase for the separation of chloroform fraction was found to be 3: 1: 0.2; ethyl acetate: benzene: ammonia that yielded measure spots. Two measure spots were collected separately. It was dissolved in chloroform and was given to NMR for farther study. Extraction of Lipids: The extraction of lipids was done by Bligh & Dyer (1964) method. Estimation of Carbohydrates: The estimation of carbohydrates was done by Dubois et al, 1956 method.

Antimicrobial activity:

Microbial samples: The bacterial and fungal cultures were obtained from the microbial type culture collection of Department of Microbiology, Institution of Medical Sciences, BHU, Varanasi, Uttarpradesh, India, and experiments were performed in the same lab. Microorganisms used were Escherichia coli (12207), Klebshiella pneumoniae, Staphylococcus aureus, Pseudomonas aurogenosa (ATCC27853), Enterococcus fecalis, Vibrio choleriae, Salmonella typhymurium,

Proteus mirabilis, Salmonella enturidis and Staphylococcus aureus, and Candida albicans, Candida krusie, Candida tropocalis and Candida paropulosis.

Culture media: Mueller-Hinton agar media (Hi Media laboratories, Mumbai) was used for the growth of bacterial cultures, and Sabouraud dextrose agar for fungal growth.

Screening for Antibacterial Activity: Antibacterial activity of all eluted extracts was tested employing agar disc diffusion method. The culture plates were prepared by pouring 10-15 ml of Mueller-Hinton agar medium into sterile Petri plates. The discs of 5 mm diameter were used. The test bacteria were then swabbed over the agar media using sterile cotton swabs to get uniform distribution of the bacterial cultures. The discs were loaded with sample extracts. The 100 ml eluted samples were evaporated to 5 ml at room temperature, from which 5 µl extract was used for loading the discs. Corresponding solvent served as control. The antibacterial assay plates thus prepared were incubated at 37 °C for 24 h. The diameter of the zone of inhibition was recorded as a measure of antibacterial activity.

Screening for Antifungal Activity: To evaluate the antifungal activity, sterile agar plates were used according to disc diffusion assay. Activated fungal cultures in Sabouraud broth were adjusted to 1 × 108 cfu/m2 as per McFarland standard. Separate sterile filter paper discs of 5 mm diameter were loaded with 5µl sample extracts (as described before) and corresponding solvents served as control. The plates were incubated at 37 °C for 24 h, and diameter of the inhibition zone was recorded.

Result: Table 1: Phytochemical screening: Solvent Phytochemicals Alkaloids Flavonoids Triterpenes Phenols Cumarins Saponins Proteins Glycosides

Petroleum ether

Chloroform

++ + ++

Ethyl acetate

Acetone

+++ ++ + ++ ++ +++

+++

+++ + +++ +++ ++

Ethyl alcohol +

Water + +++ -

+++ -strongly positive, ++ -positive, + -trace, - negative.

Table 2. Antibacterial activity: Inhibition zone diameter (mm)

Solvent extract

Petroleum Ether

Chloroform

Ethyl Acetate

Acetone

Ethanol

Escherichia coli (12207)

12.0000

2.0000

0.0000

8.0000 

1.0000

Klebshiella pneumonia

13.0000

0.0000

0.0000

0.0000 

0.0000

Staphylococcus aureus

8.0000

0.0000

0.0000

1.0000 

3.0000

12.0000

0.0000

0.0000

0.0000 

0.0000

Enterococcus fecalis

14.0000

0.0000

4.0000

1.0000 

0.0000

Vibrio cholariae

18.0000

0.0000

2.0000

4.0000 

3.0000

Salmonella typhymurium

11.0000

2.0000

2.0000

0.0000 

0.0000

Proteus mirabilis

10.0000

0.0000

4.0000

10.0000  1.0000

Pseudomonas enturidis

10.0000

0.0000

2.0000

1.0000 

Bacteria strains

Pseudomonas

aurogenosa

(ATCC27853)

Water extract did not show any activity.

0.0000

Figure 1 A and B. NMR data

A 

B 

Figure 2. Antibacterial activity of sample extracted with different solvents measured in terms of inhibition zone diameter (mm)

20 Petroleum Ether Chloroform Ethyl Acetate Acetone extract Ethanol

18

inhibition zone (mm)

16 14 12 10 8 6 4 2

Ps e

sm

on m ud o

Pr o

te u Ps e

ell a on

bil is as en tu rid is

ira

ur

ium

e ym

typ h

ch io Vi br Sa lm

us

fe

ca

ola ria

lis

) 78 53 oc c

En te r

a( os ge n

oc

s eu au r

cu s oc

hy loc

ud om

on

as

au ro

St ap

AT CC 2

on m

pn eu

iel la

Kl eb sh

Es c

he

ric

hia

co

li (

12

20

7)

ia

0

Bacteria strains

Table 3: Antifungal activity in terms of inhibition zone diameter (mm) Solvent

Petrolium ether

Chloroform

Ethyl acetate

Acetone

Ethanol

Candida albicans

24.0000

1.0000

1.0000

0.0000

1.0000

Candida parapulosis

24.0000

9.0000

0.0000

7.0000

0.0000

Candida tropocalis

44.0000

10.0000

1.0000

8.0000

0.0000

Candida krusie

19.0000

4.0000

2.0000

9.0000

0.0000

Bacteria strains

Figure 3. Antifungal activity (inhibition zone diameter (mm)

50 Petroleum ether Chloroform Ethyl acetate Acetone Ethanol extract

inhibition zone (mm)

40

30

20

10

                 

kr us ie an di da C

tro po ca lis an di da C

pa ra pu lo si s C an di da

C an di da

al bi ca nc e

0

Fungal strain

Extraction of lipids: The extraction of lipids was done as described by Bligh & Dyer (1959) and modified by Kates (1964). Crude extract (30 mg) was taken for extraction of lipids. The amount recovered was 30 mg indicating 100 % lipid in the sample. Estimation of Carbohydrates: The estimation of carbohydrates was done according to Dubois et al. (1956). The amount of carbohydrates present was 0.71 mg/mg of crude extract. Zone of inhibition of Bacteria: Figure3: The spots marked 1a, 2a, 3a, 4a and 5a are control, while 1, 2, 3, 4, 5, 6, 7 represent the sample extract.

Zone of inhibition of fungus: Figure 4: The spots marked 1a, 2a, 3a, 4a and 5a are control, while 1, 2, 3, 4, 5, 6, 7 represent the sample extract.

Conclusion: The secondary metabolite secreted by Ervatamia species was extracted with various solvents and showed the presence of cumarins, flavonoids, triterpenes, phenols, glycoproteins. Petroleum Ether extract exhibited higher activity to that of other extracts against the microbial strains tried and was more active against fungal activity compared to that of bacterial strains. The extract is highly active against Candida tropocalis, a fungus that grows in chocolates and spoils it. The extract may be used as preservative in chocolate industry. The acetone extract showed moderate antibacterial and antifungal activity. The water extract contains high percentage of saponins Thus, the bioactive molecules secreted by Ervatamia species may be used in pharma and other industries.

References: Gislene GF, Nascimento, Juliana Locatelli., Paulo C.Freitas., Giuliana L.Silva. (2000) Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria Brazilian Journal of Microbiology. 31: 247-256. Hitesh K. Dhamija and Ankit S. Chauhan. (2011) Herbs now in arena of Hyperlipidaemia, Der Pharmacia Sinica, 2 (3):51-59. Lotempio MM, Veena MS, Steele HL, Ramamurthy B, Ramalingam TS, Cohen AN, Chakrabarti R, Srivatsan ES, Wang MB, Curcumin suppresses growth of head and neck squamous cell carcinoma. Clin Cancer Res 11, 6994–7002 (2005). Majumdar AM, Naik CN, Dandge and Puntambekar HM. (2000) Antiinflammatory activity of Curcuma amada Roxb. In albino rats, Indian Journal of Pharmacology. 32: 375377. Manimegalai V, Ambikapathy V, Panneerselvam A (2011) Biological control of paddy brown spot caused by Bipolaris Oryzae (Breda de Haan), European Journal of Experimental Biology, 2011, 1 (4):24-28 Menon VP, Sudheer AR. (2007) Antioxidant and anti-inflammatory properties of curcumin Adv.Eep.Med.Biol. 595: 105-125.

Nair R, Kalari T and Sumtra Chanda. (2005) Antibacterial Activity of Psoralea corylifolia L. Seed and Aerial Parts with Various Extraction Methods Turk.J.Biol. 29: 41-47. Pathak P, Saraswathy Dr, Vora A, Savai J, In vitro antimicrobial activity and phytochemical analysis of the leaves of Annona muricata. International Journal of Pharma Research and Development. 2010; 2(5). Chulet Rahul, Pradhan Pankaj, Sharma K Sarwan, Jhajharia K Mahesh (2010) Phytochemical screening and antimicrobial activity of Albizzialebbeck J. Chem. Pharm. Res., 2(5): 476-484476 Arutselvi R, Balasaravanan

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Phytochemical screening and comparative study of anti microbial activity ofleaves and rhizomes of turmeric varieties Asian Journal of Plant Science and Research, 2 (2): 212-219 Lgbinosa OO, Igbinosa EO and Aiyegoro OA (2009) Antimicrobial activity and phytochemical screening ofstem bark extracts from Jatropha curcas (Linn) African Journal of Pharmacy and Pharmacology Vol. 3(2). pp. 058-062,