Chapter4shodhganga.inflibnet.ac.in/bitstream/10603/27168/8/08... · 2018. 7. 9. · Chapter4 IsolationandScreening. 59 | Chapter 4 Isolation and Screening Natural products, either

Chapter 4

Isolation and Screening

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Chapter 4 Isolation and Screening

Natural products, either as pure compounds or as standardized extracts mainly of plant

resources, provide unlimited opportunities for new discoveries in health care products because of

the unmatched availability of chemical diversity (Cos et al. 2006). Plants used for traditional

medicine contain a wide range of substances that can be used to treat chronic as well as

infectious diseases (Duraipandiyan et al. 2006). The increasing prevalence of multidrug resistant

strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics

adds urgency to the search of new infection fighting strategies. The use of plant waste extracts

having antifungal and antibacterial properties can be of great significance in therapeutic

treatments (Hsieh et al. 2001; Arias and Ramon-Laca 2005).

Plants are capable of synthesizing a diverse array of secondary metabolites which include

tannins, terpenoides, coumarins, alkaloids and flavonoids (Perez and Anesini 1994). These may

be produced constitutively (preformed antimicrobial compounds or phytoanticipins) or in

response to pathogen or herbivore attack or stress (phytoalexins) (Wittstock and Gershenzon

2002). There are reports that these secondary metabolites are present in all parts of the plant viz.

bark, stalks, leaves, fruits, roots, flowers, pods, seeds, stems, latex, hull and fruit rind (Kaneria et

al. 2009; Aref et al. 2010; Rajaei et al 2010). Therefore, no part of the plant parts is considered

as wastes. Scientists have isolated antimicrobial compounds from different plants/plant parts

such as from the leaves of Psidium guajava (Burkill 1966), Vaccinium oxycoccos (Senchyuk and

Demkevich 1974), Garcinia mangostana (Sundram et al. 1983), Ziziphus spinachristi (Shah et

al. 1986), Annona montana (Wu et al. 1987), Carica papaya (Rajashekhara et al.) and

Passiflora edulis (Jensen et· al. 1990). Though the search for new compounds with antimicrobial

activity from plants/plant parts has been the subject of intense research since time immemorial

but there has been renewed interest over the last two decades (Cowan 1999; Hostettmann et al.

2003; Petiers and Vleitinck 2005; Harvey 2007; Lee et al., 2007).

. Therefore, screening of plant/plant parts for the therapeutically important

phytochemicals would be rewarding. Still many plants/plant parts remain unexplored. One of the

possible methodologies that can be used for the discovery of antimicrobials from these plant

parts is the screening of their crude extracts for the activity followed by bioassay.

This chapter deals with isolation and screening of antimicrobials from plant wastes.

.

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Methodology

Extraction of antimicrobial compound :

Commonly available plant wastes such as - Hay, Rice husk, Coconut fibre, Potato peel,

Pumpkin peel, and Betel leaf stalk were collected from Bolpur, West Bengal within a radius of

50 km (Fig 1). After repeated washing the wastes were shade dried and crushed into fine powder.

The powdered samples were sealed in polythene bags and were stored in dessicator at room

temperature.

Hay Rice husk Coconut fibre Potato peel Pumpkin peel Betel leaf stalk

The dried and powdered wastes (1kg) were extracted successively with 500ml of solvent

non-polar to polar seperately using soxhlet extractor (Lin et al. 1999). The extract was

concentrated to dryness in rotary vacuum evaporator below 50oC.

The crude extracts were transferred to different glass vials and kept at 4oC for further use.

For antimicrobial testing the different extracts were dissolved in DMSO (1%), a non toxic

solvent.

Screening for antimicrobial activity:

Screening of waste samples for their antimicrobial activity was done against, randomly

selected, two Gram positive, two Gram negative bacteria and three spore forming fungi by agar

cup assay (Mbata et al. 2006), paper disc assay (Freixa et al. 1996) using Nutrient agar (NA) for

bacteria and Malt agar plate (MA) for fungi.

The test organism used for the purpose were Bacillus subtilis (MTCC 121),

Staphylococcus aureus (MTCC 1430), Escherichia coli (MTCC 1610), Pseudomonas

aeruginosa (MTCC 424), Penicillium chrysogenum (MTCC 161), Alternaria solani (MTCC

2101) and Aspergillus niger (MTCC 1344).

Fig 1: Wastes used for antimicrobial screening

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Stock cultures were maintained at 4oC on nutrient agar medium and malt agar medium.

Active cultures were prepared by inoculating fresh nutrient broth and malt extract broth with a

loopful of cells from the stock and incubated at 37oC for 24h (Bacteria) and 25oC for 96h

(Fungi), to get a desirable cell/spore count (106 CFU/mL) for bioassay.

Antimicrobial activity of the crude extracts was made against the test bacteria and fungi

using agar diffusion. The agar plates were prepared by pouring 20mL of molten nutrient agar

medium into sterile petri plates. The plates were allowed to solidify and 0.1 % cell/spore

suspension (106 CFU/mL) of test organisms were spread uniformly and kept undisturbed for

15mins. Crude extracts were used in wells or as paper discs. Whether in wells or in paper discs

the plates were kept at low temperature in a refrigerator for 20mins and then incubated at 37oC

for 24h (Bacteria) and 25oC for 96h (Fungi). At the end of incubation, inhibition zones formed

around the wells/discs were recorded.

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Results:

The dried waste samples (1kg each) were powdered and attempted to extract with polar to

non-polar solvents. The extracted samples were filtered and concentrated in vacuum evaporator

to dryness and stored at 4oC for further use. The concentrates were redissolved in DMSO (1%)

and used for antimicrobial testing. All the samples showed positive activity against the test

bacteria both in agar cup and in disc assay, showing an inhibition zone diameter between 9-

27mm. It appeared that the solvent used were with certain role in the process of extraction and

diethylether showed the highest inhibition followed by those extracted with chloroform and

methanol. However, among all the extracted samples of different solvent system, the Betel Leaf

Stalk (BLS) sample exhibited highest activity (Fig 2-5). But it did not show inhibition against the

fungal strains used (Fig 6) Studies showed that suitability of extraction of antimicrobial

metabolites depended on polarity of solvents but the activity reciprocated with test organisms

(Khan and Kumar, 2011) Table (2-5).

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DEE – Diethylether, CHL- Chloroform, MET- methanol, C- Control

1 INHIBITION ZONE (dia in mm)

HE BE DEE EA CHL ACE MET ETHBLS 14±0.55 14 ±0.49 26±0.26 10 ±0.45 21±0.4 14 ± 0.26 20 ± 0.1 16±0.5

PP 10±0.26 12±0.26 14±0.26 9±0.26 15±0.43 12±0.37 14±0.45 14±0.15

HY 0 0 0 0 0 0 0 0

PK 12±0 13±0.23 13±0 11±0.1 14±0 15±0.05 16±0.17 13±0.05

RH 0 0 0 0 0 0 0 0

CF 12±0.1 11±0.23 13±0.1 14±0.1 12±0.05 12±0.05 11±0.46 11±0.1


HE BE DEE EA CHL ACE MET ETHBLS 12±0.1 15±0.15 18±0.1 9±0.1 16±0.05 12±0.26 15±0.1 12±0.05

PP 9±0.1 10±0.1 12±0.1 9±0.1 12±0.1 10±0.05 12±0.05 12±0.11

HY 0 0 0 0 0 0 0 0

PK 10±0.07 10±0.07 10±0 10±0.14 12±0.07 13±0.07 13±0.07 11±0

RH 0 0 0 0 0 0 0 0

CF 11.1±0.05 10±0.1 12±0.05 12±0.1 10±0.1 9±0.1 10±0 10±0.05

Fig 2: Effect of crude extract against Pseudomonas aeruginosa using (1) agar cup (2) paper disc assay

1 22

DEE DEE

MET MET

MET

CHL CHL

Table 2: Effect of crude extract against Pseudomonas aeruginosa using (1) agar cup (2) paperdisc assay

C C

HE-Hexane, BE-Benzene, DEE-Diethylether, EA-Ethylacetate, CHL-Chloroform, ACE-Acetone, MET-Methanol, ETH –Ethanol

Betel leaf stalk –BLS, Potato peel- PP, Hay – HY, Pumpkin peel – PKP, Rice husk – RH, Coconut fibre -CF

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PP 10±0.26 12±0.26 14±0.26 9±0.26 15±0.43 12±0.37 14±0.45 14±0.15

HY 0 0 0 0 0 0 0 0

PK 12±0 13±0.23 13±0 11±0.1 14±0 15±0.05 16±0.17 13±0.05

RH 0 0 0 0 0 0 0 0

CF 12±0.1 11±0.23 13±0.1 14±0.1 12±0.05 12±0.05 11±0.46 11±0.1



PP 9±0.1 10±0.1 12±0.1 9±0.1 12±0.1 10±0.05 12±0.05 12±0.11

HY 0 0 0 0 0 0 0 0

PK 10±0.07 10±0.07 10±0 10±0.14 12±0.07 13±0.07 13±0.07 11±0

RH 0 0 0 0 0 0 0 0

CF 11.1±0.05 10±0.1 12±0.05 12±0.1 10±0.1 9±0.1 10±0 10±0.05


1 22

DEE DEE

MET MET

MET

CHL CHL


C C



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PP 10±0.26 12±0.26 14±0.26 9±0.26 15±0.43 12±0.37 14±0.45 14±0.15

HY 0 0 0 0 0 0 0 0

PK 12±0 13±0.23 13±0 11±0.1 14±0 15±0.05 16±0.17 13±0.05

RH 0 0 0 0 0 0 0 0

CF 12±0.1 11±0.23 13±0.1 14±0.1 12±0.05 12±0.05 11±0.46 11±0.1



PP 9±0.1 10±0.1 12±0.1 9±0.1 12±0.1 10±0.05 12±0.05 12±0.11

HY 0 0 0 0 0 0 0 0

PK 10±0.07 10±0.07 10±0 10±0.14 12±0.07 13±0.07 13±0.07 11±0

RH 0 0 0 0 0 0 0 0

CF 11.1±0.05 10±0.1 12±0.05 12±0.1 10±0.1 9±0.1 10±0 10±0.05


1 22

DEE DEE

MET MET

MET

CHL CHL


C C



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HE BE DEE EA CHL ACE MET ETHBLS 15±0.1 14±0.05 25±0.05 13±0.1 19±0.46 14±0.05 20.1±0.05 16±0

PP 11±0.05 13±0.05 15±0 9±0.05 17±0.05 13±0.05 15±0.05 16±0.05

HY 0 0 0 0 0 0 0 0

PK 12±0 15±0 15±0 12±0 14±0.07 15±0 17.±0.07 14±0.07

RH 0 0 0 0 0 0 0 0

CF 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05



PP 11±0.05 13±0.1 15±0.05 9±0.1 17±0.1 13±0.1 15±0.1 16±0.1

HY 0 0 0 0 0 0 0 0

PK 13±0.07 15±0.07 15±0.07 12±0.07 14±0 15±0.07 17±0.07 14±0.07

RH 0 0 0 0 0 0 0 0

CF 13±0.05 13±0.05 15±0 15±0.05 14±0.05 14±0.05 15±0.05 12±0.05

Fig 3: Effect of crude extract against Escherichia coli using (1) agar cup (2) paper disc assay

DEE DEE

CHL CHL

MET MET

C C

Table 3: Effect of crude extract against Escherichia coli using (1) agar cup (2) paperdisc assay

1 2


HE-Hexane, BE-Benzene, DEE-Diethylether, EA-Ethylacetate, CHL-Chloroform, ACE-Acetone, MET-Methanol, ETH–Ethanol


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PP 11±0.05 13±0.05 15±0 9±0.05 17±0.05 13±0.05 15±0.05 16±0.05

HY 0 0 0 0 0 0 0 0

PK 12±0 15±0 15±0 12±0 14±0.07 15±0 17.±0.07 14±0.07

RH 0 0 0 0 0 0 0 0

CF 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05



PP 11±0.05 13±0.1 15±0.05 9±0.1 17±0.1 13±0.1 15±0.1 16±0.1

HY 0 0 0 0 0 0 0 0

PK 13±0.07 15±0.07 15±0.07 12±0.07 14±0 15±0.07 17±0.07 14±0.07

RH 0 0 0 0 0 0 0 0

CF 13±0.05 13±0.05 15±0 15±0.05 14±0.05 14±0.05 15±0.05 12±0.05


DEE DEE

CHL CHL

MET MET

C C


1 2




64 |




PP 11±0.05 13±0.05 15±0 9±0.05 17±0.05 13±0.05 15±0.05 16±0.05

HY 0 0 0 0 0 0 0 0

PK 12±0 15±0 15±0 12±0 14±0.07 15±0 17.±0.07 14±0.07

RH 0 0 0 0 0 0 0 0

CF 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05 13±0.05



PP 11±0.05 13±0.1 15±0.05 9±0.1 17±0.1 13±0.1 15±0.1 16±0.1

HY 0 0 0 0 0 0 0 0

PK 13±0.07 15±0.07 15±0.07 12±0.07 14±0 15±0.07 17±0.07 14±0.07

RH 0 0 0 0 0 0 0 0

CF 13±0.05 13±0.05 15±0 15±0.05 14±0.05 14±0.05 15±0.05 12±0.05


DEE DEE

CHL CHL

MET MET

C C


1 2




65 |




PP 12±0.1 12±0.05 20±0.1 11±0.1 18±0.1 12±0.05 15.1±0 12±0

HY 0 0 0 0 0 0 0 0

PK 12±0.07 13±0.07 15±0.07 12±0 14±0 13±0.07 12.±0 11±0.14

RH 0 0 0 0 0 0 0 0

CF 12±0.05 11±0.05 15±0.05 12±0.05 15±0.05 13±0.1 12±0.05 11±0.1


HE BE DEE EA CHL ACE MET ETHBLS 11±0.05 12±0 19±0.05 12±0.1 16±0.05 11±0 14±0.1 11±0.05

PP 10±0.05 12±0.58 13±0.1 11±0.1 13±0.45 12±0.5 13±0.11 13±0.05

HY 0 0 0 0 0 0 0 0

PK 11±0.07 12±0.07 14±0.07 12±0 13±0.07 13±0.5 12±0 11±0.07

RH 0 0 0 0 0 0 0 0

CF 12±0.11 11±0.05 15±0.05 11±0.1 14±0.05 13±0.5 12±0.05 10±0.05

Fig 4: Effect of crude extract against Bacillus subtilis using (1) agar cup (2) paper disc assay

DEE DEE

MET MET

CHL CHL

C C

Table 4: Effect of crude extract against Bacillus subtilis using (1) agar cup (2) paper discassay

1 2




65 |




PP 12±0.1 12±0.05 20±0.1 11±0.1 18±0.1 12±0.05 15.1±0 12±0

HY 0 0 0 0 0 0 0 0

PK 12±0.07 13±0.07 15±0.07 12±0 14±0 13±0.07 12.±0 11±0.14

RH 0 0 0 0 0 0 0 0

CF 12±0.05 11±0.05 15±0.05 12±0.05 15±0.05 13±0.1 12±0.05 11±0.1



PP 10±0.05 12±0.58 13±0.1 11±0.1 13±0.45 12±0.5 13±0.11 13±0.05

HY 0 0 0 0 0 0 0 0

PK 11±0.07 12±0.07 14±0.07 12±0 13±0.07 13±0.5 12±0 11±0.07

RH 0 0 0 0 0 0 0 0

CF 12±0.11 11±0.05 15±0.05 11±0.1 14±0.05 13±0.5 12±0.05 10±0.05


DEE DEE

MET MET

CHL CHL

C C


1 2




65 |




PP 12±0.1 12±0.05 20±0.1 11±0.1 18±0.1 12±0.05 15.1±0 12±0

HY 0 0 0 0 0 0 0 0

PK 12±0.07 13±0.07 15±0.07 12±0 14±0 13±0.07 12.±0 11±0.14

RH 0 0 0 0 0 0 0 0

CF 12±0.05 11±0.05 15±0.05 12±0.05 15±0.05 13±0.1 12±0.05 11±0.1



PP 10±0.05 12±0.58 13±0.1 11±0.1 13±0.45 12±0.5 13±0.11 13±0.05

HY 0 0 0 0 0 0 0 0

PK 11±0.07 12±0.07 14±0.07 12±0 13±0.07 13±0.5 12±0 11±0.07

RH 0 0 0 0 0 0 0 0

CF 12±0.11 11±0.05 15±0.05 11±0.1 14±0.05 13±0.5 12±0.05 10±0.05


DEE DEE

MET MET

CHL CHL

C C


1 2




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HE BE DEE EA CHL ACE MET ETHBLS 14±0.05 13±0.05 18±0.1 12±0.05 16±0.05 13±0.2 15±0.1 12±0

PP 11±0.05 13±0.05 13±0.11 9±0.05 14±0.05 12±0.05 10±0.05 10±0

HY 0 0 0 0 0 0 0 0

PK 12±0.07 12±0.21 12±0.28 11±0 11±0.49 10±0.07 10±0.7 10±0.07

RH 0 0 0 0 0 0 0 0

CF 11±0.46 12±0.1 10±0.1 11±0.05 11±0.05 11±0.05 10±0.47 10±0.05



PP 10±0.05 12±0.05 12±0.05 13±0.05 9±0.05 9±0.05 11±0.43 10±0.05

HY 0 0 0 0 0 0 0 0

PK 10±0.7 9±0.14 11±0.07 10±0 11±0.07 10±0 10±0.07 10±0.07

RH 0 0 0 0 0 0 0 0

CF 9±0.58 10±0.1 9±0.51 10±0.1 11. ±0.45 9±0.15 10±0.05 10±0

Fig 5: Effect of crude extract against Staphylococcus aureus using (1) agar cup (2) paper disc assay

DEE DEE

MET MET

CHL CHL

C C

Table 5: Effect of crude extract against Staphylococcus aureus using (1) agar cup (2) paper discassay.

1 2




66 |




PP 11±0.05 13±0.05 13±0.11 9±0.05 14±0.05 12±0.05 10±0.05 10±0

HY 0 0 0 0 0 0 0 0

PK 12±0.07 12±0.21 12±0.28 11±0 11±0.49 10±0.07 10±0.7 10±0.07

RH 0 0 0 0 0 0 0 0

CF 11±0.46 12±0.1 10±0.1 11±0.05 11±0.05 11±0.05 10±0.47 10±0.05



PP 10±0.05 12±0.05 12±0.05 13±0.05 9±0.05 9±0.05 11±0.43 10±0.05

HY 0 0 0 0 0 0 0 0

PK 10±0.7 9±0.14 11±0.07 10±0 11±0.07 10±0 10±0.07 10±0.07

RH 0 0 0 0 0 0 0 0

CF 9±0.58 10±0.1 9±0.51 10±0.1 11. ±0.45 9±0.15 10±0.05 10±0


DEE DEE

MET MET

CHL CHL

C C


1 2




66 |




PP 11±0.05 13±0.05 13±0.11 9±0.05 14±0.05 12±0.05 10±0.05 10±0

HY 0 0 0 0 0 0 0 0

PK 12±0.07 12±0.21 12±0.28 11±0 11±0.49 10±0.07 10±0.7 10±0.07

RH 0 0 0 0 0 0 0 0

CF 11±0.46 12±0.1 10±0.1 11±0.05 11±0.05 11±0.05 10±0.47 10±0.05



PP 10±0.05 12±0.05 12±0.05 13±0.05 9±0.05 9±0.05 11±0.43 10±0.05

HY 0 0 0 0 0 0 0 0

PK 10±0.7 9±0.14 11±0.07 10±0 11±0.07 10±0 10±0.07 10±0.07

RH 0 0 0 0 0 0 0 0

CF 9±0.58 10±0.1 9±0.51 10±0.1 11. ±0.45 9±0.15 10±0.05 10±0


DEE DEE

MET MET

CHL CHL

C C


1 2




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Penicillium chrysogenum Alternaria solani

Aspergillus niger

Fig 6: Effect of crude extract against test fungi

67 |



Aspergillus niger


67 |



Aspergillus niger


68 |


Discussion:

Since long, there is exhaustive search for antimicrobial properties from plants. Successful

isolation of biologically active compounds from plant material is largely dependent on the type

of solvent used in the extraction procedure. Properties of a good solvent in plant extraction

include low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption

of the extract, preservative action and inability to cause the extract to complex or dissociate

(Houghton and Raman 1998).

Scientists have used different types of solvent for the extraction of biologically active

compound from plants. Acamovic and Brooker (2005) used aqueous acetone although it is not a

very commonly used solvent but has been used by a number of workers (Dilika et al. 1996;

Mathkega et al. 2000; Lourens et al. 2004; Basri and Fan, 2005). Harmala et al. (1992) used

chloroform for the extraction of biologically active compounds. Hammer et al. (1999) used polar

solvents such as methanol to extract polyphenolic compounds such as flavonols and other

bioactive compounds reported. The most commonly used solvents for investigations of

antimicrobial activity in plants are methanol, ethanol and water (Salie et al. 1996; Bisignino et

al. 1999; Parekh et al. 2005; Rojas et al. 2006). Dichloromethane has also been used by a

number of researchers (Freixa et al. 1996). In a study by Masoko and Eloff (2006) both acetone

and methanol were found to extract saponins which have antimicrobial activity. Eloff (1998) and

Nostro et al. (2000) reported that the use of combination of all these solvents for better extraction

of biologically active substances.

Different workers use separate methods to screen antimicrobial potentialities of plants.

For instance Pavithra et al. (2010) used disc diffusion and broth dilution techniques to test the

antimicrobial activity of Delonix elata, Enicostemma axillare, Merremia tridentata, Mollugo

cerviana and Solanum incanum against Gram-positive bacterial strains and Gram-negative

bacterial strains. Agnihotri and Vaidya (1996) developed a novel approach for studying

antibacterial properties of certain plants like Eugenia caryophyllus, Thymus vulgaris,

Cinnamonum zeylanium and Cuminum cyminum. Volatile components of the hexane extracts of

these plants were tested against standard Gram positive and Gram negative bacteria grown on

agar slants and the results were expressed as percentage inhibition of the area of the slants. Gupta

et al. (2010) used Lowenstein Jensen (L-J) medium and colorimetric BacT/ALERT 3D system to

69 |


test antituberculosis activity of Acalypha indica, Adhatoda vasica, Allium cepa, Allium sativum

and Aloe vera.

To test the antifungal activity different workers used different methods. Nair et al. (1991)

and Nene and Thapliyal (2000) used spore germination assay and poison food technique to test

the antifungal activity of essential oil isolated from the leaves of Aegle marmelos and Cassia

alata.

All these results suggests that proper choice of solvents and application of appropriate

antimicrobial technique might be useful in obtaining the compound with bioactivity which

supports the present study.

The result (Table 2-5) shows that among all the wastes samples used BLS has the highest

activity. As found positive, BLS was selected for further study.

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