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Natural products as potential sources of inhibitors of bacterial quorum-sensing

von Assistant Professor Vijay Kothari (Autor) Haren Gosai (Autor) Shreya Raval (Autor) Vimla Chaudhary (Autor)

Wissenschaftliche Studie 2014 32 Seiten

Biologie - Mikrobiologie, Molekularbiologie





3. Results

4. Discussion



Authors thank Nirma Education and Research Foundation (NERF), Ahmedabad for financial and infrastructural support. All authors thank their respective family members for their unconditional support.


Nature has been a source of medicinal agents for thousands of years since it comprises of compounds that are highly diverse and often provide highly specific biological activities. This follows from the proposition that essentially all natural products have some receptor binding capacity (Verdine GL., 1996).To promote the proper use of herbal medicine and to determine their potential as sources for new drugs, it is essential to study medicinal plants, which have folklore reputation in a more intensified way( Awadh NA et al., 2001).

Some natural products have been approved as new antimicrobial drugs, but there is a continuous and urgent need to screen more and more plant species and discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action to combat new and re-emerging infectious diseases of today’s era( Rojas R et al.,2003). Plant based natural constituents can be derived from any part of the plant like bark, leaves, lowers, roots, fruits, seeds, etc( Gordon MC et al., 2001).i.e. any part of the plant may contain active components. World Health Organization has also approved the study of medicinal plants for the development of new drug lead (WHO, 2000).

There is an evolving group of chronic infectious diseases and condition in which bacteria is present and they are difficult to culture and demonstrate consistently hence resistant to current antimicrobial and tools and often require surgical removal to resolve. Despite adequate drainage antibiotics are not the “silver bullet” once envisaged for this chronic infectious condition (Hayes GW et at., 1993). The use of antimicrobials from natural sources could have been a great impact for preserving food storage from contamination, and in controlling plant and human diseases of microbial origin (Balandrin et al., 1985).The continued evolution of infectious disease and the resistance offered by the pathogens to the existing pharmaceuticals have led to an intensified search for the new availability of antimicrobials against fungal, bacterial and viral particles as plants constantly interact with the rapidly changing and damaging environmental conditions. Being organism devoid of mobility plants has developed a strong immunity against the metabolites and chemical stress which come across their life. The ability of plant to carry out combinatorial chemistry by mixing matching and evolving the gene products required for certain secondary metabolite biosynthetic pathway create an unlimited pool of chemical compound, which humans have exploited to their benefit.

Many pathogenic bacteria trigger the production of their virulence factors in a population density-dependent manner, a cell-to-cell communication mechanism known as quorum sensing (Vandeputte et al., 2009).Quorum sensing (QS) is mediated through small signal molecules called auto inducers that regulate the target gene expression responsible for the phenotypes essential to pathogenicity/symbiosis. In Gram negative bacteria, QS is mediated through N-acyl homoserine lactones (AHL) (Bassler BL., 2002).

QS has led to its role in controlling the production of virulence factors such as exopolysaccharide synthesis, biofilm formation, swarming motility, pigment production and antibiotic production in some pathogenic bacteria (Smith JL et al.,2011, Schonewille E et al., 2012). Increasing emergence of antibiotic resistance in Gram negative bacteria also demands alternative strategies to combat bacterial infections and anti-QS approach is being viewed as an attractive alternative.

Plant metabolites having the ability to control the growth of microbes have been traditionally used to treat human diseases including microbial infections (Shahid M et al., 2009). There are certain plants such as carrot, garlic, habanero (chilli), and water lily produce compounds that interfere with bacterial QS. Other plants found to be having anti-quorum sensing potential were pea (Pisum satavium) seedlings, crown vetch, soybean, and tomato (Rasmussen et al., 2006). Plants have recognised symbiotic relationship with colonizing bacteria. There may be plant based natural quorum sensing signals to control pathogens. It was observed that certain seaweed plants never became cover with bacteria or higher organism ( called bio fouling, which is also observed on sub surfaces of ships) because the plant produced halogenated furanones, which have quorum sensing inhibitors activity(QSIs), but these halogenated furanones contain halogen which make them restricted to human use. Therefore, large scale screening of QSIs in nature was therefore performed. In recent years multiple drug resistance occur against commercially available antimicrobials which has forced scientist to search for new antimicrobials from plants which has vast source of bioactive agents in it.

Plant materials:

1. Pongamia pinnata

Family- Fabaceace

Common name- karanj, and is also called as Derris indica in India and Myanmar.

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Fig 1 – Pongamia pinnata: A. growing in natural habitat; B. Matured seeds.

Pongamia pinnata (Syn. Milleta pinnata) is a species of tree in the pea family. It is native in tropical and temperate Asia including parts of India, China, Japan, Malaysia, and Australia. Pongamia pinnata also called as Derris indica is a monotypic genus and grows abundantly along the coasts and riverbanks in India and Myanmar. The tree is known for its multipurpose benefits and as potential sources of biodiesel (Kesari V et al., 2009, Naik M et al., 2008). The seeds are reported to contain on an average about 28–34% oil with high percentage of polyunsaturated fatty acids. Historically, Pongamia has been used as folk medicinal plant, particularly in Ayurvedha and Siddha systems of Indian medicine (Meera B et al., 2003). All parts of the plant have been used as a crude drug for the treatment of tumours, piles, skin diseases, itches, abscess, painful rheumatic joints wounds, ulcers, diarrhea etc.

Besides, it is well known for its application as animal fodder, green manure, timber and fish poison.

It recognized to possess applications in agriculture and environmental management, with insecticidal and nematicidal activity. More recently, the effectiveness of P. pinnata as a source of biomedicines has been reported (Brijesh S et al., 2006), specifically as antimicrobial and therapeutic agents.

2. Manilkara hexandra

Family- Sapotaceae

Common name- rayan (Hindi), raina, khirni(Marathi), Ceylon Iron Wood

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(Fig 2 –Manilkara hexandra)

Manilkara hexandra(Roxb.) Dubard belonging to family Sapotaceae, is a socio-economically important underutilized fruit species of western-central India. It is locally known as ‘Khirni’, ‘Raina’ and ‘Rayan’ by tribal people of different states of India. It is believed to be originated in India. The tree is found as natural wild in the south, north and central India mostly in the states of Rajasthan, Gujarat, Madhya Pradesh and Maharashtra. It is a commercially and medicinally important tropical tree species and is a significant source of livelihood and nutritional support species for local tribal population. In India, this species is occasionally cultivated in backyards, homestead gardens, public parks as avenue tree and in farmer’s fields near villages due to its economic importance as fruit tree having nutritional and medicinal properties.

Fruits of Khirni have high economic value as mature fresh fruits which are sweet and a good source of minerals, sugars, protein, carbohydrate and vitamin A (Pareek J et al. 1998). Whole partially matured fruits are dried in the sunlight to reduce the chances of infection and deterioration. Tribal people sell the fresh as well as dried fruits in the local market at the cost of Rs. 30–40 per kg. Each tree provides fruits worth of Rs. 500–2,000/- to a tribal family, which is a substantial livelihood support to them. Fresh fruits are generally consumed by entire tribal family which provides good nutritional support in the form of vitamin A, hence whole fruit works as ‘‘vitamin A capsule’’ for the nutritionally deficient tribal population especially children and women. Bark and fruits are also used for several medicinal purposes like treatment of ulcers, dyspepsia, opacity of the cornea, bronchitis, urethrorrhea, leprosy, etc. (Pareek et al. 1998;Chanda and Parekh 2010).

The seeds contain approximately 25 % oil, which is used for cooking purposes. The bark also contains 10 % tannin, which is used for treatment of fever and may be utilized in tanning purposes. The wood of this tree is very hard, tough and durable and is used for oil presses, house building and turnery.

3. Pyruspyrifolia


Common name- nashpati, pear, Chinese pear, apple pear, sand pear

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(Fig 3 – pyrus pyrifolia (

The sand pear is larger than most kinds of pear trees, reaching as high as 40 ft. (12 m), with a rounded crown that may spread 20 feet (6 m) or more across. The tree is almost completely covered with white flowers, putting on a spectacular show in early spring. Pyrus pyrifolia is native to China and Japan. Dozens of cultivars are grown in Asia. The fruits are hard and the flesh is grainy, some say "sandy" in texture. They are most useful for making pear butter, preserves, pies, and for canning. Pears are a good source of dietary fiber and vitamin C. Pear wood is one of the preferred materials in the manufacture of high-quality woodwind instruments and furniture. pears are used to treat nausea.

Test microorganisms

Chromobacterium violaceum (MTCC:2656)

Chromobacterium violaceum is a Gram-negative rod which is found in the soil and water of tropical and subtropical areas. Chromobacterium violaceum (Cv) appears to be an opportunistic pathogenic bacterium, which affects humans and animals in subtropical and tropical areas. The low infectious capability of Cv is evidenced by the fact that rivers such as Negro River, in the Amazon region of Brazil, where the bacteria is highly abundant, are sources of drinking water, without the occurrence of widespread infection among the local population( Groves M et al,. 1969).


Plant material

The plants were selected on the basis of earlier reported antimicrobial activity of their other parts (pulp, stem, leaf, fruits, etc.), (Arote SR et al., 2009; Kesari V et al., 2009), and same parts against other organisms. The seeds of three plants Pongamia pinnata (Karanj), Pyrus Pyrifolia (Nashpati), Manilkara hexandra (Rayan) were collected during months of October, 2013 to November, 2013. They were authenticated for their unambiguous identity by Dr.Himanshu Pandya, Department of Botany, Gujarat University, Ahmedabad. They were thoroughly washed with tap water, air-dried in shade and stored in opaque air-tight containers (at room temperature) to avoid photo-oxidation(Houghton and Raman, 1998). The seeds were checked at regular intervals for any physical or biological damage. Damaged seeds were removed from the collection.

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Table1. Details of plant seeds selected

Test organisms:

Following test organisms were procured from Microbial Type Culture Collection (MTCC), Chandigarh.

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Table 2.Test organisms

#Antibiotic susceptibility pattern determined by macro broth dilution assay in our lab

Microwave Assisted Extraction (MAE):

Seeds were extracted by MAE method earlier published by us (Kothari et al., 2009, Kothari et al., 2011; Ramanuj et al., 2012; Darji et al., 2012). Seeds were crushed twice in grinder (Maharaja whiteline bonus grinder) to coarse powder at knob 1 for 60 sec. One gram of seed powder was soaked into 50 ml of respective solvent in brown coloured screw capped bottle (250 ml, Merck, Mumbai, India), cap was loosened slightly to avoid pressure build up. Solvents used for extraction were acetone, methanol (Merck, Mumbai) and ethanol (50%; Ureca consumers Co. Op Stores Ltd., Ahmedabad India). Then this was subjected to microwave assisted extraction (MAE). For this, bottle was kept in microwave oven (Electrolux, EM30EC90SS) for extraction at 720W. At a time only one bottle was kept for procedure. The time for extraction of all seeds for respective solvent is reported in Table 3. The extraction was followed by macrofiltration (nylon strainer), which was further subjected to centrifugation at 7,500 rpm for 20 min. Then after microfiltration was carried out using Whitman filter paper no. 1 (Whatman International Ltd., England) to remove the fine particulate matter and was allowed to evaporate in pre-weighed petridishes (in weighing balance, Setra, BL-410S) a temperature below the boiling point of respective solvents. For determination of antimicrobial activity, extract were reconstituted in dimethyl Sulfoxide (DMSO, Merck, Mumbai) as it possesses the ability to dissolve polar as well as non-polar compounds. Extract was collected in sterile flat bottom glass vials (15ml, Merck, Mumbai) which were protected from light to avoid photo-oxidation of light sensitive compounds (Houghton and Raman, 1998). The internal surface of vial cap was wrapped with aluminium foil to avoid leaching of vial cap material and their absorption by extract (Houghton and Raman, 1998). They were then stored in refrigerator at 4˚C.



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Titel: Natural products as potential sources of inhibitors of bacterial quorum-sensing