Samah O. Noor

Biology Department, Faculty of Science, King Abdulaziz University,
P.O. Box 42805, Jeddah 21551, Saudi Arabia.

Abstract

All surfaces of human body are colonized by many microbial communities but
gut is colonized by greater densities known as the microbiota or commensally microflora which is mainly influenced by the plant extracts in the diet. In this study, 4 different plant materials, the leaves of Camellia sinensis, Mentha piperita and Petroselinum crispum in addition to the Pimpinella anisum seeds were collected and extracted with either hot water or methanol. The antimicrobial activity was determined using agar well diffusion method. All the extracts showed antibacterial activity against some bacterial pathogens including Escherichia coli, Salmonella typhimurium, Pseudomonas
earuginosa, Enterococcus faecalis, E. faecium Staphylococcus aureus and Streptococcus agalactiae which was used as a control. The water and methanol extracts of Camellia sinensis and the water extract of Pimpinella anisum and Petroselinum crispum showed significant lower antibacterial activity against all the tested probiotic bacteria like Lactobacillus and Bifidobacteria. MICs values of the water extracts of the 4 tested plants
were recorded for all the tested bacterial pathogens in addition to the tested probiotic bacteria. Concerning the pathogenic bacteria, MIC was ranged from 50-250 μg/ml, 100-150 μg/ml, 150 μg/ml and 75-125 μg/ml for Camellia sinensis, Pimpinella anisum, Petroselinum crispum, respectively. Concerning the probiotics, the MIC of the 4 tested plants was greater than 250 μg/ml except for L. plantarum, where the MIC of Camellia sinensis was 250μg/ml. The presence of plant extracts slightly decrease the rate of growth L. acidophilus and the decrease was clear in case of Camellia sinensis> Mentha piperita > Pimpinella anisum> Petroselinum crispum. In conclusion, the tested plant extracts affect significantly the growth of pathogenic bacteria but the effect was lower on the tested gut bacteria, thus they can be used safely to improve human health.

Keywords:  Plant extract, gut microbiota, Menthapiperita, Pimpinellaanisum, Petroselinum, Camellia sinensis

Introduction

More than 1000 different bacterial species and 1014 bacterial cells colonised the adult human gastrointestinal (GI) tract and among individuals, the count vary greatly according to dietary components and host ageand health (Hooper et al. 2002, Eckburget al. 2005).There isinteractive associations and symbiotic relationship between these bacteria and the host cells. Within the tract, they have critical roles inmotility, nutrient absorption, protection from invading GI pathogens,fermentation the unused substrates, production vitamins and conservation of mucosal immune function(Flintet al. 2007, Ley et al. 2008, Zoetendal et al. 2008).The bacteria break down the ingested polysaccharides to monosaccharides, which are then fermented to form short-chain fatty acids as a final metabolic product. In this shared environment, the host gains carbon and energy, and the bacteria are provided with glycans and protection.Unbalanced bacterial community in the gut plays a role in some diseases and gut disorders. There is an interest to remove gut disorders by influencing the composition and activities of resident microflora. Probiotics, are a group of bacterial genera, found mainly in some foods, known as functional foods, that protect against gut disorder by targeting particular groups of bacteria in the gut.  Probiotics already present in the GI tractand prebiotic supplements as dietary substancesmay enhance their growth and development (Probert 2004). Certain carbohydrates such as arabinoxylan or other plant materials or extracts, which are prebiotics, are selectively metabolised by gut bacteria, thereby changing the gut ecosystem towards a more beneficial structure (Swennenet al. 2006).Bruneet al. (2000) described the gut ecosystem as a programmed bioreactor with bacteria that degrade indigestible polysaccharides and alteration of the microbiota composition of theintestinal allowed to some bacterial pathogens to grow, multiply, colonize and initiate intestinal disorders.  Some plant extracts and antibiotic mayaffect the intestinal physiology and   inhibit or enhance microbiota in human and animals (Manzanillaet al., 2004).  The aim of the present study was to evaluate the inhibitory effect of some used tradition plants on microbiota and some bacterial pathogens.

 

Material and Methods

Plant collection and extraction:

In this study, four medicinal plants   including  the leaves of Camellia sinensis, MenthapiperitaandPetroselinumcrispumin addition to the Pimpinellaanisum seeds were collected and identified at Biology Department, Faculty of Science, KAU, Saudi Arabia. The collected samples were washed, dried, grinded and extracted with either hot water or methanol (Aly and Bafeel, 2010). About 50 g of each sample were macerated in 200 ml hot water or 200 ml methanol for 4 hours at room temperature, 22°C, and then the mixtures were filtered through muslin cloth filter paper. Further extraction of the residue was carried out and all extracts were collected and dried using either rotary evaporator for organic extracts orlyophilizer for water extracts. All dried extracts were kept in the deep freezer at -80°C until used for the antimicrobial susceptibility studies.

The tested bacteria

Clinical isolates of the Gram negativeEscherichia coli, Salmonellatyphimuriumand Pseudomonas earuginosa, and the Gram positive Enterococcusfaecalis,  Enterococcusfaecium, Staphylococcus aureusand Streptococcus agalactiaewere obtained from the culture collection from King Fahd General Hospital, Jeddah, Saudi Arabia. Gut normal flora strains Lactobacillus acidophilus, L. bulgaricus, L. plantarum, Bifidobacteriumand Streptococcus thermophiluswere obtained from Culture collection Unit, Faculty of Agriculture, Ain Shams, Egypt.

The identification of all the used bacteria was confirmed using microscopic examination, Gram and spore stains, growth on selective medium and API20.Selective Strep Agar (Cat. no. A70) was used for Streptocooccus growth in presence of CO for 24 hr., MRS medium was used for Lactobacillus and Bifidobacteriumstrains growth under aerobic and anaeronic conditions, respectively at 37°C for 2 days for Lactobacillus and 4 days for Bifidobacterium

The antibacterial activity

All extracts were dissolved in DMSO and bacterial growth inhibition was tested by using   agar well diffusion method while the MIC was determined by the micro dilution method. For each of the extract, multiple plates (3replications) were prepared and the plates were then maintained for 2h at room temperature to allowextract diffusion, incubated at 37°C for 24 h and the zones of inhibition were subsequently measured in mm (Mukherjee et al., 1995a, b). Dilution micromethod for MIC determination was used (de Paivaet al, 2003). The water extracts of the fourtested plants were selected for further tests and to calculate their MIC by dilution method. This test was performed in sterile 96-well microplates the g dilution procedure. The microdilution was performed in 96-well microtiter plates with U-shaped wells. Each culture was grown inMüeller-Hinton broth for 12 h and the absorbance was adjusted to 0.5 McFarland turbidity (5×105 CFU/ml). Controls with 0.5 ml of only culture medium  without plant extractwas  used. The wells were filled with 100 µl of sterile H2O and 100 µl of the plant extracts were added to the wells by serial two fold dilution from the suspension of plant extract stock solution. Each well was inoculated with 100 µl of 0.5 McFarland standard bacterial suspensions and one drop of phenol red solution was added to each well. All plates were covered, placed in plastic bags and incubated at 37°C for 24 h. The MIC was the lowest concentration of plant extracts that exhibited no bacterial growth (yellow color and followed by red color) by visualobservations.

The effect of different water extracts of the 4 tested plants (250 µg/ml) on the growth of Lactobacillus acidophilus, grown in MRS broth medium (de Man et al., 1960) was determined  by measuring the growth (absorbance at 540 nm ) each 2 hours up to 24 h by spectrophotometer and calculating log cfu/ml.

Results

In Arab area, different plants were used traditionally as hot drinks.  In this study, 4 different plant materials, the leaves of Camellia sinensis, MenthapiperitaandPetroselinumcrispumin addition to the Pimpinellaanisum seeds were collected and extracted with either hot water or methanol. Table 1 showed the studied plants, their common names, the used plant part and the type of extraction. The antimicrobial activities of the two extracts of the tested plants were determined for some Gram positive and negative pathogens (Figure 1). The antibacterial activity of the tested aqueous extracts were determined against the tested bacterial pathogens and compared with that of Streptococcus agalactiae which was used as a control (Table 2). The water extract of Camellia sinensisshowed significant antibacterial activity against both Enterococcus faecalisand E. faecium, whilePimpinellaanisumextract showedsignificant antibacterial activity against SalmonellatyphimuriumandPseudomonas  earuginosa. The water extract ofPetroselinumcrispum has antibacterial activity against Escherichia coli, S.typhimurium, P. earuginosaandE. faecalis. Moreover,Menthapiperita recorded significantantibacterial activity against E. coli, S.typhimurium,P.earuginosa. The bacterial index showed that the maximum activity was recorded for water extract of Petroselinumcrispum, followed byPimpinellaanisumand finally  Camellia  sinensisandMenthapiperita. Similarly, the methanolic extract of Camellia sinensisshowed significant antibacterial activity against both E. faecalisand E. faecium, while the methanol extract of Pimpinellaanisum  showed significant abtibacterial activity against E. coli and Staphylococcus aureus (Table 3).Furthermore,Petroselinumcrispum extract showed antibacterial activity against E. coli, P. earuginosaandStaphylococcusaureus, whileMenthapiperita recorded significantantibacterial activity against E. coli, S.typhimurium, P. earuginosa, E. faeciumandS. aureus. Bacterial index showed that the maximum activity was recorded for the methanol extract of Menthapiperita  andPetroselinumcrispum, followed by Camellia sinensisand finally  Pimpinellaanisum. 

 

 

Table 1.The Used Medicinal Plants

Scientific name
Common name
Family
Used part
Type of extraction
Camellia  sinensis
Green tea
Theaceae
Leaves
Hot water, methanol
Menthapiperita
Mint
Labiatae
Leaves
Hot water, methanol
Petroselinumcrispum
parsley
Apiaceae
Leaves
Hot water, methanol
Pimpinellaanisum
Anise
Umbelliferae
Seeds
Hot water, methanol

 

Figure 1: Inhibitory effect of water extract Petroselinumcrispumon Escherichia  coli(A) and Streptococcus agalactiae(B).

Lower activity of the water and methanolic extracts of the four tested plants was recorded against the probiotic bacteria (Lactobacillus acidophilus, L. bulgaricus, L. plantarum, Bifidobacteriumand Streptococcus thermophilus) as shown in Table 4. Significant lower antibacterial activity was recorded  for all tested  water extracts against the tested probiotic bacteria  compared to Streptococcus  agalactiaeas a control bacterium. Moreover, the methanol extract of Camellia sinensis, Pimpinellaanisum  andPetroselinumcrispum  showed significant lower antibacterial activity  compared to control while Menthapiperita extract showed no significant  antibacterial activity against all tested bacteria except Lactobacillus acidophilus.The bacterial index was higher for the methanol extracts compared to water extracts, thus water extracts was selected for determination of the MIC values for all the tested bacteria.  The water and methanol extract of Camellia  sinensisand the water extract ofPimpinellaanisumandPetroselinumcrispumshowedsignificant lower antibacterial activity against all the tested probiotic bacteria. MICs values of the water extracts of the 4 tested plants were recorded for the test bacterial pathogens in addition to the tested probiotic bacteria (Table 5) using micromethod (Figure 2). Concerning the pathogenic bacteria, MIC was ranged from 50-250 µg/ml,100-150 µg/ml, 150 µg/ml and 75-125 µg/ml for Camellia  sinensis, Pimpinellaanisum, Petroselinumcrispum, respectively. Concerning the probiotics, the MICs of the 4 tested plants were greater than 250 µg/ml except for L. plantarum, the MIC of Camellia sinensis was 250 µg/ml.

Table 2.The Antibacterial activity of the aqueous extractsof 4tested plant extracts against some bacterial pathogens

Tested extract

 

Bacterial pathogen

 Diameter of the inhibition zone (mm)
 Camellia

sinensis

Pimpinella

anisum

Petroselinum

crispum

Menthapiperita 
Escherichia  coli 11.4±0.0 14.0±0.7 21.6±0.1* 11.0±0.1*
Salmonellatyphimurium 11.6±0.5 16.3±1.6* 18.6±1.9* 11.3±0.5*
Pseudomonas  aeruginosa 10.6±1.5 16.6±1.6* 20.0±1.4* 10.7±0.4*
Enterococcus faecalis 15.0±1.0* 12.6±0.9 19.3±1.0* 12.7±0.1
Enterococcus faecium 15.3±1.1* 12.3±1.0 14.3±1.8 13.6±1.6
Staphylococcus aureus 11.3±0.5 11.3±1.7 14.0±1.8 13.0±1.4
Streptococcus agalactiae

(control)

11.6±0.5 11.6±0.7 14.3±1.0 13.3±1.5
Bacterial index 12.4 13.5 17.4 12.2

*: significant difference at p< 0.05

Figure 2:MIC of the water extracts of Petroselinumcrispumon Some Bacterial Pathogens using micromethod technique

 

 

 

Table 3.Antibacterial activity of the methanolic extracts of the 4  tested plant extracts against some tested bacterial pathogens

Tested extract

 

 Bacterial pathogen

 Diameter of the inhibition zone (mm)
 Camellia

sinensis

Pimpinella

anisum

Petroselinum

crispum

Menthapiperita 
Escherichia  coli 17.6±0.0 16.0±0.0* 19.6±0.5* 17.0±1.0*
Salmonellatyphimurium 17.6±0.5 13.3±1.5 14.6±1.5 23.3±1.5*
Pseudomonas  aeruginosa 14.6±1.5 13.6±1.5 23.0±1.0* 19.3±2.0*
Enterococcus faecalis 13.0±1.0* 13.6±0.5 14.3±1.5 14.6±1.1
Enterococcus  faecium 12.3±1.1* 15.3±2.0 14.3±1.5 17.0±1.0*
Staphylococcus aureus 14.3±0.5 18.3±1.1* 18.0±1.7* 18.0±1.0*
Streptococcus agalactiae

(control)

17.0±0.5 15.6±0.5 13.3±1.5 12.3±0.5
Bacterial index 15.2 14.5 16.6 17.3

*: significant difference at p< 0.05

The effect of different water extracts of the 4 tested plants (250 µg/ml)on the growth of Lactobacillus acidophilus grown in MRS medium was determined each 2 hours up to 24 hours and compared to control medium, without plant extract (Figure 3). The used water extracts increase bacteria growth. In the control medium, the bacterial growth increased to 12 hours, then the rate of growth was decreased. The presence of plant extracts slightly decrease the rate of growth ofL. acidophilus and the decrease was clear in case of Camellia sinensis>Menthapiperita >Pimpinellaanisum>Petroselinumcrispum(Figure 3).

 

 

 

Tested

extract

Gut

bacteria

 Diameter of the inhibition zone (mm)
 Camellia

sinensis

Pimpinella

anisum

Petroselinum

crispum

Menthapiperita 
  Aqueous methanol Aqueous methanol Aqueous methanol Aqueous methanol
Lactobacillus acidophilus 5.3±0.1* 7.1±0.1* 7.0±1.5* 7.3±1.0* 7.3±0.5* 7.6±0.5* 7.3±0.1* 10.0±1.0*
Lactobacillus  bulgaricus 7.3±1.5* 11.1±1.0* 7.0±1.5* 12.3±1.0 7.3±1.5* 11.6±1.5 10.3±1.0* 14.0±1.5
Lactobacillus  plantarum 7.3±2.3* 11.0±0.5* 7.0±0.1* 12.4±1.3 7.3±2.3* 10.6±0.5* 11.0±1.0 13.3±1.3
Bifidobacterium 9.9±1.1* 11.6±1.5* 7.0±1.5* 12.6±1.0 9.9±1.1* 11.0±1.0 10.3±0.3* 13.9±1.5
Streptococcus thermophilus 9.0±0.5* 10.6±1.0* 9.0±1.2* 10.6±0.9 7.0±0.5* 11.3±1.5 11.0±0.3 14.0±0.2
Streptococcus agalactiae

(control)

11.6±0.5 17.0±0.5 11.6±0.7 15.6±0.5 14.3±1.0 13.3±1.5 13.3±1.5 12.3±0.5
Bacterial index 8.6 11.0 9.0 11.8 7.8 11.2 10.6 13.5

Table 4.The Antibacterial activity of the aqueous  andmethanolic extracts of the 4  tested plant extracts against some gut bacteria

*: significant difference at p< 0.05

 

 

Table 5:   MIC of the aqueous extracts of the 4  testedplant extracts against some tested bacterial pathogens and gut microbiota

Methanolic

extracts

M IC (µg/ml)
 Camellia

sinensis

Pimpinella

anisum

Petroselinum

crispum

Menthapiperita  Control antibiotic
Escherichia  coli 150 100 150 100 3
Salmonellatyphimurium 75 150 150 75 3
Pseudomonas  aeruginosa 75 150 150 125 3
Enterococcus faecalis 125 150 150 75 1
Enterococcus faecium 250 100 150 75 1
Staphylococcus aureus 50 100 150 125 2
Streptococcus agalactiae 50 100 150 75 2
Lactobacillus acidophilus 250 >250 >250 >250 <1
L. bulgaricus >250 >250 >250 >250 1
L. plantarum 250 >250 >250 >250 1
Bifidobacterium >250 >250 >250 >250 <1
Streptococcus thermophilus >250 >250 >250 >250 1

 

Figure 3: Effect of different water extracts on the growth of Lactobacillus acidophilus, grown in MRS medium after 24 hours.

 

Discussion

Intestinal normal flora are belonging to several major bacterial divisions, Firmicutes, Spirochaeates, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria (Rajilic-Stojanovicet al. 2007)  and flora composition  may be influenced by diet and stress (Mitsuoka, 1984). Previous investigations have shown that differences in intestinal bacteria cause many diseases (Hill, 1995). Plant extracts and/or probiotics are the most probable alternatives to antibiotics to the establishment of a beneficial intestinal population which antagonistic to harmful microbes (Gunalet al, 2006). Recently, much interest has focused on medicinal plants in relation to human health because they are largely free from harmful adverse effects and many plants are being investigated as natural sources of biologically important substances that may positively influence animal and human health (Aly and Bafeel, 2010, Alyet al., 2013). In this study, Camellia sinensis, Menthapiperita, Petroselinumcrispumand Pimpinellaanisum are traditionally used as drinks at least three times per day in Arabic region to improve human health. They are used to treat high cholesterol levels, cancer, rheumatoid and repair immune function due to the presence of powerful antioxidant e.gcatechin polyphenols especially epigallocatechingallate.  Menthapiperitahas many health and medicinal uses for thousands of years as a popular flavoring for food and drink.  The infusions prepared with peppermint leaves was used in complementary and alternative medical therapy include: biliary disorders, dyspepsia, enteritis, flatulence, gastritis, intestinal colic, and spasms of the bile duct, gallbladder and gastrointestinal tract (McKay and Blumberg, 2006). Parsley leaves and root are high in iron content and rich in vitamins A, B, C, trace minerals, boron and fluoride might help against bone thinning and osteoporosis. Aniseeds are used as flavouring, digestive, carminative, and relief of gastrointestinal spasms. Consumption of aniseed in lactating women increases milk and also reliefs their infants from gastrointestinal problems (Zargari, 1996).

The aqueous extracts of the four studied plants showed antibacterial activity against the tested bacterial pathogens with MIC values ranged from 50-250 µg/ml. Higher activities were recorded for the methanolic extracts for the  selected gut pathogens and probiotics, thus aqueous extracts were selected for more detail studies. Many others reported that extraction with organic solvents was more effective as compared to aqueous extraction and many previous studies reported that methanol was a better solvent for more consistent extraction of antimicrobial substances from medicinal plants as compared to other solvents such as water and ethanol (Ahmad et al., 1998, El Sayed and Aly, 2014).

 

The antimicrobial activity against bacterial pathogens was due to large number of active components in medical plants which may be effective against bacteria. Plants have several major components, including allicin, ajoene, thymol, and carvacrol, that have many biological activities e.g., antimicrobial, antioxidant, and antiseptic activities (Lee and Ahn, 1998) and the use of these material or the whole plant extract as food additives can decrease the number of intestinal pathogens (Pelicanoet al., 2005). Camellia sinensis  (Green tea) is a heterogeneous product, rich in various components such as caffeine, tannins, amino acids, vitamins and saponins,  and increase Intestinal microorganisms that participate in normal physiological functions and  decrease significantly  that cause various diseases (Ahnet al., 1990 b). Extracts of leaves from the tea plant Camellia sinensis contain polyphenolic components with activity against a wide spectrum of microbes (Ahnet al., 1990a). Studies conducted over the last 20 years have shown that the green tea polyphenoliccatechins   can inhibit the growth of a wide range of Gram-positive and Gram-negative bacterial species. They are useful in   control of common oral infections, such as dental caries and periodontal disease, the β-lactam resistant Staphylococcus aureus, methicillin-resistant S. aureus (MRSA). Catechingallates  from green tea intercalate into phopsholipid bilayers  and affect the bacterial cytoplasmic membrane functions.

In this study, no significant effects of the tested aqueous extracts were recorded against the microbial probiotics Lactobacillus and Bifidobacteriumand theMIC values were more than 250 µg/ml.The two previous benefit bacterial isolates may have an antagonistic effect against human pathogens, thus their protection from the plant extract action is very important. They are   important probiotics and used in the food industry.  They have a range of beneficial health effects, including the inhibition of harmful bacteria and pathogens, the modulation of systemic and local immune responses,   the vitamins production  and   improve the gut mucosal barrier. Many studies reported that the microbial probiotics have many beneficial effects (Gusilset al., 1999) including the competitive exclusion of pathogenic strains of Campylobacter jejuniMorishitaet al., 1997) and E. coli (Watkins et al., 1982).  In chickens, Lactobacillus acidophilus enhance the growth and viability of the other beneficial gut microflora (Hosoi et al., 2000), inhibit the pathogenic Escherichia coli and Salmonella entericaserovarEnteritidis (Pascualet al., 1999); and improved digestion and absorption of nutrients (Thomke and Elwinger, 1998).   Previous studies have shown that Lactobacillus rhamnosus (Alanderet al., 1999), Lactobacillus plantarum (West  and   Warner,  1988), Lactococcuslactis (Spelhaug and Harlander, 1989), and Pediococcuspentosaceus (Graham  and McKay. 1985)  inhibited the growth and development of Clostridium spp.    Teo and Tan   (2005) found five strains of lactic acid bacteria were antagonistic  toward C. perfringens ATCC 13124 without production of a zone of inhibition while two strains of Bacillus subtilis, PB3 and PB6, exhibited antimicrobial activity against C. perfringens ATCC 13124. Moreover, Noodles from red bean stimulated the growth of microflora in the large intestine to 109-1010cfu/ml but in the presence of plant extracts, garlic, onion and oregano, the count decreased to 108cfu/ml (Gumiennaet al., 2007).

Antimicrobial mechanisms of natural compounds found in herbs or spices have been discussed (Brul and Coot, 1999). Cinnamomumextract has excellent antibacterial activities and an inhibitory effects on the growth of enteric bacteria (E. coli O157:H7 and Salmonella typhimurium) due to prominent outer membrane disintegration activity and the increase in cytoplasmic membrane permeability to ATP (Helanderet al., 1998).  It has been reported that extracts from Panax ginseng   not only enhanced the growth of bifidobacteria, but also inhibited selectively various clostridia (Ahn, 1990 b)    Green tea extract has selective growth-inhibitory activity against various strains of clostridia including C. perfringens, C. dificileand C. paraputrijjicum (Ahnet al., 1990 a). They added that the daily intake of green tea might be expected to alter the growth and composition of the microbial community and to modulate the genesis of potentially harmful products such as carcinogenic N-nitroso compounds or aromatic steroids within the intestinal tract, thus protecting from a variety of diseases and helping to maintain optimal human health. In conclusion, the most common aqueous extracts of Camelliasinensis, Menthapiperita, Petroselinumcrispumand Pimpinellaanisumcan be used to inhibit gut pathogens without any bad effects on gut benefit bacteria, Lactobacillus and Bifidobacterium.

  Acknowledgements

This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant no. (54/247/1433), the author, therefore, acknowledge with thanks DSR technical and financial support.

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