Ethnobotanical Leaflets 14: 84- 94, 2010.

 

 

Antimicrobial Properties of Selected Indian Medicinal Plants Against Acne-Inducing Bacteria

 

*Harisaranraj, R., S. Saravana Babu and Suresh, K.

 

*Department of Plant Biology and Plant Biotechnology

Chikkaiah Naicker College, Erode. (Tamil Nadu) INDIA

*hari83biotech@yahoo.com

 

Issued: January 01, 2010

 

Abstract

 

Propionibacterium acnes and Staphylococcus epidermidis have been recognized as pus-forming bacteria triggering an inflammation in acne. The present study was conducted to evaluate antimicrobial activities of Indian medicinal plants against these etiologic agents of acne vulgaris. Ethanolic extracts of Rauwolfia serpentina (roots), Piper nigrum (seeds), Azadirachta indica (leaves), Cardiospermum halicacabum (leaves), Momordica charantia (fruits), Casuarina equisetifolia (fruits), Cynodon dactylon  (leaves), Ficus religiosa (leaves), Euphorbia hirta (roots), Ocimum sanctum (leaves), Phyllanthus niruri (Whole plant), and Jasminum sambac (flowers) were tested for antimicrobial activities by agar well diffusion and broth dilution methods. The results from the disc diffusion method showed that 07 medicinal plants could inhibit the growth of Propionibacterium acnes. Among those Azadirachta indica, Momordica charantia, Casuarina equisetifolia, Rauwolfia serpentina, Cardiospermum halicacabum, Phyllanthus niruri and Piper nigrum had strong inhibitory effects. Based on a broth dilution method, the Rauwolfia serpentina extract had the greatest antimicrobial effect. Piper nigrum and Momordica charantia showed outstanding antimicrobial properties against Propionibacterium acnes based on the agar well diffusion assay, each had a MIC value of 2.8 and 0.862 mg/ml and a MBC of 2.45 and 0.461 mg/ml for Propionibacterium acnes respectively. In bioautography assay, the Rauwolfia serpentina extract produced strong inhibition zones against Propionibacterium acnes. Phytochemical screening of Rauwolfia serpentina revealed the presence of alkaloid which could be responsible for activity. Taken together, our data indicated that Rauwolfia serpentina had a strong inhibitory effect on Propionibacterium acnes and Staphylococcus epidermidis.

 

Keywords: Acne; Propionibacterium acnes; Staphylococcus epidermidis; Antimicrobial activity

 

 

Introduction

            Acne vulgaris is a common skin condition, caused by changes in the pilosebaceous units, skin structures consisting of a hair follicle and its associated sebaceous gland via androgen stimulation. It is characterized by non inflammatory follicular papules or comedones and by inflammatory papules, pustules, and nodules in its more severe forms. Acne vulgaris affects the areas of skin with the densest population of sebaceous follicles; these areas include the face, the upper part of the chest, and the back. Severe acne is inflammatory, but acne can also manifest in non inflammatory forms. Acne is most common during adolescence, affecting more than 85% of teenagers, and frequently continues into adulthood. Acne affects areas containing the largest oil glands, including the face, back, and trunk (Leyden 1997). It is generally characterized by formation of seborrhea, comedone, inflammatory lesions and presence of bacteria Propionibacterium acnes, Staphylococcus epidermidis and Malassezia furfur in the follicular canal and sebum production (Leyden 2001). Propionibacterium acnes have been described as an obligate anaerobic organism. It is implicated in the development of inflammatory acne by its capability to activate complements and by its ability to metabolize sebaceous triglycerides into fatty acids, which chemotactically attract neutrophils. On the contrary, Staphylococcus epidermidis, an aerobic organism, usually involves in superficial infections within the sebaceous unit (Burkhart et al., 1999). These factors provide a potential target for treatment. Propionibacterium acnes and Staphylococcus epidermidis are the target sites of antiacne drugs (Leyden 2001 and Hamnerius  1996).

 

            Long term use of antibiotics against acne is outdated because of exacerbated antibiotic resistance (Degroot et al., 1998 and Swanson 2003). The development of antibiotic resistance is multifactorial, including the specific nature of the relationship of bacteria to antibiotics, how the antibacterial is used, host characteristics, and environmental factors. To overcome the problem of antibiotic resistance, medicinal plants have been extensively studied as alternative treatments for diseases. In the present study, 12 medicinal plants, which have been traditionally used as antimicrobial and anti-inflammatory agents were examined for antimicrobial activities against microorganisms frequently involved in acne inflammation, Propionibacterium acnes and Staphylococcus epidermidis.

 

Materials and Methods

Plant material

        The 12 plant materials used in this study were collected from various locations in Tamil nadu, India. The plant was identified with the Herbarium of Botanical Survey of India, Southern Circle, Coimbatore, India (Accession No. 32561-65)

 

Microorganisms and media

      The test organisms used in this study were as followed: Propionibacterium acnes (MTCC 1951) and Staphylococcus epidermidis (MTCC 931). These bacteria were obtained from the Microbial Type Culture Collection and Gene Bank, Chandigarh, India All media were purchased from Himedia.

 

Preparation of plant extracts

      Dried parts of the plants were made into fine powder. 400 g Rauwolfia serpentina (roots, 15.2% w/w), Piper nigrum (seeds, 14.1% w/w), Azadirachta indica (leaves, 25.4% w/w), Cardiospermum halicacabum (leaves, 20.9% w/w), Momordica charantia (fruits, 5.2% w/w), Casuarina equisetifolia (fruits, 10.9% w/w), Cynodon dactylon  (leaves, 15.9.1% w/w), Ficus religiosa (leaves, 21.0% w/w), Euphorbia hirta (roots, 9.2% w/w), Ocimum sanctum (leaves, 15.4% w/w), Phyllanthus niruri (Whole plant, 19.6% w/w), and Jasminum sambac (flowers, 15.5% w/w) were macerated in ethanol. The macerate was filtered after seven consecutive days, filtrate was dried under reduced pressure and finally under vaccum desicator.

 

Antimicrobial susceptibility testing Agar well diffusion method

        This experiment was performed by the method of (Bauer AW et al., 1966) with some modifications. Propionibacterium acnes was incubated in brain heart infusion medium (BHI) with 1% glucose for 48 h under anaerobic conditions and adjusted to yield approximately 1.0×108 CFU/ml. Aliquots of molten BHI with glucose agar were used as the agar base. A prepared inoculum was added to the molten agar, mixed, poured over the surface of the agar base and left to solidity. For agar well diffusion method, a well was prepared in the plates with the help of a cork-borer (0.85 cm). 100 µl of the test compound was introduced into the well. The plates were then incubated at 370C for 48 h under anaerobic conditions in an anaerobic jar (Hi-Media) with gas pack and indicator strip and the jar was kept in an incubator for 48 h at 37 ± 10C. Gas packs containing citric acid, sodium carbonate and sodium borohydride were used to maintain and check the anaerobiosis, where citric acid releases carbon dioxide and sodium borohydride releases hydrogen when they come in contact with oxygen. An indicator strip of methylene blue, when introduced into the jar, changes in colour from white to blue in the absence of anaerobiosis. Staphylococcus epidermidis was incubated in tryptic soy broth (TSB) for 24 h at 370C and adjusted to yield approximately 1.0×108 CFU/ml. The procedures were the same as mentioned above except the plates were incubated at 370C for 24 h under aerobic conditions. All agar well diffusion tests were performed in three separate experiments and antibacterial activity was expressed as the mean of inhibition diameters (mm) (see Table 1).

 

Determination of minimum inhibitory and bactericidal concentrations

        The minimal inhibitory concentration (MIC) values were determined by broth dilution assay (Isao Kubo et al., 1994, Sahin et al., 2003 and Kumar  et al., 2004). The cultures were prepared at 24 h and 48 h broth cultures of Staphylococcus epidermidis and Propionibacterium acnes, respectively. The MIC was defined as the lowest concentration of the compound to inhibit the growth of microorganisms. 3ml of the Nutrient yeast glucose broth (NYG) for Propionibacterium acnes, and Nutrient broth for Staphylococcus epidermidis, in 10 ml glass screw cap test tube was sterilised by autoclaving at 1210C for 15min. The medium was cooled and inoculated with 50 μl of the bacterial suspension containing 1 x 108 cells/ml. 1 ml of the plant extracts (100mg/ml) was added to corresponding test tubes under anaerobic condition. 3 ml of NYG broth inoculated with 50μl of organisms was taken as positive control. The test tubes were then kept in anaerobic jar (Hi- Media) with gas pack and indicator strip and the jar was kept in incubator for 48 h at 37 + 10C. Gas packs containing citric acid, sodium carbonate and sodium borohydride were used to maintain and check the anaerobiosis, where citric acid releases carbon dioxide and sodium borohydride releases hydrogen when they come in contact with oxygen. An indicator strip of methylene blue, when introduced into the jar, changes in colour from white to blue in the absence of anaerobiosis. For Staphylococcus epidermidis, the test tubes were incubated at 37 ± 10C for 24 h aerobically and growth of Propionibacterium acnes and Staphylococcus epidermidis was measured as a function of turbidity at 660 nm using (Systronics 131) Nephaloturbidometer. The MIC and MBC values of 12 medicinal plant extracts against Propionibacterium acnes and Staphylococcus epidermidis were determined. The results are shown in Table 2 as average values from three separate experiments per medicinal plants and indicate the susceptibility of bacteria to the medicinal plant extracts.

 

Phytochemical screening (Ravishankara  et al., 2002)

       The ethanolic extract was subjected to preliminary phytochemical testing for the detection of major chemical groups. The details of the tests are as follows:

 

1. Phenols

            The ethanolic extract was spotted on a filter paper. A drop of phoshomolybdic acid reagent was added to the spot and was exposed to ammonia Vapours (Blue coloration of the spot indicates the presence of phenols).

 

2. Braemer’s test for tannins

            To a 2–3 ml of ethanolic extract, 10% alcoholic ferric chloride solution was added. (Dark blue or greenish grey coloration of the solution indicate the presence of tannins in the drug).

 

3. Liebermann-Burchardt test for steroids and terpenoids

            To 1 ml of ethanolic extract of drug, 1 ml of chloroform, 2–3 ml of acetic anhydride and 1 to 2 drops of concentrated sulfuric acid were added. (Dark green coloration of the solution indicate the presence of Steroids and dark pink or red coloration of the solution indicate the presence of terpenoids).

 

4. Alkaloids

            A drop of ethanolic extract was spotted on a small piece of precoated TLC plate and the plate was sprayed with modified Dragendorff’s reagent. (Orange coloration of the spot indicates the presence of alkaloids).

 

5. Borntrager’s test for anthraquinones

            About 50 mg of ethanolic extract was heated with 10% ferric chloride solution and 1 ml of concentrated hydrochloric acid. The extract was cooled, filtered and the filtrate was shaken with diethyl ether. The ether extract was further extracted with strong ammonia. (Pink or deep red coloration of aqueous layer indicate the presence of anthraquinones).

 

6. Shinoda test for flavonoids

            To 2–3 ml of ethanolic extract, a piece of magnesium ribbon and 1 ml of concentrated hydrochloric acid were added. (Pink red or red coloration of the solution indicate the presence of flavonoids in the drug).

 

Bioautography

            Bioautography was performed with bacterial cultures exhibiting high sensitivity to the extracts. Developed TLC plates were carefully dried for complete removal of solvent, overlaid with agar containing an aliquot of an overnight culture and incubated at 37 ◦C. The plates were run in duplicate; one set was used as the reference chromatogram and the other was used for bioautography.

 

Results

            In the present study, 12 medicinal plant extracts were examined for antimicrobial activity against Propionibacterium acnes and Staphylococcus epidermidis. The results showed that 07 extracts could effectively inhibit the growth of Propionibacterium acnes. Among these, ethanolic extracts of Azadirachta indica, Momordica charantia, Casuarina equisetifolia, Rauwolfia serpentina, Cardiospermum halicacabum, Phyllanthus niruri and Piper nigrum showed strong inhibitory effects (Table 1). Interestingly, Azadirachta indica, Cardiospermum halicacabum, Momordica charantia and Piper nigrum  extracts showed promising antibacterial activities against both Propionibacterium acnes and Staphylococcus epidermidis. The remaining 5 plant extracts had considerable activity against both microorganisms. Subsequent experiments were conducted to determine inhibitory concentrations of all selected plant extracts. Rauwolfia serpentina showed the potent antimicrobial effect. The MIC and MBC values against both organisms were more than 5 (Table 2). Further, the plant extracts was subjected to preliminary Phytochemical screening for the presence and absence of different chemical groups (Table 3).

 

 

Table 1: Antimicrobial Activity of Medicinal Plant Extracts.

 

Plant extracts

Susceptibility of bacteria to medicinal plant extracts

Zone of inhibition (mm)a*

 

Propionibacterium acnes

Staphylococcus epidermidis

Rauwolfia serpentina

18

20

Azadirachta indica

15

16

Cardiospermum halicacabum

16

17

Momordica charantia

15

17

Casuarina equisetifolia

13

12

Cynodon dactylon

11

09

Euphorbia hirta

10

11

Ficus religiosa

08

09

Ocimum sanctum

13

12

Jasminum sambac

10

11

Phyllanthus niruri

14

16

Piper nigrum

15

14

Clindamycin

22

24

aConcentration of the extract used: 100 mg / ml, Clindamycin: 100 μg / ml

*Mean of triplicate measurements

 

 

Table 2: The MIC and MBC values of 12 medicinal plant extracts against Propionibacterium acnes

and Staphylococcus epidermidis. The results are shown as mean of triplicate measurements.

 

Plant extracts

Susceptibility of bacteria to medicinal plant extractsa

 

Propionibacterium acnes

Staphylococcus epidermidis

 

MIC (mg/ml)

MBC (mg/ml)

MIC (mg/ml)

MBC (mg/ml)

Rauwolfia serpentina

>5

>5

>5

>5

Azadirachta indica

0.046

0.025

0.265

0.215

Cardiospermum halicacabum

1.006

1.013

0.524

0.212

Momordica charantia

0.862

0.799

0.461

0.488

Casuarina equisetifolia

0.26

0.28

0.325

0.354

Cynodon dactylon

0.541

0.365

0.159

0.357

Euphorbia hirta

1.011

0.962

0.451

0.265

Ficus religiosa

0.538

0.462

0.268

0.164

Ocimum sanctum

1.064

1.068

1.344

0.542

Jasminum sambac

0.132

0.145

0.212

0.253

Phyllanthus niruri

>4

>4

2.1

2.3

Piper nigrum

2.8

2.9

2.45

2.81

Clindamycin

76

80

72

69

aThe results indicate of average of 3 separate experiments

*Clindamycin- All values are in μg/ml

 

Discussion

            The similar values of MIC and MBC obtained from this plant against Propionibacterium acnes suggest that the ethanolic extract of Rauwolfia serpentina could possibly act as a bactericidal agent to this microorganism. In addition, the Cardiospermum halicacabum extract also showed good antimicrobial effects against Propionibacterium acnes with a MIC of 1.006 mg/ml but a high concentration was required to kill both Propionibacterium acnes and Staphylococcus epidermidis as compared to the ethanolic extract of Rauwolfia serpentina. Piper nigrum and Momordica charantia showed outstanding antimicrobial properties against Propionibacterium acnes based on the agar well diffusion assay, each had a MIC value of 2.8 and 0.862 mg/ml and a MBC of 2.45 and 0.461 mg/ml for Propionibacterium acnes respectively. The plant extracts were further analyzed by phytochemical screening for detection of phytoconstituents. The assay for bioautography demonstrated strong inhibition zones of Rauwolfia serpentina extract against the growth of Propionibacterium acnes.

 

Table 3: Preliminary phytochemical screening of 12 medicinal plant extracts.

 

Plant extracts

Phenols

Tannins

Steroids

Alkaloids

Glycosides

Flavonoids

Terpenoids

Rauwolfia serpentina

+++

-

+

+++

++

++

-

Azadirachta indica

+

++

+++

++

++

+

-

Cardiospermum halicacabum

+

+

+++

+

++

++

+

Momordica charantia

++

+

++

++

+++

++

+

Casuarina equisetifolia

++

++

++

+

++

+

++

Cynodon dactylon

++

+

+

++

+++

++

++

Euphorbia hirta

+++

++

++

++

+

-

+

Ficus religiosa

-

-

++

++

+

-

++

Ocimum sanctum

++

++

+

++

++

-

-

Jasminum sambac

-

-

++

++

+

-

-

Phyllanthus niruri

++

++

++

-

-

++

++

Piper nigrum

+++

++

++

+++

-

+

+

– = absent; + = Trace; ++ = moderate, +++ = Abundant

           

       The clear zones were located in separate places on the TLC plate, suggesting that more than one compound possessed an antimicrobial effect. There was no inhibition zones presented above the bands of the other plant extracts covered with Propionibacterium acnes. This implied that the strongest effect of the Rauwolfia serpentina extract was against Propionibacterium acnes. Phytochemical screening of Rauwolfia serpentina extract showed positive results for the presence of alkaloids. Alkaloid and its derivatives have activities against Staphylococcus aureus and methicillin-resistant S. aureus (Valsaraj R et al., 1997). The mechanism of action of highly aromatic planar quaternary alkaloids such as berberine and harmane (Hopp et al., 1976) is attributed to their ability to intercalate with DNA (Phillipson et al., 1987). It is possible that berberine an alkaloid present in Coscinium fenestratum may act in the same mechanism to inhibit Propionibacterium acnes and Staphylococcus epidermidis. Therefore, the active component of the Coscinium fenestratum extract could be of interest for further development as an alternative treatment for acne.

 

 

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