Antibacterial activity of some bryophytes used traditionally for the treatment of burn infections

Abstract

Context: Plagiochasma appendiculatum L. & L. (Aytoniaceae), Conocephalum conicum (L.) Necker (Conocephalaceae), Bryum argenteum Hedw. (Bryaceae), and Mnium marginatum (With.) P. Beauv. (Mniaceae) are bryophytes (liverworts and mosses) used by traditional healers for the treatment of burn, cuts, wounds, and skin disorders.

Objective: This study evaluated the antibacterial activity of four bryophytes against some common bacteria responsible for burn infections.

Materials and methods: Different fractions of bryophytes were screened using the disc diffusion (qualitative) and broth microdilution (quantitative) methods, according to the guidelines of the National Committee for Clinical and Laboratory Standards.

Results and discussion: Chloroform fractions of liverworts were more active against Gram negative strains while butanol fractions of mosses had significant activity against Gram positive bacteria. Staphylococcus aureus was the most sensitive strain of those tested with the butanol fraction of M. marginatum (moss), with the strongest inhibition zone of 102.92% and minimum inhibitory concentration of 30 μg mL⁻¹.

Conclusion: Our findings support the use of the bryophytes in traditional medicine for burn infections because of their significant antibacterial activity.

Keywords::

• Antibacterial
• bryophytes
• burn
• National Committee for Clinical Laboratory Standards
• traditional healers

Introduction

Plagiochasma appendiculatum L. & L., Aytoniaceae, Conocephalum conicum (L.) Necker, Conocephalaceae (Liverworts), Bryum argenteum Hedw., Bryaceae, and Mnium marginatum (With.) P. Beauv., Mniaceae (Mosses) are bryophytes used by traditional healers for the treatment of boils, burns, cuts, eczema, skin disorders, and wounds and are also reported as an antimicrobial, antioxidant, antipyretic, and antidotal agent (Crum, 1973; Ding, 1982; Pant and Tewari, 1989; Zehr, 1990; Toyota and Asakawa, 1999; Kumar et al., 2000). The use of bryophytes in herbal medicines has been common in China, India, and among Native Americans since ancient times. Numerous compounds, including oligosaccharides, polysaccharides, sugar alcohols, amino acids, fatty acids, aliphatic compounds, prenylquinones, and aromatic and phenolic compounds occur in bryophytes, but few links have been made between any medical effects and specific bryophyte species or compounds as compared with vascular plants (Flowers, 1957; Huneck, 1983; Asakawa, 1984; Pant and Tewari, 1990).

Burn injury is a major concern in many parts of the world: approximately 75% of all deaths are reported to be due to infections. Burn wounds consisting of moist necrotic tissue represent an ideal culture medium for a wide variety of microorganisms (Altoparlak et al., 2004). The most common pathogens causing serious infection in burn patients are Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus mirabilis, and Clostridium spp. (Al-Akayleh, 1999; Lawrence, 1999; Gnanamani et al., 2003; Soares De Macedo and Santos, 2006).

Bryophytes (liverworts and mosses) are traditionally used in India by different cultural groups for burns, cuts, wounds, and skin diseases, suggesting that they protect the skin and open wounds from microbial pathogens (Pant & Tewari, 1990; Saxena & Harinder, 2004; Subhisha & Subramoniam, 2005). Some reports are available for the chemical constituents of these four bryophytes such as sesquiterpenoids and cyclic bis-bibenzyls (Toyota & Asakawa, 1999) isolated from P. appendiculatum; sesquiterpene lactones, lunularic acid, and flavonoids (apigenin, luteolin, etc.) (Connolly, 1994) isolated from C. conicum; and apigenin, apigenin-7-O-triglycoside, vitexin and luteolin-7-O-neohesperidoside, and saponarin (Basile et al., 1999) isolated from both B. argenteum and M. marginatum.

The main purpose of this study was to identify and obtain information on the bryophytes that are used by traditional healers in the management of burn infections.

Materials and methods

Plant material and extraction

The bryophytes, including liverworts and mosses, were collected during the month of September and October 2006, from different places of Uttaranchal, India. The plants were identified by Dr. Virendra Nath, and voucher specimens were deposited in the Bryophytes Herbarium at the National Botanical Research Institute, Lucknow. The voucher specimen numbers are LWG-226840, 247532, 228212, and 226950 for P. appendiculatum, C. conicum, B. argenteum, and M. marginatum, respectively. Air-dried and powdered materials were successively extracted by cold percolation in polar and non-polar organic solvents. The extracts were decanted, filtered with Whatman No. 1 filter paper, concentrated at reduced pressure below 40°C through a Rotavapor, and lyophilized (Labcanco, Kansas, MO). The extract yields are given in Figure 1.

Figure 1.  Yield percentage of different fractions of plant extracts.

Antimicrobial activity assays

Two different methods were used for the determination of antimicrobial activities: disc diffusion and microdilution assays as recommended by the National Committee for Clinical Laboratory Standards (CLSI/NCCLS, 2006). All tests were performed in triplicate.

Microbial strains

Bacillus cereus (MTCC, 430), Bacillus subtilis (MTCC, 121), Escherichia coli (MTCC, 443), Klebsiella pneumoniae (MTCC, 109), Pseudomonas aeruginosa (MTCC, 424), S. aureus (MTCC, 96), S. pyogenes (MTCC, 1927), Enterococcus faecalis (MTCC, 439), Enterobacter aerogenes (MTCC, 111), and P. mirabilis (MTCC, 1429) were used as test microorganisms. These test organisms were grown on Muller Hinton Broth (MHB; Oxoid, Basingstoke, UK) at 37°C for 6–12 h.

Inocula preparation

Stock bacterial inocula suspensions were obtained from the culture grown on MHB at 37°C for 6–12 h. These final suspensions are used for the inocula preparation. The cell density of each suspension was determined using a counting chamber adjusted to 0.5 McFarland turbidity at the concentration of 10⁵–10⁶ colony forming units mL⁻¹ by dilution with MHB, as per the NCCLS guidelines.

Antimicrobial assay by disc diffusion assay

The dried extracts and oil were dissolved in 2.5% dimethylsulfoxide to a final concentration of 30 mg mL⁻¹ and sterilized by filtration through 0.45-μm Millipore filters. The disc (6 mm in diameter; HiMedia, Mumbai, India) was impregnated with 10 μL of 30 mg mL⁻¹ crude and placed on seeded agar. Erythromycin (30 μg per disc) was used as a positive control, and test plates were incubated at 37°C for 18–24 h depending on incubation time required for visible growth. Antimicrobial activity was evaluated by measuring the zone of inhibition against test organisms with the help of Hi Antibiotic zone scale (HiMedia).

Standard microdilution method (NCCLS)

The test is performed in sterile U bottom 96-well plates by dispensing 95 μL of MHB and 5 μL of inoculum into each well (0.5 McFarland turbidity). Finally, 100 μL of test materials was added to appropriate well. The final volume in each well was 200 μL. A standard antibiotic, erythromycin (Sigma, St. Louis, MO), was used as the positive control. The plates were covered with sterile sealer and incubated at 37°C for 18–24 h. To indicate bacterial growth, 40 μL of 0.2 mg mL⁻¹ p-iodonitrotetrazolium violet (Sigma) solution was added to each well and incubated for further 30 min. Inhibition of bacterial growth was visible as a clear well and the presence of growth was detected by the presence of pink-red color. In this study, we tested concentration of fractions ranging from 10,000 to10 μg mL⁻¹ for mosses, 15,000 to10 μg mL⁻¹ for liverworts, and 1000 to10 μg mL⁻¹ for erythromycin.

Determination of minimum inhibitory and bactericidal concentrations

Inhibition of bacterial growth was visible as clear wells, and the presence of growth was detected by the appearance of pink-red color. The minimum inhibitory concentration (MICs) and the minimum bactericidal concentration (MBCs) of extract and standard drugs were determined by the microdilution method (well showing the minimum turbidity) and streaking a 5-μL sample of the same well onto an over-dried agar (MH) plate and then incubating at 37°C for 18–24 h.

Results and discussion

The antibacterial activity of hexane, chloroform, butanol, and methanol extracts of four bryophytes including two liverworts (P. appendiculatum and C. conicum) and two mosses (B. argenteum and M. marginatum) against common flora of burn infection tested in this study were qualitatively (disc diffusion) and quantitatively (microdilution) assayed (Tables 1 and 2). The results showed that almost all the extracts of these four different bryophyte species have antibacterial activity against most of the tested bacterial strains with the strongest being M. marginatum, followed by P. appendiculatum, B. argenteum, and C. conicum. In the case of the liverworts, the chloroform fraction showed significant activity, whereas in the case of mosses, the butanol fraction was most active. Most of the liverworts reportedly contain sesqui- and diterpenoids in major amount along with lipophilic aromatic compounds, which are mainly constituted in the oil bodies, whereas in mosses a variety of flavonoids have been isolated (Asakawa, 1981, 1990; Connolly, 1994; Basile et al., 1999).

Table 1.  Antibacterial activity* of some fractions (conc. 30 mg mL⁻¹) of Indian bryophytes.

Bacterial strains	Fractions	Pa	Ba	Mm	Cc
Gram positive
Staphylococcus aureus (MTCC, 96)	Hexane	37.73	43.48	42.85	–
	Chloroform	64.82	82.23	72.1	–
	Butanol	48.99	92.14	102.92	–
	Methanol	30.03	43.36	42.66	–
Escherichia coli (MTCC, 443)	Hexane	56.23	63.32	62.32	–
	Chloroform	72.43	92.48	86.48	–
	Butanol	43.28	100.23	92.39	–
	Methanol	21.12	–	23.62	–
Streptococcus pyogenes (MTTC,1927)	Hexane	40.58	–	48.32	–
	Chloroform	72.45	–	59.48	–
	Butanol	48.48	–	98.92	70.32
	Methanol	12.93	–	23.78	–
Bacillus subtilis MTCC (121)	Hexane	28.32	–	58.32	–
	Chloroform	58.43	–	57.82	–
	Butanol	48.43	–	92.42	–
	Methanol	–	–	–	–
Bacillus cereus MTCC (430),	Hexane	41.32	42.32	19.37	70.83
	Chloroform	60.87	58.82	43.42	75.0
	Butanol	53.07	69.48	80.57	47.2
	Methanol	–	–	–	66.6
Gram negative
Pseudomonas aeruginosa (MTCC 424)	Hexane	40.62	28.67	43.49	80.42
	Chloroform	92.52	41.33	53.48	120.32
	Butanol	72.43	52.66	80.28	72.49
	Methanol	19.37	17.33	12.37	32.47
Klebsiella pneumoniae MTCC (109)	Hexane	41.32	–	42.32	47.37
	Chloroform	87.38	30.69	53.72	83.89
	Butanol	56.42	81.33	72.82	70.02
	Methanol	21.33	45.66	–	36.62
Enterococcus faecalis (MTCC, 439)	Hexane	40.58	51.67	21.13	56.96
	Chloroform	82.42	71.33	28.17	80.19
	Butanol	48.45	92.43	53.28	60.19
	Methanol	12.93	–	–	39.06
Enterobacter aerogenes (MTCC, 111)	Hexane	43.48	43.42	43.28	46.15
	Chloroform	87.32	62.42	52.49	76.92
	Butanol	21.43	72.78	68.48	68.46
	Methanol	–	–	–	34.61
Proteus mirabilis (MTCC, 1429)	Hexane	40.03	23.29	78.72	58.62
	Chloroform	83.37	69.11	48.42	83.89
	Butanol	70.38	83.23	91.32	58.42
	Methanol	32.48	–	–	39.06

*Inhibition zone in percentages in relation to the standard drug erythromycin. Pa, Plagiochasma appendiculatum; Ba, Bryum argentium; Mm, Mnium marginatum; Cc, Conocephalum conicum; –, not determined.

Table 2.  MIC and MBC values of liverwort (chloroform) and mosses (butanol) fraction of Indian bryophytes (μg mL⁻¹).

Bacterial strains	Pa		Ba		Mm		Cc		Erythromycin
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
Gram positive
Staphylococcus aureus (MTCC, 96)	930	1870	70	150	30	70	–	–	90	180
Escherichia coli (MTCC, 443)	460	1870	30	70	70	150	–	–	40	90
Streptococcus pyogenes (MTTC,1927)	460	930	–	–	70	310	460	1870	90	180
Bacillus subtilis (MTCC,121)	1870	3750	–	–	150	310	–	–	20	40
Bacillus cereus (MTCC,430),	930	1870	620	1250	150	310	460	1870	20	40
Gram negative
Pseudomonas aeruginosa (MTCC, 424)	110	230	1250	2500	150	620	20	110	20	90
Klebsiella pneumoniae (MTCC, 109)	230	930	310	620	620	1250	230	1870	20	40
Enterococcus faecalis (MTCC, 439)	230	460	150	310	1250	2500	230	930	40	180
Enterobacter aerogenes (MTCC, 111)	460	930	620	1250	620	1250	460	930	90	180
Proteus mirabilis (MTCC, 1429)	230	460	150	310	150	310	110	460	10	20

MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; Pa, Plagiochasma appendiculatum; Ba, Bryum argentium; Mm, Mnium marginatum; Cc, Conocephalum conicum; –, Not determined.

The results indicate that Gram positive S. aureus was the most sensitive strain of those tested with the butanol fraction of M. marginatum (moss), with the strongest inhibition zone of 102.92% (in relation to standard drug erythromycin). The butanol fraction also exhibited significant antimicrobial activity against E. coli (106.84%), S. pyogenes (92%), B. cereus (92.48%), and B. subtilis (94.73 %). P. aeruginosa was found to be more sensitive among the Gram negative bacteria tested with an inhibition zone of 92.52 and 120.32% in chloroform fractions of liverworts (P. appendiculatum and C. conicum). Thus, the significance of the activity of liverwort extracts for medical application lies in its very selective inhibition of specific Gram negative bacteria, without affecting other bacteria, which is very rarely seen in higher plants and needs to be highlighted and further studied. The production of antibacterial substances is a wide-spread phenomenon in bryophytes, as different literature data have shown (Asakawa, 1982a, 1982b).

The MIC values were also recorded for the bacterial strains that were found to be most sensitive to the extracts in disc diffusion assay. There are no validation criteria for MIC and end points for in vitro testing of plant extracts. Based on the above observation, all bryophyte extracts showed strong activity except P. appendiculatum (1870 μg mL⁻¹), B. argenteum, and M. marginatum (1250 μg mL⁻¹) against B. subtilis, P. aeruginosa, and E. faecalis.

This study shows that the information obtained from traditional healers can lead to discovery of therapeutically useful antibacterial agents against common bacteria responsible for burn infection. As infection is a major cause of morbidity and mortality in burn patients, these bryophyte extracts may prevent infection that could lead to high risk of sepsis, and thereby prevent the prolongation of the inflammatory phase. Thus, the results of this study suggest a fairly good correlation between traditional therapeutic use and in vitro antibacterial activity.

Conclusions

Bryophytes including liverworts and mosses that are traditionally used in the treatment of burn infections or related ailments could be a good source for new, safe, biodegradable, and renewable antimicrobial agents.

Acknowledgment

The authors are grateful to the Director of the National Botanical Research Institute for his kind support and encouragement.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.