Antibacterial activity of resin rich plant extracts.

BACKGROUND: The in vitro antibacterial activity of resin rich methanolic extracts (RRMEs) of Commiphora myrrha, Operculina turpethum, and Pinus roxburghii.
MATERIALS AND METHODS: Different concentration were studied by agar-well diffusion method against Gram-positive (Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Enterococcus faecalis) and Gram-negative bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae).
RESULTS: Among all the bacterial strains tested, E. faecalis was most sensitive and S. typhi was resistant to C. myrrha and P. roxburghii. The extracts of O. turpethum were active against all tested strains in which B. subtilis and S. aureus were the most sensitive.
CONCLUSION: This suggested that the antibacterial activity of RRMEs of O. turpethum was more than C. myrrha and P. roxburghii. This probably explains the potential of these plants against a number of infections caused by bacterial strains tested.

Finding healing powers in plants is an ancient idea. It is estimated that there are 250,000-500,000 species of plants on the earth. A relatively low percentage of these are used as foods by both human and other animal species.[1] Plants and plant products have been in existence for 1000s of years almost from the beginning of human civilization and continue to provide mankind with the new remedies. Medicinal plants have importance as well as great economic status around the globe. Multiple drug resistance has occurred consequently due to inappropriate use of commercially available antibacterial drugs that lead to repeated use of antibiotics and insufficient control of the disease.[2] At the same time, patients come across unavoidable antibiotic associated adverse effects. This leads to the development of alternative medicines from plants for the prevention and treatment of bacterial infections.[3456] Phytoconstituents showed enormous therapeutic potentials and minimal or lower side-effects as compared to synthesized drugs. Phytoconstituents such as alkaloids, tannins, polyphenols, quinines, flavonoids, coumarins, terpenoids, lectins, and polypeptides are responsible for activity concern.[78] Plants showed their useful properties such as anticancer, anti-tumor, anti-mutagenic, antioxidant, hepato-protective, antiviral, antimalarial, anti-dysenteric, antiseptic, anti-stress, immunotherapeutic, antibacterial, antifungal, and several more pharmacological actions.[9] Natural resins are known for their antiseptic and antibacterial effects since from ancient history of human civilization. However, fewer reports are available regarding resins and resin containing plants for antibacterial activity. Resins are complex mixtures of resin acids, resin alcohols, resin phenols, and their esters. Resins associated with volatile oils are oleoresins, with gums are gum-resins or with oil and gum are oleo-gum-resins. These products are usually contained in schizogenous or schizolysigenous ducts or cavities.[1011] Commiphora myrrha has many medicinal powers and has been used to treat various diseases, such as fever, ache, amenorrhea, tumors, stomach complaints, chest ailments, and skin infections in ancient India, China, and Greece.[1213] The characteristic constituents of oleo-gum-resins of C. myrrha are furanosesquiterpenes such as furanoelemanes, furanoeudesmanes, and furanogermacranes.[1415] The crude extracts and some constituents of the C. myrrha exhibited diverse biological activities, such as cytotoxic, anaesthetic, anti-inflammatory, antioxidant, and antimicrobial effects.[16171819] In Indian traditional system of medicine, Operculina turpethum is used internally to treat fevers, edema, anemia, constipation, hepatitis, ulcers, skin disorders, obesity, hemorrhoids, cough, asthma, paralysis, gout, and rheumatism.[202122] It is proved to have anti-secretory and ulcer protective, anti-inflammatory,[23] hepatoprotective,[24] anti-microbial,[25] anticancer, and antioxidant activities.[26] Glycosidic resins of O. turpethum constitutes terpethin, α-terpethein and β-terpethein, turpethic acids A-C, and two intact resin glycosides, turpethosides A and B, jalapinolic acid, and operculinolic acid.[2728] Pinus roxburghii oleo-resin mainly consists of α and β-pinene, Δ-3-carene, longifolene, abietic acid and isopimaric acid and have been used as antiseptic, expectorant, carminative, antioxidant, anthelmintic, and analgesic.[2930] The main objective of the work was to screen and evaluate antibacterial activity of methanolic extracts of C. myrrha (oleo-gum-resin), O. turpethum (glycosidic resin), P. roxburghii (oleo-resin) against both multidrug resistance Gram-positive as well as Gram-negative bacteria.

Materials and Methods

Plant materials

The C. myrrha (oleo-gum-resin), O. turpethum (glycosidic resin) and P. roxburghii (oleo-resin) were procured from local market of Delhi and identified by Dr. H.B. Singh, Scientist F and Head, Raw Materials Herbarium, and Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. Voucher specimens (Nos. N/R/C/06-07/803/120; National Research Council (NRC)/-2007/08/842/26; N/R/C/-R007/08/851/35) were deposited in the NISCAIR, New Delhi.

Preparation of methanolic extracts

The dried material of C. myrrha, O. turpethum and P. roxburghii were cleaned, and grounded into a coarse powder by a Multi-Mill and passed through a sieve (20 mesh). Dried plant samples were further air-dried in a ventilated oven at 45°C for 24 h, then grounded into a coarse powder and passed through a sieve as above. Each Powdered sample (100 g) was extracted separately with methanol (500 ml) for 12 h using Soxhlet apparatus. The methanolic extracts were filtered by a Millipore filter with a 0.45 μm nylon membrane under vacuum. The filtrates were concentrated under reduced pressure and freeze dried. The yield of methanolic extract of C. myrrha, O. turpethum and P. roxburghii was 7.4 g, 8.33 g and 93 g, respectively. The samples were stored at 4°C until use.

Microbial cultures and growth conditions

Cultures of the Gram-positive and Gram-negative bacteria were kindly provided by ITL LABS Pvt. Ltd, New Delhi. Gram-positive bacteria were Staphylococcus aureus (American Type Culture Collection (ATCC) 6538), Bacillus subtilis (ATCC 6633), Micrococcus luteus (MTCC 2470), Enterococcus faecalis (Microbial Type Culture Collection (ATCC) 439). Gram-negative bacteria were Escherichia coli (MTCC 452), Pseudomonas aeruginosa (ATCC 25619), Salmonella typhi (ATCC 786), Shigella dysenteriae (MTCC 1457). Cultures of bacteria were grown on a nutrient broth at 37°C for 12 h and were maintained on respective agar slants at 4°C.

Antimicrobial assay by agar diffusion method

Antimicrobial activity of resin rich methanolic extracts (RRMEs) was evaluated by agar diffusion method.[31] The freeze dried RRMEs of tested plants in concentration 100, 250, 500, and 1000 μg/ml were prepared in phosphate buffered saline (Phosphate Buffer Solution, pH 7.0) and sterilized by filtration through 0.22 μm sterilizing Millipore express filter. All bacteria were suspended in sterile water and diluted to ~106 Colony Forming Unit (CFU)/ml. The suspension (100 μl) was spread onto the surface of plate count agar medium. Wells (4.6 mm in diameter) were cut from the agar with a sterile borer and extract solutions (60 μl) were delivered into them. Negative controls were prepared using PBS solution. Tetracycline (30 μg/well) and streptomycin (30 μg/well) were used as a positive reference standards to determine the sensitivity of Gram-positive and Gram-negative bacterial species tested, respectively. The inoculated plates were incubated at 35°C for 24 h. Antibacterial activity was evaluated by measuring the diameter of inhibition zone of the tested bacteria, which was expressed in millimeters.

Results and Discussion

The RRMEs of C. myrrha, O. turpethum and P. roxburghii were tested in different concentration for antibacterial effect against Gram-positive (S. aureus, B. subtilis, M. luteus, E. faecalis) and Gram-negative bacterial strains (E. coli, P. aeruginosa, S. typhi, S. dysenteriae). When tested by agar diffusion method the results obtained in the study showed that the RRMEs of C. myrrha showed significant activity against all Gram-positive bacterial strains while variable effects against Gram-negative bacterial strains [Table 1 and Figure 1]. The RRMEs of C. myrrha (100 μg/ml) exhibited maximum inhibition against P. aeruginosa that measured and inactive against E. coli and S. typhi. RRMEs of C. myrrha (250 μg/ml) showed maximum zone of inhibition against E. faecalis (12.9 mm) while inactive against S. typhi. Higher concentration of RRMEs of C. myrrha 500 μg/ml and 1000 μg/ml observed maximum activity against E. faecalis, which were 15.3 and 17.5 mm, respectively and minimum against S. typhi, which were 4.6 and 8.1 mm, respectively. The RRMEs of O. turpethum showed significant activity against all strains of both Gram-positive and Gram-negative bacteria at different concentrations [Table 2 and Figure 2]. The RRMEs of O. turpethum (100 μg/ml) observed maximum activity against B. subtilis (12.3 mm) and minimum against S. dysenteriae (6.0 mm). The RRMEs of O. turpethum (250 μg/ml) exhibited maximum activity against P. aeruginosa (15.9 mm) and B. subtilis (15.8 mm) while the minimum against M. luteus measured 7.8 mm. On increasing the concentration the RRMEs of O. turpethum 500 μg/ml and 1000 μg/ml showed the highest zone of inhibition toward B. subtilis (17.1 mm) and (18.4 mm), respectively and minimum against M. luteus (10.8 mm) and E. coli (14.5 mm), respectively when compared to other tested bacterial strains. The RRMEs of P. roxburghii exhibited better activity against Gram-positive bacteria than Gram-negative bacterial strains in a concentration dependent manner [Table 3 and Figure 3]. The RRMEs of P. roxburghii (100 μg/ml) did not exhibited inhibition against E. coli, P. aeruginosa and S. typhi, while in concentration (250 μg/ml) showed significant inhibition in S. aureus (12.3 mm), M. luteus (12.1 mm) and E. faecalis (12.3 mm) on comparison with standard. The RRMEs of P. roxburghii 500 μg/ml and 1000 μg/ml exhibited maximum inhibition against E. faecalis (15.6 mm) and (18.4 mm) respectively while the minimum against S. typhi measured 3.5 mm and 6.8 mm, respectively.

Table 1

Antibacterial activity of RRMEs of Commiphora myrrha against Gram-positive and Gram-negative bacteria

Figure 1

Zones of inhibition produced by resin rich methanolic extracts of Commiphora myrrha at the tested concentrations (Bl = control; S = standard; A = 1000 μg/ml; B = 500 μg/ml; C = 250 μg/ml; 100 μg/ml)

Table 2

Antibacterial activity of RRMEs of Operculina turpethum against Gram-positive and Gram-negative bacteria

Figure 2

Zones of inhibition produced by resin rich methanolic extracts of Operculina turpethum at the tested concentrations (Bl = control; S = standard; A = 1000 μg/ml; B = 500 μg/ml; C = 250 μg/ml; 100 μg/ml)

Table 3

Antibacterial activity of RRMEs of Pinus roxburghii against Gram-positive and Gram-negative bacteria

Figure 3

Zones of inhibition produced by resin rich methanolic extracts of Pinus roxburghii at the tested concentrations (Bl = control; S = standard; A = 1000 μg/ml; B = 500 μg/ml; C = 250 μg/ml; 100 μg/ml)

In the present observations, C. myrrha and P. roxburghii extracts showed maximum activity against E. faecalis while the minimum against S. typhi. The extracts of O. turpethum were active against both Gram-positive and Gram-negative bacteria in which B. subtilis and S. aureus were the most sensitive. The maximum sensitivity of Gram-positive bacterial strains toward plant extracts may be due to its cell wall structure and outer membrane.[32] Gram-negative bacteria have an outer membrane and a unique periplasmic space, which is absent in Gram-positive bacteria.[3334] In Gram-negative bacteria, the hydrophilic surface of outer membrane, which is rich in lipopolysaccharide molecules, providing a barrier against various antibiotics and is also associated with the enzymes that present in the periplasmic space capable of breaking down the molecules introduced from outside.[353637] Gram-positive bacteria do not have such a protective outer membrane and cell wall configuration. Antibacterial substances cause destruction of the cytoplasmic membrane that result in a leakage of the cytoplasm and its coagulation.[3138] This study suggested that the RRMEs of C. myrrha and P. roxburghii were more potent against Gram-positive than Gram-negative bacterial strains while RRMEs of O. turpethum were active against all tested microbial strains. The antibacterial activity of RRMEs of O. turpethum against both Gram-positive and Gram-negative bacteria may be due to its glycosidic resins.

Conclusion

Our results allow us to conclude that the RRMEs of C. myrrha, O. turpethum and P. roxburghii exhibited some degree of antimicrobial activity against Gram-positive (S. aureus, B. subtilis, M. luteus, E. faecalis) and Gram-negative bacterial strains (E. coli, P. aeruginosa, S. typhi, S. dysenteriae). This probably explains the use of these plants by the indigenous people against a number of infections since generations. The comparable activity of RRMEs to some antibiotics may help to discover new chemical classes of antimicrobials that may be used for the topical treatment of disorders resulted from tested microbial strains. Further research should be highlighted on exploring the phytochemicals and medicinal worth of above tested plants.