Evaluation of Hydrogen peroxide Scavenging and Antibacterial activity of successive extracts of Luisia tenuifolia Blume against skin and wound infections

 

Sakthipriyadarsini S, Kumar PR*

Department of Pharmacognosy, SRM College of Pharmacy, Faculty of Medicine and Health Sciences, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur - 603203, Kanchipuram, Chennai TN, India.

*Corresponding Author E-mail: sakthivendan@gmail.com

 

ABSTRACT:

Skin and wound infections might trigger severe secondary complications when left untreated. A proper balance between reactive oxygen species generation and antioxidant formation is necessary to achieve redox haemostasis. Luisia tenuifolia Blume (Orchidaceae) has been traditionally applied on cuts, boils and wounds in regions of Western Ghats. The present study was aimed to validate the antioxidant and antibacterial effect of the whole plants of Luisia tenuifolia against five pathogenic bacteria often responsible for skin and wound infections The whole plants of L.tenuifolia, after drying under shade, was pulverised, successively extracted with solvents of different polarity and evaporated to dryness. The hydrogen peroxide scavenging potential of all the four extracts was determined by H2O2 assay.  The agar-well diffusion technique was employed for the preliminary screening of antibacterial activity. Further, the minimum inhibitory concentration was determined by broth-dilution assay. Among the four extracts, the ethanol extract of L. tenuifolia exhibited a strong antioxidant activity with an IC50 value of 42.61μg/mL compared to the standard Ascorbic acid (IC50 value of 21.56μg/mL). Moreover, the n-hexane extract was found to be more effective against Staphylococcus aureus whereas the ethanol extract exhibited highest antibacterial effect on Streptococcus oralis, Pseudomonas aeruginosa, Aeromonas hydrophila and Escherichia coli. Furthermore, the ethanol extract was found to be a promising antibacterial candidate with an MIC value of 0.312μg/mL against S.oralis and A.hydrophila. Thus the current study validate the antioxidant and antibacterial potential of L. tenuifolia whole plant extracts and further studies were aimed at identification of active components responsible for the activity.

 

KEYWORDS: Antibacterial, hydrogen peroxide assay, Luisia tenuifolia, Orchidaceae, skin and wound infections.

 

 


INTRODUCTION:

Skin and topical wounds are often prone to common pathogens including Staphylococcus spp., Streptococcus spp., Pseudomonas aeruginosa, Enterobacter, Klebsiella, Fusobacterium, Candida, Aeromonas and cause delayed healing.  Moreover, this demands special attention as they might result in secondary complications1. Also, a proper balance between reactive oxygen species generation and antioxidant formation is necessary to achieve redox haemostasis and this play a crucial role in wound healing2.

 

Plant extracts and its phytoconstituents remain as a promising source of novel and potential anti-microbials from time immemorial3,4.

 

Orchids are known for their traditional uses particularly in the treatment of skin ailments including cuts, burns, wounds, abscess and tumors5. Additionally several orchids have been a scientifically proven source of antimicrobials6. The epiphyte, Luisia tenuifolia Blume belongs to the family of Orchidaceae.  In India, the plant is mainly distributed in the Western Ghats at an altitude of 1500m. The plant is therapeutically used for treating boils, cuts, wounds and abscess7

 

Literature studies revealed that very few research works were reported in this epiphytic orchid. A study by Balachandar et al., 2019 has reported the anatomical characteristics of the vegetative structures and mycorrhizal morphology of various species of Luisia including L. tenuifolia7.  Also, another study on dermal anatomy of two corticolous orchids, investigated by Mulgaonkar et al., 2005 has observed the paracytic stomata found in L. tenuifolia and the leaves were found to be hypostomatic8. Due to its promising traditional claims and its uninvestigated scientific validations, the current study was carried out to evaluate the antioxidant and antibacterial potential of the whole plant extracts against skin and wound infection causing pathogenic bacteria.

 

MATERIALS AND METHODS:

Plant material:

The L. tenuifolia whole plants collected during February 2019 in and around Yercaud, TamilNadu, were authenticated by Botanical Survey of India, Coimbatore, BSI/SRC/5/23/2019/Tech/3443.  The voucher specimens were preserved in the Department of Pharmacognosy, SRM College of Pharmacy, Kattankulathur, India.

 

Extraction procedure:

The whole plants of L.tenuifolia after drying under shade were coarsely powdered and successively extracted with n-hexane, chloroform, ethyl acetate and ethanol. The extracts obtained were further concentrated using a rotary vacuum evaporator and stored9.

 

Test organisms, culture media, chemicals and reagents:

Five pathogenic bacterial species used for the study namely, Staphylococcus aureus- 902, Streptococcus oralis- 2696, Pseudomonas aeruginosa- 424, Aeromonas hydrophila and Escherichia coli- 443 were purchased from MTCC, Chandihar, India. Nutrient Agar medium, Nutrient broth, Gentamicin antibiotic solution was purchased from Himedia, India. All the chemicals and solvents used in the study were of high analytical grade.

 

Hydrogen peroxide assay:

The hydrogen peroxide scavenging effects of the successive extracts of L.tenuifolia was estimated in accordance with the method described by Ruch et al. with minor modifications. Different concentrations of all the four extracts as well as standard ascorbic acid (500 µg/mL, 250µg/mL, 100µg/mL, 50µg/mL and 10µg/mL) was added to 0.6mL of 43mM hydrogen peroxide in 1M phosphate buffer solution (pH 7.4). The absorbance was determined at 230nm at the end of 10min with phosphate buffer as blank10. The hydrogen peroxide scavenging activity was determined by using the formula,

                                         Control - Test

Percentage inhibition = --------------------------------------- X 100

                                             Control

 

Antibacterial assay:

The successive extracts at different concentrations (500, 250,100 and 50μg/mL) in their respective solvents were evaluated for antibacterial effect in comparison with the standard antibiotic, Gentamycin (10μg/mL) by agar-well diffusion method. Each solvent of the extracts were made the negative control. The medium was prepared by dissolving 2.8g of the commercially available Nutrient Agar Medium (HiMedia) in 100ml of distilled water and the dissolved medium was autoclaved at 15 lbs pressure at 121°C for 15 minutes. Petri plates (25-30ml/plate) containing 20ml of autoclaved nutrient agar medium were seeded with 0.1mL of 24hr culture of bacterial strains (Staphylococcus aureus, Streptococcus oralis, Pseudomonas aeruginosa, Aeromonas hydrophila and Escherichia coli with an optical density of approximately 0.1 at 600nm. Wells were cut and different concentrations of n-hexane, chloroform, ethyl acetate and ethanol extracts of L. tenuifolia were added and incubated at 37°C for 24 hours. The zones of inhibition formed around the wells were measured. Gentamicin antibiotic (10μg/mL) was used as the positive control11-13.

 

Determination of minimum inhibitory concentration:

Based upon the results of antibacterial assays, the most active extract was further selected for the determination of MIC. The minimum inhibitory concentration was determined in accordance with the method followed by Vianney et al., 2018. Accordingly, 50µL of the serially diluted extracts in the respective solvents, two-fold, at concentration ranging from (0.312-10µg/mL) and 50µL of bacterial suspensions with a 100-dilution from OD600 value of approximately 0.1 were added into a microplate well. Then the microplates were agitated at 700rpm for 1 minute and incubated at 37°C for 24 hours on a microplate reader (ROBONIK, India). Further, in order to determine the viability, 10µL of 2, 3, 5 – triphenyl tetrazolium chloride (TTC) at 20mg/mL was added to each well and incubated at 37°C for 30 minutes in dark. The formation of red formazan precipitate indicates the viability. Then the precipitate formed was resuspended with 20µL of absolute ethanol and the difference in the absorbance between the blank and sample was noted at a wavelength of 485nm. The MIC was determined as the lowest concentration at which no viability was observed. The blank was prepared using the sterile nutrient broth without the bacterial suspension13.

 

Statistical analysis:

Statistical analysis was performed by two-ways ANOVA followed by Bonferroni’s multiple comparison test using GraphPad Prism ver. 7.03 and any value of p<0.05 was considered as statistically significant.

 

RESULTS AND DISCUSSION:

H2O2 assay is a widely accepted method in determining the radical scavenging ability of plant extracts. The principle is based on the measuring the decrement of H2O2 when incubated with antioxidants using UV spectroscopy at 230nm14,15. Hydrogen peroxides are capable of crossing membranes and generate hydroxyl radicals that are detrimental to cells and tissues. Hence, scavenging of hydroxyl radicals is important and antioxidants play a powerful role in it16. The hydrogen peroxide scavenging activity of the different extracts of L. tenuifolia, at various concentrations were given in figure 1. All the four extracts showed a dose dependent scavenging effect on hydrogen peroxide. The order of scavenging potential of the extracts with IC50 values were as follows – ethanol extract (42.61μg/mL) > n-hexane extract (43.82μg/mL) > chloroform (51.81 μg/mL) > ethyl acetate (60.8μg/mL) The ethanol extract showed the highest scavenging ability and was comparable with the ascorbic acid which possessed the least IC50 value of 21.56μg/mL.

 

Many scientific reports have proved the antibacterial potential of plant extracts including Clerodendrum infortunatum, Jatropha curcas, Terminalia chebula, Annona squamosa, Casuarina equisetifolia, Spinacia oleracea, Cassia tora against a wide range of pathogens17-23. A study by Reshma et al., (2018) has carried out the synthesis of copper nanoparticles from the leaf extracts of Catharanthus roseus and Azadirachta indica for antibacterial and antioxidant activity24. Another study by Abhijeet et al., (2011) has formulated and evaluated a herbal gel from Aloe vera, Azadirachta indica and Lycopersicon esculentum seed extracts25. Also, in a study by Thanaporn et al., (2012) antibacterial films loaded with mangosteen peel extract was formulated and evaluated for its physicochemical characteristics, mechanical properties and antibacterial activity26. Furthermore, another study carried out by Shruthi et al., (2014) has reported the antibacterial effect exerted by the essential oils of Cinnamon, Clove and Rosemary over Streptococcus mutans, against dental caries27. Furthermore, Krishnaveni et al., (2015) has investigated the volatile components in the ethanol extract of stems of Parthenium hysterophorus by GC-MS/MS and evaluated its antimicrobial activity28.

 

Figure 1: H2O2 scavenging activity of various extracts of L. tenuifolia. Ascorbic acid was used as standard drug. Values were expressed as Mean+SEM; n=3, Two way ANOVA followed by Bonferroni's multiple comparison test. ***p<0.001 compared with standard.

 

Several orchids including Spiranthus mauritianum, Galeola foliate, Bletilla striata, Cypripedium macranthos var. rebunense, Acanthephippium bicolor Lindley have been reported for their antimicrobial potential against a broad spectrum of pathogens29-33. The antibacterial effect of the different extracts of L.tenuifolia is presented in Table.1-5. The results were evident that all the extracts exhibited antibacterial activity against the tested organisms and were comparable with the standard Gentamycin. The n-hexane extract was found to be more active against S. aureus. The ethanol extract showed the highest antibacterial activity against on S.oralis, P.aeruginosa, A.hydrophila and E.coli. At the highest concentration of 500μg/mL, the zone of inhibition exhibited by the ethanol extract against S.oralis, P.aeruginosa, A.hydrophila and E.coli were (16.5±1.5)mm, (12± 2.0)mm, (17.7±1.7)mm and (14.9±1.9)mm, respectively.

 

Table 1. Antibacterial effect of different extracts of whole plants of Luisia tenuifolia against Staphylococcus aureus (Mean ± SD)

Concentration of the extract

Zone of inhibition (mm)

n-hexane extract

Chloroform extract

Ethyl acetate extract

Ethanol extract

50 μg/mL

3.2±0.2

2.1±0.1

0±0.0

2.2±0.2

100 μg/mL

4.7±0.7

4.6±0.6

0±0.0

4.5±0.5

250 μg/mL

8±1.0

7.8±0.8

9.8±0.8

6.8±0.8

500 μg/mL

10.5±1.5

10±1.0

12.5±1.5

11±1.0

Gentamycin (10 μg/mL)

12±2.0

12±1.0

12.5±0.5

10±1.0

Values were expressed as mean + SD (n=3).



Table 2. Antibacterial effect of different extracts of whole plants of Luisia tenuifolia against Escherichia coli (Mean ± SD)

Concentration of the extract

Zone of inhibition (mm)

n-hexane extract

Chloroform extract

Ethyl acetate extract

Ethanol extract

50 μg/mL

0 ± 0.0

2.4±0.4

2.1±0.1

3.2±0.2

100 μg/mL

5.9±0.9

7.6±0.6

5.4±0.4

4.5±0.5

250 μg/mL

8±1.0

10.8±0.8

9.9±0.9

12.6±0.6

500 μg/mL

9.5±1.5

13±1.0

13.5±1.5

14.9±1.9

Gentamycin

(10 μg/mL)

13±1.0

14.5±1.5

13±1.0

11±1.0

Values were expressed as mean + SD (n=3).

 

Table. 3 Antibacterial effect of different extracts of whole plants of Luisia tenuifolia against Pseudomonas aeruginosa (Mean ± SD)

Concentration of the extract

Zone of inhibition (mm)

Mean ± SD

n-hexane extract

Chloroform extract

Ethyl acetate extract

Ethanol extract

50 μg/mL

0 ± 0.0

0 ± 0.0

0 ± 0.0

3.3±0.3

100 μg/mL

4.5±0.5

0 ± 0.0

4.7±0.7

6.5±0.5

250 μg/mL

6±1.0

6.7±0.7

9.8±0.8

9.9±0.9

500 μg/mL

11±2.0

10±1.0

11.9±0.9

12±2.0

Gentamycin

(10 μg/mL)

11±1.0

7±1.0

10±1.0

10±1.0

Values were expressed as mean + SD (n=3).

 

Table. 4 Antibacterial effect of different extracts of whole plants of Luisia tenuifolia against Streptococcus oralis (Mean ± SD)

Concentration of the extract

Zone of inhibition (mm) Mean ± SD

n-hexane extract

Chloroform extract

Ethyl acetate extract

Ethanol extract

50 μg/mL

0 ± 0.0

0 ± 0.0

0 ± 0.0

2.3±0.3

100 μg/mL

4.5±0.5

3.4±0.4

0 ± 0.0

4.5±0.5

250 μg/mL

6.7±0.7

5.8±0.8

9±0.7

13±1.0

500 μg/mL

10±1.0

9±2.0

13±1.0

16.5±1.5

Gentamycin

(10 μg/mL)

13±1.0

8±1.0

13±1.0

10±1.0

Values were expressed as mean + SD (n=3).

 

Table. 5 Antibacterial effect of different extracts of whole plants of Luisia tenuifolia against Aeromonas hydrophila (Mean ± SD)

Concentration of the extract

Zone of inhibition (mm) Mean ± SD

n-hexane extract

Chloroform extract

Ethyl acetate extract

Ethanol extract

50 μg/mL

0 ± 0.0

5.4±0.4

3.5±0.5

3.4±0.4

100 μg/mL

2.5±0.5

6.6±0.6

4.7±0.7

4.6±0.6

250 μg/mL

4.7±0.7

13.8±0.8

10.9±0.9

13±1.0

500 μg/mL

6.5±1.5

15.7±1.7

12.5±1.5

17.7±1.7

Gentamycin

(10 μg/mL)

16±2.0

11±1.0

15±1.0

14±1.0

Values were expressed as mean + SD (n=3).

 


The strong antibacterial effect exerted by the ethanol extract in turn directed the study for MIC determination. The MIC of ethanol extract of L.tenuifolia against S.aureus and P.aeruginosa was found to be 1.25μg/mL while the MIC against E.coli was 0.625μg/mL. Furthermore, the ethanol extract exhibited the least MIC of 0.312μg/mL against S. oralis and A.hydrophila.

 

CONCLUSION:

Thus the current study concludes that among the four successive extracts of L. tenuifolia, the ethanol extract possessed an efficient hydrogen peroxide scavenging and antibacterial activity against the tested organisms. Further studies can be focused on the investigation of the active phytocomponent responsible for the therapeutic activity.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

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Received on 14.07.2021            Modified on 09.09.2021

Accepted on 18.10.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(10):4565-4569.

DOI: 10.52711/0974-360X.2022.00766