The document summarizes a study that investigated the antimicrobial and enzyme inhibition activities of Triphala, a traditional Ayurvedic herbal formulation. Key findings include: 1) Triphala extract showed antibacterial activity against Pseudomonas aeruginosa as demonstrated by a 18mm clear zone in a disc diffusion assay and a minimum inhibitory concentration of 7.8125mg/ml. 2) Zymography analysis showed Triphala reduced metalloprotease activity from P. aeruginosa at concentrations ≥1500μg/ml. 3) The results highlight the potential of topically applying Triphala to accelerate wound healing and control infection by inhibiting P. aeruginosa and its proteases.

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The control and eradication of wound infection is a challenging
aspect in the management of wounds. Despite the use of synthetic antimicrobial agents,
drug resistance and toxicity hinder the activity of these antimicrobial agents and thereby
increase the chances of infection and delay wound healing processes. Additionally, the
microbial enzymes secreted by these pathogens degrade the extracellular matrix at wound
site. The present study investigates the influence of Triphala on antimicrobial activity and
enzyme inhibition activity which will be used in wound healing studies. The methanolic
extract of triphala was prepared and its antimicrobial activity was tested against
Pseudomonas aeruginosa. The activity of triphala extract against metalloprotease were
studied by Zymography. Enzymatic activities were detected as clear bands of gelatin lysis
against a dark background. The 18 ± 2mm clear zone in disc diffusion assay and minimal
inhibitory concentration (MIC) of 7.8125mg/ml against pseudomonas control strains
clearly showed the antibacterial activity of Triphala. Zymography analysis exhibited
greater reduction in d metalloprotease activity at ≥1500μg/ml. By virtue of the inhibitory
effect of Triphala on Pseudomonas aeruginosa strains and their enzymes such as proteases,
it could be, potentially used as a new therapeutic agent for pseudomonas infected dermal
wounds. The results hence highlighted the beneficial effects of the topical application of
Triphala in the acceleration of wound healing and its effect on controlling wound infection
at wound site.
ABSTRACT
INTRODUCTION
RESULTS
Protease Inhibition By Methanolic Extract of Triphala
DISCUSSION
Inhibitory activity of triphala against P. aeruginosa and provides some scientific rationale
for its use as antimicrobial agents on infected dermal wounds. As an important observation
in our study, we have found both methicillin resistant and sensitive S. aureus were also
inhibited at the same concentration of triphala extract. Although the triphala has rich in
polyphenols and ascorbic acid. The poly phenols might be the responsible for
antimicrobial action due the presence of EGCG (epigallocatechin gallate) as one of the
condensed tannins. The literature proved that Epigallocatechin gallate has the specific
mechanism for antimicrobial activity. The epigallocatechin gallate more amount binds to
S. aureus than that of gram negative bacterium E.coli and sensitivity of EGC treated S.
aureus to high ionic strength was more than towards and low osmotic pressure. The
epigallocatechin gallate binds to the peptidoglycan layer in the cell wall of S.aureus.
Peptidoglycan is a cross-linked complex formed from various polysaccharides and
peptides. Thirty to fifty layers of peptidoglycan form part of the cell wall of S.aureus
offering osmotic protection, assisting in cell division and also act as a primer for further
biosynthesis of peptidoglycan. In the case of pseudomonas as gram negative bacterium,
The thin layer of peptidoglycan is available for binding with active constituents in triphala.
However, The mechanism of antimicrobial action remains obscure. The reduction in the
activity of pseudomonas metalloprotease by the triphala extract gives additional support to
its potential use as a therapeutic agent for wound treatment.
ACKNOWLEDGEMENT
Bharathi Ravi would like to express her gratitude to her organization Lifecell International
private limited for presenting this work in this international conference.
As the development of bacterial resistance to antibiotics and
the controversies regarding the use of topical antiseptics persists, thousands of
phytochemicals were sucessfully isolated from plants that inhibit all types of
microorganisms yet safe and broadly effective without inducing microbial resistance.
According to WHO, nearly 20,000 medicinal plants are present in 91 countries. New
compounds inhibiting microorganisms such as benzoin and emetine have been isolated
from plants . The antimicrobial compounds from plants may inhibit bacterial growth by
different mechanisms than those presently used antimicrobial agents and may have a
significant clinical value in treatment of resistant microbial strains . To date, widespread
opinion among wound care practitioners is that aerobic or facultative pathogens such as
Staphylococcus aureus, Pseudomonas aeruginosa, and beta-hemolytic streptococci are the
primary causes of delayed healing and infection in both acute and chronic wounds .
Triphala is a traditional Ayurvedic herbal formulation that
consists of the dried fruits of three medicinal plants, Terminalia chebula, Terminalia
bellirica, and phyllanthus emblica. Triphala and/or its constituent plants have been
reported to possess numerous biological and pharmacological activities such as
antioxidant, antibacterial, antifungal, antiviral, antimalarial, anti-mutagenic, anticancer,
radio protective, anti-allergic, cardiotonic, hypocholesterolaemic, capillary strengthening
and hepatoprotective. The antimicrobial potential of Triphala is tested against various
wound pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and beta-haemolytic
streptococci. But the antimicrobial activity of Triphala against Pseudomonas aeruginosa is
focused in this work and presented in this conference.
*Centre for Biotechnology, Anna University, Chennai-600025, India.
# Lifecell International Private Ltd, Chennai, India.
Email:bharathi.p.ravi@gmail.com
S.Kirubanandan*, Bharathi Ravi# and S.Renganathan*
Enzyme Inhibition and Antimicrobial Activity of Triphala against
Pseudomonas aeruginosa
EXPERIMENTAL METHODS
Microorganisms Triphala extract Antibiotic
S. aureus
P. aeruginosa
18 mm
20 mm
34 mm (methicillin)
30 mm (ciprofloxacin)
Fig 1- Enzyme Inhibition by Triphala Extract
Microorganisms Minimum concentration
S. aureus
P. aeruginosa
Streptococcus pyogenes
7.8 mg
7.8 mg
31.25mg
Table 1 - Disc Diffusion Assay by Triphala Extract
Fig 2- Disc Diffusion Assay by Triphala Extract
Table 2 – Minimum Inhibitory Triphala Extract
Preparation of alcohol extract of Triphala
100 g of powder was extracted in 500 mL of methanol by stirring overnight and was
centrifuged at room temperature. The supernatant was collected and evaporated to dryness
under reduced pressure in a rotary evaporator. The yield of this methanolic extract was
12.5%. The concentrated extract was aliquoted in amber-colored bottles and kept in
desiccators for further use. The dried extract was dissolved in 10% Dimethyl Sulfoxide
(DMSO) and used to assay the antibacterial activity.
Determination of antibacterial activity
The minimal inhibitory concentration (MIC) of the extract was determined by the broth
tube dilution method and The antibacterial sensitivity test was performed by disc diffusion
method. Sterile blank discs (6 mm diameter) were impregnated with minimum inhibition
concentrations of triphala extract against standard strains. Standard methicillin disc and
discs treated with DMSO were used as control. Inhibition zone diameters around each of
the disc were measured and recorded at the end of the incubation time.
Enzyme inhibition assay
The overnight microbial cultures were transferred in to sterile 50 mL conical tubes and
centrifuged at 5000 rpm for 5 minutes. To the culture supernatant, solid ammonium
sulfate was added slowly with stirring to achieve 80% saturation. The resulting precipitate
was collected by centrifugation and dissolved in 0.02 M phosphate buffer, pH 6.8. From
this, 1mL was transferred in to sterile vials and incubated with different concentration of
triphala extract (100µg-2000µg/mL) for overnight at 37oC. 1mL of 10% DMSO was used
as control. The activity of triphala extract against metalloprotease was studied by
Zymography. Enzymatic activities were detected as clear bands of casein/gelatin lysis
against dark background..