Biological and Clinical Sciences Research Journal
ISSN:
2708-2261
www.bcsrj.com
DOI:
https://doi.org/10.47264/bcsrj0101037
Biol. Clin.
Sci. Res. J., Volume, 2020: e037
Original
Research
ANTI-BIOFILM
POTENTIAL OF MENTHOL PURIFIED FROM MENTHA
PIPERITA L. (MINT)
1EJAZ
R*, 2MALIK S, 1AHMAD M, 3ALI H, 1CHOUDHRY
S
1Department
of Biotechnology, Kinnaird College for Women, 93 Jail Road, Lahore, Punjab, Pakistan
2Center of
Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
3College
of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China
Abstract
Menthol, a bioactive compound of Mentha piperita (mint) with antibacterial
properties was purified by column chromatography to determine its anti-biofilm potential. After phytochemical
analysis, TLC was carried out using n-hexane: ethyl acetate: methanol: water
(2:2:2:1) as the solvent system for ethanolic extract
of mint. TLC achieved the maximum separation of mint constituents with Rf value of 0.68. A purified
menthol fraction was obtained after silica gel column chromatography using four
different eluting solvents. The menthol obtained was then used to perform biofilm inhibition assay to establish its antibacterial
potential. Percentage inhibition was highest for bacillus subtilis (79.4%), as opposed to Pseudomonas aeruginosa
(33.6%) and the combination of both bacteria (20%). ELISA reader was used
to measure absorbance at 450-620nm and 630 nm. Using 450-620nm filter the
values for percentage inhibition lies between 48.6-95% for standard and crude
menthol samples. Similarly, at 630nm the values of inhibition lie between
23.4-70.6%. This anti-biofilm property of menthol can
be utilized in antibacterial drug formulations.
Keywords: menthol, Mentha piperita,
anti-bioflim, Pseudomonas
aeruginosa, inhibition
Introduction
Herbal
medicines are an effective remedy for many infectious diseases since ancient
times. Even today, several drug constituents are obtained from plant sources.
One such plant of medicinal importance is Mentha
piperita which is commonly referred as peppermint
or mint (Al-Bayati, 2009; Snoussi et al.,
2015). M. piperita (peppermint)
is a member of family Lamiaceae and is found growing in moist
habitats. Mint not only serves as the flavoring agent for many food products
and drinks, but it also imparts fragrance. It has been used to treat common
colds, inflammation and gastrointestinal tract problems even by earlier
civilizations (Bupesh et al.,
2007). Menthol is a terpenoid
which is the major bioactive agent of mint family. It is a waxy, crystalline,
white color substance which is solid at room temperature and has a sweet,
minty, refreshing odor (Mikaili et al.,
2013). The extraction of menthol for pharmaceutical
purposes is made possible using different techniques; like colorimetric
methods, gas-liquid chromatography, column chromatography and High-performance
liquid chromatography (HPLC). Its quantification using UV-spectrophotometer has
not been reported (Parkin, 1984).
The
researchers have investigated different methods by which terpenoids
can be isolated. Several different solvents were used for extraction process
like acetone, 80% methanol, ethanol, hexane, ethyl acetate and chloroform.
However, gas liquid chromatography proved to be the most efficient method for
purification of terpenoids (Liu et al., 2014). Menthol has antiseptic, antibacterial, antitumor and
antiallergenic properties. Its antibacterial property has been extensively
studied against a few pathogenic and non-pathogenic strains. Due to this
property, it has been used for the treatment of sore throat, common cold,
coughing and mouth, throat irritation (Chandki et al.,
2011; Husain et al., 2015). A breakthrough in the antibacterial properties of
menthol is its ability to disrupt or inhibit biofilms.
This has been confirmed by the work of Husain and his colleagues who reported 64.8 % retardation of A. hydrophila biofilm at
a concentration of about 800 μg/mL of menthol (Husain et al.,
2015). Similarly, The researchers have assessed the
formation of S. aureus biofilm through biofilm assay at different pH
levels (Doughari, 2012; Zahra et al.,
2011). Qualitative analysis showed that the percentage of biofilm formation was about 60 %. It also showed that the
formation of biofilm was dependent on pH. Very acidic pH (Sun et al., 2013) and very basic pH (Skalicka-Woźniak and Walasek, 2014) showed lower growth of biofilms
whereas as neutral pH showed moderate growth. The bacteria living in biofilms interact with each other via a mechanism known as
Quorum sensing. Menthol extracted from peppermint inhibits quorum sensing
mechanism in bacteria. Inhibition of quorum sensing can reduce pathogenicity and modify antibiotic resistance of bacteria.
Owing to its antibiofilm potential menthol can be
used to modify the antibiotics used to treat oral problems (Chusri et al.,
2012; Saharkhiz et al., 2012; Wakimoto et al., 2004).
Materials
and methods
Preparation of
crude extract
Fresh
mint was air dried for about two days and the leaves were finely grounded to
powder. Ethanolic extract was prepared by dissolving
10g of the mint powder in 80ml of the solute. The extract was kept overnight
before performing phytochemical analysis; which
included the detection of alkaloids, terpenoids,
phenols and flavonoids.
Thin layer
chromatography
For
TLC, mint extract was prepared by dissolving 50 g of mint powder in 500 ml of
70% ethanol. The extract was placed in a shaking incubator for 24 hours at 37oC
and 120 rpm. After shaking for 24 hours the solution was filtered with Whatman’s filter paper. Further washing was done with 20 ml
ethanol and 10 ml distilled water. The filtrate obtained was then concentrated
at 720C in a rotary evaporator (Rao et al., 2007; Shaikh and Patil, 2010; Still et al., 1978). The 50-100 ml
concentrated crude extract obtained had high concentration of menthol. This
concentrated extract was subjected to preparative thin layer chromatography using
n-Hexane: Ethyl acetate: Methanol: Water (2:2:2:1) as the solvent system. The
retention factor (Rf
value) was calculated:
Rf = Distance travelled
by the active fraction
Purification of
menthol by column chromatography
Purification
of menthol from the crude sample was carried out using silica gel column
chromatography. A 20-cm long plastic, reusable column with a filter paper disc
at the bottom and a yellow plastic plug which fits in the nozzle stopping the
flow of solvent was used. Slurry was prepared with a 70-230 mesh column grated
silica gel. About 20 grams of silica was dissolved in n-hexane to make free-flowing
slurry. 400µl of sample was loaded in the column. The adsorbed compound was
eluted using four different solvents i.e.
n-Hexane, Chloroform: Ethanol (10:1), Chloroform: Methanol (10:1) and Methanol
under the flow rate of 2ml/min. Seventeen different fractions (2ml volume each)
were collected after eluting the column with four different solutes. First four
fractions were obtained using n-hexane, fraction 5-9 using chloroform: ethanol
(10:1), fraction 10 to 12 using chloroform: methanol (10:1) and fraction 13 to
17 using methanol. The collected fractions were pooled together and were
subjected to TLC(n- Hexane: Ethyl acetate: Methanol: Water (2:2:2:1)) against
commercially available standard menthol for further confirmation (Çitoğlu
and Acıkara, 2012; Tang et al., 2011).
Determination of antibiofilm
potential of menthol
The antibiofilm property of menthol was established using menthol
fraction obtained after column chromatography, crude ethanolic
extract of Mentha piperita
and commercially purified menthol standard. Then the efficiency of their
percentage inhibitions was compared. Biofilms were
prepared using two bacterial strains of
P. aeruginosa and B. subtilis. To perform the biofilm inhibition assay, the biofilms
were grown on freshly prepared LB media in 300μl capacity microtiter plate. Single strain biofilms
as well as biofilms formed by the combination of P. aeruginosa
and B. subtilis
were subjected to inhibition by standard menthol, crude sample of Mentha piperita and
purified fraction from column chromatography. 5% crystal violet was used as
method of staining. Two filters of ELISA reader i.e. 630nm and a bi-chromatic
filter of 450-620nm was used to obtain absorbance. Percentage inhibition was
calculated using:
OD control
Results
and Discussion
Phytochemical analysis
The phytochemical analysis for ethanolic
extract showed positive results for flavonoids, phenols
and terpenoids whereas the test was negative for
alkaloids. Menthol
is a monoterpene having one phenolic
group. The presence of this phenolic group makes it
slightly polar. Therefore, it is soluble is non-polar solvents like chloroform,
n-hexane, toluene, ether, petroleum ether and polar solvents like glacial
acetic acid, methanol and ethanol (both absolute and 70%). Its solubility in
alcohols is due to the presence of phenolic group. It
is insoluble in water because water is a highly polar solvent which forms
hydrogen bonds. Menthol being weakly polar itself, does not dissolve in water (Lugemwa, 2012;
Still et al., 1978).
Thin layer chromatography
The
solvent system used for TLC consisted of n-Hexane: Ethyl acetate: Methanol:
Water (2:2:2:1). The Rf
value was calculated to be 0.68. This Rf value
was similar to the Rf value reported for menthol (Lugemwa, 2012). It is also important to note
that different solvent systems give different Rf values. The difference in Rf value is because of
different solubility of menthol in different solvent system. It also depends on
the amount of the sample spotted, temperature and the thickness of the TLC
plate or card (Kuehler and
Lindsten, 1983). Menthol being very slightly polar moves rapidly up the TLC card. It is because it is
more readily soluble in non-polar solvents therefore; it has a higher Rf value.
The polar compounds in the extract bind to the adsorbent (silica), so they move
slowly and have low Rf values (Kuehler and
Lindsten, 1983; Lugemwa, 2012).
Purification
of menthol by column chromatography
TLC was followed
by column chromatography to isolate and purify menthol from the crude extract. Seventeen different fractions of about 2 ml each were
collected in vials using four different elution solvents. The fractions were labelled as M1- M17 and were subjected to TLC. The same
solvent system was used i.e. n-hexane: ethyl acetate: methanol: water (2:2:2:1).
The fractions having same Rf
value were pooled (Table 1).The presence of menthol was then further made by a
test. All those spots on the TLC card were sprayed with Folin-Ciocalteu
reagent. Purple color appears due to oxidation reaction. Addition of folin reagent is a confirmatory test for the detection of phenolic compounds or group present (Figure 1).
Table 1: The Rf value of standard
and all the fractions pooled
|
FRACTION
NUMBER |
Rf Value |
|
Standard |
2.4/3.5
= 0.68 |
|
Combined
Fractions |
2.4/3.5
= 0.68 |
Figure 1: TLC card
showing the result of standard and all the fractions combined. Purple spot
appeared after spraying Folin-Ciocalteu.
Determination of
antibiofilm potential of menthol:
Anti-biofilm potential of the fraction purified by column chromatography
The
absorbance was taken at 630 nm. Acetic acid served as blank this was because
acetic acid was present in each well as biofilm
dissolving agent. 0.0294 was the reading obtained for blank. The results are
depicted in table 2.
Table
2. Biofilm inhibition
showed by purified menthol fraction and standard menthol
|
BIOFILM |
ABSORBANCE |
630nm |
|
PERCENTAGE |
INHIBITION % |
|
|
|
Control |
Menthol
Fraction |
Menthol
Standard |
Menthol
Fraction |
Menthol
Standard |
|
|
P. aeruginosa |
0.1625 |
0.1078 |
0.0622 |
33.6
% |
62% |
|
|
B. subtilis |
0.9552 |
0.1966 |
0.1423 |
79.4% |
85.1% |
|
|
P. aeruginosa + B.subtilis |
1.1241 |
0.9021 |
0.6720 |
20% |
40.2% |
|
Biofilm Inhibition assay
The
biofilm ring was visible on the microtiter
plate after drying. The absorbance for the inhibition was taken using two
filters of the ELISA reader i.e., one
on a bichromatic filter and the other one using 630nm
filter.
Using 630nm
wavelength
The
results obtained with 630nm filter (Table 3) showed that 100µl of each sample
either crude, standard menthol, or purified menthol fraction showed maximum
inhibition. Also, the biofilms formed by single
strains are more easily inhibited than the biofilm
formed by both Pseudomonas and Bacillus. Crude extract has better
ability to inhibit biofilms because it has severalphytochemical constituents like phenols, terpenoids and flavonoids all of
which exhibit antimicrobial property (Djordjevic et al., 2002; Sasidharan et al., 2011).
Table
3.
Mean percentage inhibition values of Biofilm
inhibition assay using 630nm Eliza filter
|
|
P.aeruginosa + B.subtilis Biofilm
Percentage Inhibition % with sample volume |
P.aeruginosa Biofilm Percentage Inhibition % with sample volume |
B.subtilis Biofilm Percentage
Inhibition % with sample volume |
||||||
|
50µl |
75 µl |
100 µl |
50 µl |
75 µl |
100 µl |
50 µl |
75 µl |
100 µl |
|
|
Crude Sample |
29.7 |
34.8 |
52 |
66 |
70.3 |
70.6 |
20.1 |
28.5 |
33.2 |
|
Menthol
Standard |
39.5 |
42 |
43 |
37 |
58.7 |
62 |
13.1 |
20.1 |
23.4 |
Using a bi-chromatic
filter (450-620nm)
The
second set of absorbance was taken with a bi-chromatic filter (450-620nm),
showed almost similar results (Table 4). The working of a bichromatic
lens is different than that of a single wavelength filter. The bichromatic lens takes the absorbance at two wavelengths
which in this case is 450 and 620 nm. The ELISA reader disperses the light at
both wavelengths then shows the result after subtraction. It is because of this
reason, the values of absorbance obtained are much lower than those obtained
using a single wavelength filter (Donlan, 2001;
Elvers et al., 1998; Odeyemi and Oluwajoba, 2011). A higher percentage
inhibition was therefore calculated using a bichromatic
lens, but a similar trend of inhibition was observed.
Table
4. Mean percentage
inhibition values of Biofilm inhibition assay using a
bichromatic filter (450-620nm).
|
|
P.aeruginosa + B.subtilis Biofilm
Percentage Inhibition % with sample volume |
P.aeruginosa Biofilm Percentage Inhibition % with sample volume |
B.subtilis Biofilm Percentage
Inhibition % with sample volume |
||||||
|
50µl |
75 µl |
100 µl |
50 µl |
75 µl |
100 µl |
50 µl |
75 µl |
100 µl |
|
|
Crude Sample |
5.5 |
45.8 |
48.6 |
42 |
68.4 |
80 |
80 |
93 |
93.5 |
|
Menthol
Standard |
54 |
94 |
94 |
71.5 |
75 |
95 |
16 |
89.7 |
94.4 |
The
basic mechanism behind the inhibition of biofilms has
been studied for many medicinal plant extracts and their components like
alkaloids, flavonoids etc. Biofilms are inhibited because these
antimicrobial compounds either inhibit the mechanism of Quorum sensing within
the biofilm, prevent the formation of flagella, pili or fimbriae, or prevent the
formation of nucleotides (Kelly et al., 1979; Sun et al., 2013). Antimicrobial agents also
affect the membrane fluidity and permeability of bacterial cell wall resulting
in the disruption of cells (Delwiche and
Gaines, 2005). Menthol also works in similar
fashion. It prevents the attachment of bacteria to the surface of the
substratum by affecting the flagella of the bacterial strains of Pseudomonas and Bacillus. It also interferes with the extracellular matrix
(consisting of DNA, proteins and polymers) necessary for biofilm
formation and its attachment, thus preventing the formation of biofilm. It also
interferes with the quorum sensing mechanism (Qiu et al., 2011; Uzair et al., 2008). Hence, it was established after extensive and repeated research
that crude extract of mint as well as the purified form of menthol exhibit antibiofilm property. This property can be exploited to
produce number of antimicrobial drugs as well as for the modification of
antibiotics. Using compounds derived from medicinally important plants can
redefine and reshape the formation of drugs in future.
Conclusion
The
qualitative analysis proved ethanol to be the best solvent for the detection of
phytochemicals. The purified menthol fraction
obtained after column chromatography has the Rf value of 0.68 using n-hexane: ethyl
acetate: methanol: water as solvent system. The purified menthol as well as its
crude extract when subjected to biofilm inhibition
assay shows a reasonable inhibition of biofilm,
whereas the maximum inhibition of 99.5% was achieved by crude menthol extract. This
result illustrates that menthol is a medicinal plant and can be used to alter
the antibiotic resistance of many disease-causing microbes.
Conflict of
interest
The
authors declared absence of any conflict of interest.
References
Elvers, K.,
Leeming, K., Moore, C., and Lappin‐Scott, H. (1998). Bacterial‐fungal biofilms in flowing water photo‐processing tanks. Journal of Applied Microbiology 84, 607-618.
Rao, M. V.,
Al‐Marzouqi, A. H., Kaneez, F. S., Ashraf, S. S.,
and Adem, A. (2007). Comparative evaluation of SFE and solvent extraction
methods on the yield and composition of black seeds (Nigella Sativa). Journal of liquid chromatography &
related technologies 30,
2545-2555.