Volume 3, Issue 8, August – 2018 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Formulation of Herbal Sanitizers and Determining

Their Antimicrobial Activities Against Skin
Pathogens
Rutuja Sunil Patankar Dr. Nayna Chandak
Microbiology Dept., Patkar Varde College Head of Microbiology Dept.
Mumbai, Maharashtra, India Patkar Varde College, Mumbai
Maharashtra, India

Abstract:- Medicinal properties of plants is been studied When soap and water are not readily available, alcohol
since long back and various research on it was carried based hand sanitizers or rubs are acceptable. Hand sanitizers
out and from that various pharmaceutical, health care, are effective against bacterial and fungal infections, as well
cosmetic products are been manufactured. This research as enveloped viruses, such as the common cold and flu
was aim to formulate the herbal sanitizer. Sanitizer viruses and in preventing nosocomial infections caused by
formulated from neem and lemon juice. Phytochemical different opportunistic microorganisms. Alcohol rub
and antioxidant properties of neem extract and lemon sanitizers containing at least 70% alcohol kill 99.9% of the
juice were analysed. Their MIC was checked against bacteria on hand 30 seconds after application and 99.999%
standard culture and hand isolates from lab assistance. in 1 minute (10). Cleansing products powered by natural
MIC of neem extract and lemon juice was found. Three essences like witch hazel, bitter orange peel extract, thyme,
different sanitizers were formulated lemon, lemon-neem lavender (a popular one), and even organic alcohol are on
and neem sanitizer. Formulated sanitizer was compared the rise.
with sterillium sanitizer and it was found that sterillium
was effective against standard organism only and not Medicinal plants produce a diverse range of bioactive
against hand isolates while formulated sanitizer was molecules, making them rich source of different types of
effective against both. Even in comparison with respect medicines. Pharmacological studies have accepted the value
to zone of inhibition, formulated sanitizers were showing of medicinal plants as potential source of bioactive
more zones as compared to sterillium sanitizer. compounds (WHO survey, 1996). Phytochemicals are
Antibacterial, antifungal and antibiofilm activity of secondary metabolites, which are produced by medicinal
formulated herbal sanitizer was studied. Antibiotic plant.
potentiation activity of formulated sanitizer was tested
against Methicillin Resistant Staphylococcus aureus and So, different studies and sanitizer formulation were
isolate IV to find out change in the sensitivity pattern of carried out. Sanitizer with complex chemicals as well herbal
antibiotics. Finally lemon, lemon-neem and neem sanitizer was formulated and their efficacy were checked
sanitizer efficacy was tested. Result obtained showed against various groups of organisms. Like, in this research
reduction in bacterial growth after treatment with three herbal sanitizers were formulated from neem and
sanitizer. As neem sanitizer was tested found to be bitter lemon. As, Neem Azadirachta indica extract is an important
hence instead of using it as hand sanitizer it can be use as source of compounds having anti-microbial, anti-oxidant,
a spray on medical devices or as a floor cleaner. anti-tumor, anti-malarial, anti-fungal, anti-inflammatory and
anti-viral properties and Lemon Citrus limon is an important
Keywords:- Sanitizer, MIC, Antibacterial. medicinal plant, antibacterial potential in crude extracts of
different parts (viz., leaves, stem, root, fruit and flower) of
I. INTRODUCTION lemon against clinically significant bacterial strains has been
reported (6). Hence in this studies neem leaves and lemon
Hand hygiene is now regarded as one of the most juice is been use their antimicrobial properties were studied
important element of infection control activities. In the wake and accordingly sanitizers were formulated.
of the growing burden of health care associated infections,
the increasing severity of illness and complexity of II. MATERIALS AND METHODS
treatment, superimposed by multi-drug resistant pathogen
infections, health care practitioners are reversing back to the A. Preparation of Leaf extract and lemon juice (10):
basics of infection preventions by simple measures like hand Tulsi and neem leaves were dried in oven at 50°c and
hygiene. This is because enough scientific evidence supports coarsely grinded to get a powder. For methanol extraction,
the observation that if properly implemented, hand hygiene 10g of leaves powder and 90% methanol was added in
alone can significantly reduce the risk of cross-transmission 250ml flask (maceration) and kept on shaker for 48 hours.
of infection in healthcare facilities (7). After maceration, sample was filtered by whatman no.1
filter paper. This filtrate was placed in water bath at 60°c till
Washing hands with soap and water is the best way to thick sticky, dark coloured extract is obtained and lemon
reduce the number of microorganisms (germs) on hands. were washed, cut into half and squeeze to get a juice. This
juice was stored at 4°C for further use.

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ISSN No:-2456-2165
B. Antibacterial activity of extract against standard  Neem sanitizer: Add 18ml of 70% isopropyl alcohol in
organism: flask. In that add 2ml Glycerol and 4ml Rose water.
Antibacterial activity of extract and juice was carried Then add 1g of neem extract.
out by agar well diffusion method against standard
organism. After swabbing culture and adding extract the I. Antimicrobial and Antifungal activity of sanitizer (1):
plates were incubated at 37°C for 24hours. For this method, Mueller-Hinton agar plates were
used. Agar surface of each plate was streaked by a sterile
C. Phytochemical analysis : cotton swab with the reference bacterial strain. Then agar
Test for carbohydrates (Fehlings test), amino acid plate was punched with a sterile cork borer of 4 mm size and
(Ninhydrin Test), phenols and tannins, Flavonoids, Saponins then poured 100 µL of each sample (sanitizer) with
(Foam test), Terpenoids, steroids and sterols (Salkowski’s micropipette in the bore. Plates were allowed to stand for 30
test) was performed as per given reference (3). min. Then incubated at 37°C for 24 h. Zone of inhibition
(mm) of the bacterial strains against three herbal sanitizers
D. Radical Scavenging Activity by the DPPH Assay: was measured.
DPPH assay was performed to check radical
scavenging activity of neem extract and lemon juice. J. Effect of sanitizer on biofilm (4):
Absorbance of all the samples was taken (9). The percentage Crystal Violet assay: This is a quantitative method for
radical scavenging activity of each extracts was calculated biofilm detection. Loopful of test organisms was inoculated
using the following equation in a 10 mL of trypticase soy broth with 1% glucose in test
tubes. Tubes were incubated at 37°C for 24 h. After
Percentage radical scavenging activity (RSA %) = incubation, decant tubes was wash with sterile saline. Base
of tube was Heat fix. Tubes were then stained with crystal
(Absorbance of control – Absorbance of sample) × 100%
violet (0.1%). Excess stain washed with distilled water.
Absorbance of control Distained with 4ml of ethanol by incubating for 5mins at
room temperature. Recorded reading by taking optical
E. Collection of hand isolates from lab assistance: density at 660nm.
Hand swabs of lab assistants by using sterile cotton
swabs were taken. Plated on nutrient medium and incubated. K. Efficacy of sanitizer:
Colony characterization, gram staining and biochemical To check efficacy a 200mm size of petriplate were
were performed. Isolates were labeled as isolate I, II, and so taken. In that 200ml of LB agar were poured. After cooling
on and preserved for further use and to study the effect of of agar two plates one for control and other for test were
herbal sanitizer on hand microflora. selected. Without applying sanitizer hand of assistant were
placed on control plate and with applying sanitizer hand of
F. Antibiotic sensitivity test (AST) of pathogens: assistant were placed use as test. Then plates were incubated
To study the susceptibility of bacteria to various at 37°C for 24hours. The decrease in number of colony was
antibiotics AST was performed by referring Kirby Bauer determined. Same procedure was followed for all three
and koneman’s chart. sanitizers.

G. MIC determination by Macro agar dilution method (8): L. Antibiotic potentiation:

Minimum inhibitory concentration of all three extracts This procedure is modification from Kirby-Bauer disc
was done by macro agar dilution method. Four different diffusion method. Different dilution of the sanitizers was
dilutions of extract using DMSO as diluent was prepared. prepared ranging from 1:10, 1:20, and 1:30 with sterile
Undiluted, 1:2, 1:4 and 1:8 were the dilutions used, along distilled water. 1ml of each dilution were added to 9ml of
with control. 1ml of these dilutions was mixed with 19ml of MH agar and then poured into petriplate. MH agar plate
LB butts and was poured on petri plates. Then culture without sample was taken as control. Within 15mins after
suspension of 0.5 Mc Farland turbidity was prepared and adjusting turbidity (0.5 McFarland standards) of inoculum
5µl of culture were spot inoculated on plates. Plates were suspension, a sterile cotton swab was dipped into it and was
kept stand for 5mins and then incubated at 37°C for 24 swabbed over entire surface of MH agar medium containing
hours. MIC was determined by inhibition of culture at different dilution of sample. The antibiotic disc was placed
particular dilution. in agar plate seeded with respective bacteria. Antibiotic disc
used were Oxacillin (1mcg) for MRSA and Norfloxacin
H. Preparation of sanitizer: (10mcg) for Isolate IV. The plates were incubated at 37°C
 Lemon sanitizer: Add 19ml of 70% Isopropyl alcohol in for 24hours. After incubation each inhibition zone diameter
flask. In that add 2ml Glycerol and 4ml Rose water. (IZD) will be measured and analyzed according to clinical
Then add 25ml of Lemon juice. and laboratory standards institute guidelines and the
 Neem-Lemon sanitizer: Add 19ml 70% Isopropyl antibacterial activity will be expressed as the mean of IZD
alcohol in flask. In that add 2ml Glycerol and 4ml Rose (mm).
water. Then add 12.5g of neem extract and 12.5ml lemon
juice.

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ISSN No:-2456-2165
III. RESULTS Table 3. DPPH assay of neem extract and lemon juice

A. Antimicrobial and Antifungal of leaf extract and lemon Absorbance at 530nm

juice: Concentration
Antimicrobial and antifungal activity of extract against Neem RSA% Lemon RSA%
standard organism was performed by Agar well diffusion extract juice
method. Neem extract was found to be effective against S. Undiluted 0.28 47.16 0.42 8.69
aureus and MRSA and not against C. albican and P. 1:2 0.06 88.67 0.29 36.95
aeruginosa. Tulsi extract did not show antibacterial activity 1:4 0.05 90.56 0.28 39.13
against selected organisms. Lemon juice was inhibiting S. 1:8 0.01 98.11 0.26 43.47
aureus, MRSA, P aeruginosa and not to C. albican. Hence Control 0.53 - 0.46 -
neem extract and lemon juice was selected for further study. Blank 0.0 - 0.0 -
DMSO was use as diluent for extract which does not show
its antibacterial activity against selected organisms (Table D. Antibiotic Sensitivity Test:
1). Antibiotic sensitivity test of selected culture were
performed by reffering kirby bauer and koneman’s chart
Table 1. Antimicrobial and Antifungal activity of extract by (Table 4). Candida albican found to be resistant to
agar well diffusion method clotrimazole.

Table 4. AST of selected culture

Organism S.aureus MRSA C.albican P.aeruginos
a
Cultu Zone Of Inhibition(mm)
Zone of Inhibition (mm) re
Neem 14 10 - -
extract Iso Iso Iso Iso Iso
Tulsi - - - - S.a M P.aer I III IV VI VII
extract ure RS ugino (Gr (Gra (Gr (Gr (Gr
Lemon 16 14 - 20 us A sa am m am am am
juice pos posit pos pos pos
DMSO - - - - itiv ive itiv itiv itiv
e dipl e e e
coc ococ sho rod sho
B. Phytochemical analysis:
ci) ci) rt s) rt
Presence and absence of phytochemicals in lemon
Antib rod rod
juice and neem extract as shown in (Table 2).
iotic s) s)
Table 2. Lemon juice and Neem phytochemicals
Oxac 36 - 19 17 24 - 10 -
Result illin
Test
Lemon juice Neem extract Norfl 34 10 - 19 25 12 27 15
oxaci
Saponins - + n
Tannin + +
Steroid - - Vanc 30 14 21 16 2 23 24 16
Phenol + + omyc
Flavonoids + + in
Carbohydrate - -
Amino Acid - - Tetra 37 35 - 34 40 39 40 27
Terpenoid - + cycli
Key: +: Present / - : Absent ne
C. Free radical scavenging assay:
Free radical scavenging assay of neem extract and
lemon juice was performed by DPPH method and
absorbance was taken at 530nm. Percentage radical
scavenging activity was calculated by formula. Neem
extract has more radical scavenging activity than lemon
juice. Even diluted sample of neem extract and lemon juice
shows a radical scavenging activity (Table 3).

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Volume 3, Issue 8, August – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
F. Sanitizer:
Zone of inhibition Formualtion of sanitizers were done by using neem
Iso V extract and lemon juice with base iso-propyl alcohol, rose
Antibiotic (Gram negative water and glycerol. Three different sanitizers were
short rods) formulated named as lemon, lemon-neem and neem
sanitizer (Figure 2 a,b,c).
Ciprofloxacin 36

Norfloxacin 31

Piperacillin 27

Tetracycline 15
Key:  No inhibition

E. MIC determination:
MIC for isolate I, IV, VII and S. aureus were found to
be 1:2. While that for isolate III MIC found to be 1:4. For a. Lemon b. Lemon-neem c. Neem
isolate VI MIC found to be 1:8. No inhibition at culture V, Fig 2:- a,b,c: Formulated Sanitizer
MRSA and P. aeruginosa is seen, it means further dilutions
were needed to be performed. ( Figure 1 a). MIC for isolate
I, III, IV, V, VI, VII no growth was seen at any G. Antibacterial activity of sanitizer by agar well method:
concentration it means further dilutions were needed to be Antibacterial activity of all formulated sanitizers with
performed. MIC for S. aureus and P. aeruginosa were found positive control that is Sterillium and other control is the
to be 1:2. For MRSA it was found to be at undiluted base use to formulate sanitizer was checked. Against isolate
concentration (Figure 1 b). For C. albican no inhibition by III and isolate VI no growth on plate was seen. For all other
neem extract and lemon juice was found at any isolates inhibition was seen. It was found that all three
concentration. sanitizers were showing more inhibition than sterillium and
sanitizer base (Table 5. and Figure 3).
A. MIC of neem Extract

Fig 3:- Antibacterial activity of sanitizers by agar well

B. MIC of lemon Juice
diffusion method

Fig 1:- a,b: MIC of neem extract and lemon juice against
selected bacteria was performed by using macro agar
dilution method.

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ISSN No:-2456-2165
Table 5. Antibacterial activity by agar well diffusion method between test and control tubes was done. Antibiofilm
activity was found against all bacteria except for isolate IV
and P. aeruginosa in presence of lemon sanitizer (Graph 1).
Sanitizer L Lemon Positiv Cont In presence of lemon-neem sanitizer antibiofilm activity was
e -Neem Nee e rol- found against all bacteria (Graph 2). In presence of neem
m m control Sanit sanitizer antibiofilm activity was found against all bacteria
o - izer (Graph 3).
n Sterilli base
um Graph 1. Antibiofilm activity - lemon sanitizer
0.45

0.4
Zone of 0.35

Absorbance at 660nm
inhibition 0.3
Culture (mm) 0.25

0.2 Control
0.15 Test
0.1

0.05
Isolate I Inhi Inhi Inhi 26 22 0
biti bitio bitio
on n n

Isolate III - - - - -
Graph 2. Antibiofilm activity – lemon-neem
Isolate IV 24 23 22 8 12 Sanitizer
(12 (11) (10)
) 0.5
0.45
Isolate V 32 34 28 8 24 0.4
Absorbance at 660nm

0.35
(8) (10) (4)
0.3
0.25
Control
Isolate VI - - - - - 0.2
Test
0.15
0.1
Isolate VII 28 24 25 8 - 0.05
0
(28 (24) (25)
)

S. aureus 24 24 25 15 10
(14 (14) (15)
Graph 3. Antibiofilm activity – neem sanitizer
)

MRSA 32 28 23 12 20 0.5
(12 (8) (3) 0.4
0.3
) 0.2
0.1 Control
P. - 14 15 - 10 0
aeruginos (4) (5) Test
a

C. albican 12 10 15 10 8
(4) (2) (7)

Key:  No growth I. Antimicrobial and Antifungal activity of Sanitizer:

In bracket ( ) Difference in zone of inhibition between Antimicrobial and antifungal activity of sanitizer was
sanitizer and base. studied by agar handprint method and all three sanitizers
were found to be effective as reduction in growth was
H. Anti-biofilm activity: (Crystal violet tube method assay observed as making difference in growth of before applying
quantitative): and after applying plate (Figure 4,5,6).
After performing assay the antibiofilm activity was
studied by taking absorbance at 660nm and comparison

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Table 6. Antibiotic potentiation activity of lemon sanitizer
Before applying After applying on MRSA

Lemon sanitizer Norfloxacin-10

Control 11
MRSA
1:10 14
Fig 4:- Antimicrobial activity of lemon sanitizer
1:20 11

1:30 12

Table 7. Antibiotic Potentiation activity of lemon-neem

Sanitizer on Isolate IV

ZONE OF
Lemon-Neem INHIBITION (mm)
sanitizer
Fig 5:- Antimicrobial activity of lemon-neem sanitizer
Norfloxacin-10

Isolate Control 13
IV
1:10 24

1:20 17

1:30 14
Fig 6:- Antimicrobial activity of neem sanitizer
Table 8. Antibiotic potentiation activities lemon- neem
J. Antibiotic potentiation: sanitizer on MRSA
Antibiotic potentiation activity assay was performed
to study the ability of sanitizers to change sensitivity pattern
of antibiotics. It was performed by selecting antibiotics
against which organisms are resistant. By referring AST
performed earlier, the MRSA and isolate IV was selected
which was showing resistance against antibiotic Oxacillin,
Vancomycin and Norfloxacin. Different dilutions of
sanitizer were prepared. In presence of lemon sanitizer
against MRSA no change in sensitivity pattern of Oxacillin
were seen. Sensitivity pattern of Vancomycin and
Norfloxacin was found to be changed (Table 6) and against
isolate IV no change in sensitivity pattern was seen. Table 9. Antibiotic potentiation activities of neem sanitizer
on isolate IV
In presence of lemon-neem sanitizer, against MRSA
no change in sensitivity pattern of Oxacillin was seen. ZONE OF
Sensitivity pattern of Vancomycin and Norfloxacin found to Neem INHIBITION
be changed at dilution 1:10 (Table 7) and against isolate IV sanitizer (mm)
greater change in sensitivity pattern of Norfloxacin was
obtained at all three dilutions (Table 8). In presence of Norfloxacin-10
neem sanitizer against MRSA, no change in sensitivity Isolate
pattern of Oxacillin, Vancomycin and Norfloxacin was seen IV Control 16
and against isolate IV, change in sensitivity pattern of
Norfloxacin was seen at dilution 1:10 (Table 9). 1:10 18

1:20 15

1:30 14

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Volume 3, Issue 8, August – 2018 International Journal of Innovative Science and Research Technology
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Throughout history, human civilizations have
kinetically circumvented plants which have influenced a lot
to the humanity. Plants have the facility to endanger diverse
variety of phytochemical and biochemical compounds
which can be acclimated to perform different biological
functions. Many of these phytochemicals have salutary
effects on long-term health when consumed by the human
and can be efficaciously used to treat human diseases.

Neem (Azadirachta indica) is widely used in the

ayurvedic medicine system for treating malaria and fever.
This study was undertaken to prepare crude extracts of neem
leaves, same way in this research work, neem extract was
prepared my methanol extraction method. Neem
phytochemical screening, the total phenol content and free
radical scavenging activity were assessed by DPPH method.
Biochemical screening of the crude extracts of neem leaves
revealed positive results of flavonoids, saponins, steroids,
alkaloids, amino acid and tannins(5). Same way
phytochemical screening of neem extract was performed in
this research. Phytochemical like tannin, phenol, saponin,
terpenoid and flavonoid was found to be present. Neem
showed radical scavenging activity even at lowest dilution
effectively, by DPPH assay. Antioxidant activity at various
concentration and RSA% was calculated by using formula.
Lemon-Neem Lemon-Neem Lemon-Neem Of neem extract, RSA% found to be at concentration
1:10 1:20 1:30 undiluted - 47.16%, 1:2 - 88.67%, 1:4 - 90.56%, 1:8 –
98.11%.
Fig 7:- Antibiotic potentiation of sanitizer against MRSA
Citric acid was found to be an effective permeabilizer
and potentiating agent for VISA/MRSA isolates (11). So
lemon juice was also selected for formulation of sanitizer.
Phytochemical analysis was carried out as tannin, phenol
and flavonoids found to be present. Of lemon juice, RSA%
found to be at concentration undiluted – 8.69%, 1:2 –
36.95%, 1:4 – 39.13%, 1:8 – 43.47%.

Bohora et al., concluded that neem leaf extract has a

significant antimicrobial effect against E. faecalis, Candida
albicans and mixed culture (2). In this study MIC of neem
extract for isolate I, IV, VII and S.aureus were found to be
1:2. While that for isolate III MIC found to be 1:4. For
isolate VI MIC found to be 1:8. No inhibition at isolate V,
MRSA and P.aeruginosa is seen, it means further dilutions
needed to be performed. MIC of lemon juice for isolate I,
III, IV, V, VI, VII no growth was seen at any concentration
it means further dilutions were needed to be performed.
MIC for S. aureus and P. aeruginosa were found to be 1:2.
For MRSA, MIC was found to be at undiluted
concentration.

Lemon-Neem Lemon-Neem Lemon-Neem Other herbal hand sanitizer, incorporating the leaves
1:10 1:20 1:30 extracts of Ocimum canctum Linn.(Tulsi) and Eucalyptus
globulus (Nilgiri), the well-known herbal combination with
Fig 8:- Antibiotic potentiation of sanitizer against isolate IV multidimensional activities was formulated and their
respective antimicrobial efficacy were studied. The
IV. DISCUSSION formulation was evaluated against the specified micro-
organism by culture sensitivity test. The significance was
Different studies and sanitizer formulation were found to be more (13). Even in this research combination of
carried out. Sanitizer with complex chemicals as well herbal neem and lemon was use to formulate sanitizer. First their
sanitizer was formulated and their efficacy were checked individual effectivity was checked and after that
against various groups of organisms. combinational activity was also studied.

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Volume 3, Issue 8, August – 2018 International Journal of Innovative Science and Research Technology
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The prepared formulation of herbal hand sanitizer Pharmacological studies have accepted the value of
showed significant results against standard cultures as well medicinal plants as potential source of bioactive
as against hand isolates. The significance was found to be compounds. Phytochemicals are secondary metabolites,
more in comparison to the commercially available hand which are produced by medicinal plant.
sanitizer. Sanitizer with lemon juice and neem extract were
formulated along with alcohol, as alcohol have excellent, In this research Phytochemicals was found to be
rapid (within few seconds) germicidal activity against present in neem extract and lemon juice. Radical scavenging
vegetative bacteria, fungi, and many viruses and activity was also found to be present in both. MIC
antimicrobial activity is based on protein denaturation of concentration of neem extract and lemon juice was checked
microorganisms according to FDA. Alcohol based herbal by using different dilutions against standard culture and
hand sanitizer is more effective against microorganism than hand isolates. Three different sanitizers were formulated
commercially available hand sanitizer (8). Herbal hand named as lemon, lemon-neem and neem sanitizer. Their
sanitizer offer numerous advantages over commercially antibacterial activity was checked against culture and also
available hand sanitizer onto both hands usually requires compare with sterillium. Formulated sanitizer was found to
only 15 to 30 seconds. Whereas vigorous friction, rinsing be more effective than sterillium. Hence it can be use in
with water, and drying with a towel are not needed like hand hospitals. They also showed anti-biofilm activity. Antibiotic
disinfectants or soaps. Hence intension behind this research potentiation activity of sanitizer against pathogens is
was to formulate herbal sanitizer as it is very beneficial and changing the sensitivity pattern hence can be use in
can easily be use by those people living in slum areas where combination with antibiotic in some formulations.
water is a major issue. Secondly as herbal sanitizer
preparation is very cost effective hence it can be sold at very As more effective is the formulated sanitizer, it can be
low cost as no complex chemicals are use which ultimately brought into market. Neem sanitizer due to its bitter taste it
increases the cost of sanitizer. As three different sanitizers cannot be use as hand sanitizer but can be use as floor
were formulated lemon, lemon-neem and neem sanitizer, cleaner, as disinfectant, toilet spray or to clean medical
among this neem sanitizer has its colour and also a bitter test devices as it do not leave any colour traces. Lemon and
hence it cannot be used as a hand sanitizer but it can be use lemon-neem sanitizer can be use as hand sanitizers. As
as disinfectant or floor cleaner, toilet cleaner or to clean neem and lemon possesses bioactive molecules it can also
medical devices and it does not leave back a colour on be use to formulate cream for wound healing and also for
instrument or floor. treating other skin diseases. Due to its antibiofilm activity,
neem extract and lemon juice both can be use in toothpaste
A number of multidrug resistant microbial strains and or in mouth wash liquid for treatment of dental plaque.
the appearance of strains with reduced susceptibility to
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