NAME: RUTUJA SUNIL PATANKAR

ROLL NO. 15
CLASS: MSc PART II MICROBIOLOGY
RESEARCH PROPOSAL

1
LIST OF ABBREVIATIONS

AST : Antibiotic Sensitivity Test

AWDM : Agar Well Diffusion Method

BZK : Benzalkonium Chloride

CDC : Centers for Disease Control and Prevention

FDA : Food and Drug Administration

FRAP : Ferric Reducing Antioxidant Power Assay

HCAIs : Healthcare Associated Infections

HCFs : Healthcare Facilities

HCPs : Healthcare Practitioners

MDR : Multidrug Resistance

MIC : Minimum Inhibitory Concentration

NCCLS : National Committee for Clinical Laboratory

Standards

WHO : World Health Organization

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SERIAL NO. TOPIC PAGE NO.

1 PROJECT TITLE 3
2 INTRODUCTION 3
3 RATIONALE 7
4 LITERATURE REVIEW 9
5 AIM AND OBJECTIVES 13
6 PLAN OF WORK 13
7 MATERIALS AND METHODS 14
8 EXPECTED RESULTS 21
9 REFERENCES 22
10 APPENDIX

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1. PROJECT TITLE: Formulation of Herbal sanitizers and determining
their antimicrobial activities against skin pathogens.

2. INTRODUCTION:

The word hygiene derives from the ancient Greek goddess Hygeia, the
goddess of healing. Hygiene refers to behaviors that can improve
cleanliness and lead to good health, such as frequent hand washing, face
washing and bathing with soap and water. In many areas of world, practicing
personal hygiene etiquette is difficult due to lack of clean water and soap.
Many diseases can be spread if the hands, face, or body are not washed
appropriately at key times. Thousands of people die every day around the
world from infections acquired while receiving health care. Hands are the
main pathways of germ transmission during health care. Hand hygiene is
therefore the most important measure to avoid the transmission of harmful
germs and prevent health-care associated infections.

Hand hygiene is now regarded as one of the most important element

of infection control activities. In the wake of the growing burden of health
care associated infections (HCAIs), the increasing severity of illness and
complexity of treatment, superimposed by multi-drug resistant (MDR)
pathogen infections, health care practitioners (HCPs) are reversing back to
the basics of infection preventions by simple measures like hand hygiene.
This is because enough scientific evidence supports the observation that if
properly implemented, hand hygiene alone can significantly reduce the risk
of cross-transmission of infection in healthcare facilities (HCFs) (Mathur, P.,
2011)

Washing hands with soap and water is the best way to reduce the

4
number of microorganisms (germs) on hands. When soap and water are not
readily available, alcohol based hand sanitizers or rubs are acceptable but
only if the hands are not visibly soiled. The effectiveness of alcohol-based
hand sanitizers is reduced when used on hands that are visibly soiled or
dirty. Visible contamination and dirt should be removed before using hand
sanitizers and can be achieved by using a hand wipe.

Hand sanitizers are effective against bacterial, fungal infections, as

well as enveloped viruses, such as the common cold and flu viruses and in
preventing nosocomial infections caused by different opportunistic
microorganisms. Alcohol rub sanitizers containing at least 70% alcohol kill
99.9% of the bacteria on hand 30 seconds after application and 99.999% in 1
minute. ( Thombare, M. and Udugade,B. et al.,2015 ). To reduce infections
in health care settings, alcohol-based sanitizer is recommended as a
component of hand hygiene .The FDA recommends that alcohol-based hand
sanitizer have a concentration of 60 - 95 percent ethanol or isopropanol
which is the concentration range of greatest germicidal efficacy. If hands
arent actually grimy, the best way to clean them is to use hand sanitizer,
says James Scott, a microbiologist at the University of Torontos Dalla Lana
School of Public Health.

Most hand sanitizers active ingredients consist of either ethanol or

isopropanol, both forms of alcohol. Alcohol kills most germs on contact
without causing serious harm to the skin tissue, which makes it an effective
active ingredient for hand sanitizers. Ethanol and isopropanol are antiseptics
that kill germs by dissolving their essential proteins. This disrupts the
normal cell activity of the germ, causing it to die. To aid in application, and
increase the skin benefits of the product, hand sanitizers often use inactive

5
ingredients alongside ethanol or isopropanol. For example, humectants, such
as glycerin, work as moisturizing agents. Humectants draw moisture from
the surrounding environment and hold it close to the skin. Thickening
agents, such as polyacrylic acid, may also be used to give hand sanitizers a
gel like texture, which aids in the application and spreading of the product
on the hands. A newer development in hand sanitizers is the use of fragrance
oils to help lessen the smell of alcohol when applying the product.

The alcohol based version is on the World Health Organization's List

of Essential Medicines, the most effective and safe medicines needed in a
health system (WHO, 2015)

DO 'NATURAL' HAND SANITIZERS ACTUALLY WORK?

Since so many grocery items and household cleaners have veered to

the natural and organic end of the spectrum, it was only a matter of time
before consumers started demanding the same options for their hand
sanitizing needs. Cleansing products powered by natural essences like witch
hazel, bitter orange peel extract, thyme, lavender (a popular one), and even
organic alcohol are on the rise. They all sound way less harsh and much
more delicious than ethyl alcohol (the active ingredient in Purell) or toxic-
sounding agents like Triclosan or benzalkonium chloride (BZK).

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A proper protocol for hand sanitizing (Babeluk, R. et al., 2014)

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3. RATIONALE:

India has rich tradition of plant based knowledge of healthcare. The

use of the plant based medication is gradually becoming popular throughout
the world. Approximately, half of the worlds twenty five bestselling
pharmaceutical agents are derived from natural products (Abelson, P.H.,
1990)

As various sanitizers which are available in market are made up of

complex chemicals which can affect our skin like skin rashes and redness as
well as increase its cost. So to reduce the use of such chemicals we can try
making Herbal sanitizers. Even, traditional healers have long used plant to
prevent or cure infectious conditions. Plants are rich in wide variety of
secondary metabolites such as tannins, terpenoid, alkaloids, and flavonoids
etc. which have been found to possess in-vitro antimicrobial properties.
(Thombare, M.A. et al., 2015). There are various medicinal plants which has
antimicrobial properties and antioxidant activity, which can be used in
sanitizers to minimize their side effects and to reduce cost and also to
increase its effectiveness. Like, neem (Azadirachta indica) extract is an
important source of compounds having anti-microbial, anti-oxidant, anti-
tumor, anti-malarial, anti-fungal, anti-inflammatory and anti-viral properties.
Using plant parts of neem had beneficial effect in controlling the pathogenic
microbial organisms and thus can be used in therapeutic formulations in near
future (Raut, R.R. et al., 2014). Also, lemon (Citrus limon) is an important
medicinal plant of the family Rutaceae. It is cultivated mainly for its
alkaloids, which are having anticancer activities and the antibacterial
potential in crude extracts of different parts (viz., leaves, stem, root, fruit and
flower) of lemon against clinically significant bacterial strains has been

8
reported (Kawaii, K. et al., 2000).The essential oil extracted from
tulsi (Ocimum tenuiflorum) showed antimicrobial activity against S.
aureus (including MRSA) and E. coli, but was less active against P.
aeruginosa. Tulsi essential oil could be a valuable topical antimicrobial
agent for management of skin infections caused by these organisms.
(Yamani, H.A. et al.,2016). So such different medicinal plants can be used
for formulation of Herbal Sanitizer.

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4. LITERATURE REVIEW:

Hand hygiene is a general term referring to any action of hand

cleansing which includes washing hands with the use of water and soap or a
soap solution, either non-antimicrobial or antimicrobial or applying a
waterless antimicrobial hand rub to the surface of the hands (e.g. alcohol-
based hand rub). When performed correctly, hand hygiene results in a
reduction of micro-organism on hands.

Hand washing with soap removes the body's own fatty acids from the
skin, which may result in cracked skin that provides an entry portal for
pathogens (Larson, E.L. et al., 1998). In contrast, high-quality hand
disinfectants contain additional skin care products, like emollients.
(Lauharanta, J. et al., 1991). They also do not require the use of water, which
makes the application easy and uncomplicated. Unfortunately, clean water is
not always available. In these cases the CDC recommends using an alcohol-
based hand sanitizer, which is capable of killing most germs. (Sherwood, C.,
2017)

Different studies and sanitizer formulation were carried out. Sanitizer

with complex chemicals as well herbal sanitizer was formulated and their
efficacy were checked against various groups of organisms.

Like, in this research three chemical sanitizers were evaluated. The

products tested were: Sterillium (perfumed, liquid), desderman pure gel
(odorless, gel) and Lavit (perfumed, spray). Sterillium and desderman are
EN1500 (hygienic hand rubs) certified products (available in pharmacy) and
Lavit is non EN1500 certified and available in supermarkets. The two
EN1500 certified products were found to be significantly superior in terms

10
of reducing bacterial load. Desderman pure gel, Sterillium and Lavit reduced
the bacterial count to 6.4%, 8.2% and 28.0% respectively. After education of
the correct use of each hand rub, the bacterial load was reduced even further,
demonstrating the value of education in improving hand hygiene. Efficacy,
followed by skin compatibility was found to be the two most important
attributes of a hand disinfectant (Babeluk, R. et al., 2014).

According to the Comparative assessment of antimicrobial efficacy of

different hand sanitizers Sterillium possessed maximum antimicrobial effect
against all the Gram-positive as well as Gram-negative bacteria used in the
study, followed by Dettol, Lifebuoy, and PureHands respectively.
(Jain, V. M. et al., 2016)

But these types of sanitizers are costly as complex chemicals are use
and it can affect our skin as such products depend upon skin compatibility.
So in such case herbal products gets a more demand as they are less costly as
well more effective with no side effects.

Like, in one of the research main purpose of study was to help to create
a product which is cheaper and to help people who do not have enough
money to buy expensive hand sanitizer. The procedure in making this
product is simple. They scrape the gel inside the Aloe Vera leaves and put
into a plastic container and added 1 cup of ethyl alcohol and 2 teaspoons of
glycerin. Next, they grated the orange to get the zest and added it to the
mixture, then, mix till well blended. Lastly, the group recycled a small bottle
that was used as a container for the mixture. The researchers concluded that
the aloe vera gel can be used to make the hand sanitizer more jelly and the
zest of the orange more hydrating to the skin. It is easy to make since the

11
materials used are found in stores and have the cheapest price. The group is
confident that the product is effective because of the ethyl alcohol.
(Bernabe, et al., 2013)

Plants have medicinal, pharmaceuticals and cosmetic potential, using

it in many innovative products can be useful for humans. In this study herbal
hand wash was formulated using soxhlet fractions of different plant Ocimum
sanctum (Holy basil), Aloe Vera gel, Sapindus mukorossi (Reetha) fruit,
Azadirachta indica (Neem), Eucalyptus spp. (Nilgiri) and citrus fruit. These
plants are traditionally known to possess different medicinal properties.
Among the plant extracts used, Sapindus mukorossi (Reetha) and citrus
fruits showed potent antimicrobial activity against skin pathogen. All three
herbal hand wash formulations showed antibacterial activity higher than
commercially available standard soap solution. (Londhe, J. et al., 2015).

Same way in one of research the antimicrobial activity of the extracts of

leaves and bark of Cassia fistula (Golden rain tree) and Milletia pinnata
(Pongam tree) was evaluated against certain organisms. The extracts
showing good antimicrobial activity. The methanolic bark extract of Cassia
fistula showed maximum activity. Also the methanolic leaf extract of
Milletia pinnata showed good activity. This study concludes that these
plants can be formulated into topical disinfectant formulations and used to
control resistant microbes causing nosocomial and community acquired
infections. From same leaves Polyherbal soap and Herbal sanitizer was also
formulated and their antimicrobial activity were tested against skin
pathogens and concluded that this formulations can further be standardized
and can effectively use as an antiseptic and disinfectants.( Khanam,S. and
Afsar, Z., 2013, 2016)

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 Some other herbal hand sanitizer, incorporating the leaves extracts of
Ocimumcanctum Linn.(Tulsi) and Eucalyptus globulus (Nilgiri), the well-
known herbal combination with multidimensional activities was formulated
and their respective antimicrobial efficacy were studied. The formulation
was evaluated against the specified microorganism by culture sensitivity
test. The significance was found to be more. (Wani, N. S. et al., 2013)

This study was aimed to evaluate the antimicrobial efficacy and safety
of PureHands Herbal Hand Sanitizer in healthy volunteers and also on
inanimate objects. The present study was an open, non-comparative
prospective study. Sterile cotton swab sticks were used to take swabs from
both hands and swabs were inoculated on the petri dishes marked before
application (before) in both aerobic and anaerobic media. Approximately 0.5
ml of PureHands Herbal Hand Sanitizer was squeezed out on the palms of
the subjects and they were asked to rub the gel thoroughly on the palms,
back of the hands, fingernails until the hands became dry and inoculation
was done on the respective dishes, in the part marked (after). The same
procedure was repeated for seven consecutive days on all subjects. For
evaluating the efficacy and safety of PureHands Herbal Hand Sanitizer on
inanimate objects preparation similar procedure was followed. PureHands
Herbal Hand Sanitizer was found to be effective, safe and less likely to
cause adverse skin reactions and saves time and human resources. It was
concluded that PureHands Herbal Hand Sanitizer has a significant
bacteriostatic effect on the bacteria present on the hands surface and on the
surface of inanimate objects. (Mondal, S. and Kolhapure, S.A, 2004)

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5. AIM: To formulate Herbal sanitizers and determining their antimicrobial
activities against skin pathogens.

6. THE SPECIFIC OBJECTIVE INCLUDES:

1. To get an extract of plant by methanol extraction of neem leaves and tulsi

leaves and extraction of lemon juice.

2. Phytochemical analysis and studying the antioxidant activity of all

extracts.

3. To formulate herbal sanitizer from extract of neem, tulsi and lemon juice.

4. To study the antimicrobial and antifungal activity of herbal sanitizer.

5. To study the anti-biofilm activity of herbal sanitizer.

7. PLAN OF WORK:

1. Preparation of neem, tulsi leaf extract and lemon juice by methanol

extraction.

2. Phytochemical analysis of all three extract.

3. Antioxidant activity of extract by FRAP assay.

4. Preparation of sanitizer.

5. To study antimicrobial and antifungal activity of sanitizer,

A. Agar well diffusion method

B. MIC determination

6. Anti-biofilm activity by Tube Method.

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8. MATERIALS:

For extraction:

90% Methanol

For phytochemicals:

Fehlings A and Fehlings B solution

5% Ninhydrin solution
1% lead acetate
95% Ethanol,
Concentrated HCL
Magnesium turnings
Chloroform
Sulphuric acid
Acetic anhydride

For Antioxidant assay:

0.2 M sodium phosphate buffer (pH 6.6)

K3Fe(CN)6 solution
10% TCA solution
0.1% ferric chloride
Ascorbic acid

For Sanitizer:

70% Isopropyl alcohol

Glycerol

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For Antibiotic Sensitivity Test:

Mueller-Hinton agar
Antibiotic disc

Chloramphenicol, C- 10mcg

Trimethoprim, TMP- 5mcg

Tetracycline, Te- 10mcg

Oxacillin, OX- 10mcg

For Agar well diffusion method:

Mueller-Hinton agar

For Tube method (anti-biofilm)

Trypticase soy broth with 1% glucose

Phosphate buffer saline (pH 7.3)
Crystal violet (0.1%)

9. METHODOLOGY:

i. Preparation of Leaf extract: (Wani, N.S. et al., 2013)

Dry the Tulsi and Neem Leaves in oven at 50C and coarsely grind to
get a powder. This leaf powder is to be stored in fridge for further use.
For methanol extraction, 10g of leaves powder and 90% methanol is
added in 250ml flask (maceration) and kept on shaker for 48 hours.
After maceration, filtered the sample by whatman No.1 filter paper.
Heat this filtrate on water bath at 60C till thick sticky, dark coloured
extract is obtained. This extract is stored at 4C for sanitizer
preparation.

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ii. Preparation of Lemon extract:

Wash Lemon, cut into half and squeeze to get a juice. Store this juice at
4C for further use.

iii. Phytochemical analysis: (Choudhari, S. et al., 2016)

i. Test for carbohydrates (Fehlings test): 1 ml of Fehlings A

and 1ml of Fehlings B solution add to 0.5 mg of extract and
boil it in a water bath. The formation of yellow or red
precipitate indicated the presence of reducing sugar.

ii. Test for amino acid (Ninhydrin Test): To a small amount of

extract add a few drops of 5% Ninhydrin solution. Then heat
the solution in a water bath for 10 mins. Purple colour will
appear if amino acids are present.

iii. Test for phenols and tannins (lead acetate test): To 2-3 ml of
alcoholic extract, add 0.5 ml of 1% lead acetate and the
formation of white precipitate indicate the presence of tannins
and phenolic compounds.

iv. Test for Flavonoids (Shinoda Test): To extract add 5ml of

95% ethanol. Add few drops conc. HCL and 0.5g magnesium
turnings. Formation of pink color indicates the presence of
Flavonoids.

v. Test for Saponins (Foam test): Dilute the extraxt with distilled
water and shake well in a graduated cylinder for 15 min. The
persistent foam to a length of 1cm indicates the presence of
Saponins.

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 vi. Test for Terpenoids: Add 4 mg of extract treated with 0.5 ml
of acetic anhydride and 0.5 ml of chloroform. Then add
concentrated solution of sulphuric acid slowly and red violet
color will observe for terpenoid.

vii. Test for steroids and sterols (Salkowskis test): Dissolve 2

ml of extract in 2 ml of chloroform and 2ml of concentrated
sulphuric acid along the sides of the test tube. The upper layer
turns red and lower layer turns yellow with green fluorescence,
indicates the presence of the steroids and sterols compound in
the extract

iv. Antioxidant assay:

FRAP ASSAY (Maruthamuthu, V. and Kandasamy, R. 2016):

Different concentrations of the methanolic extract of Neem and Tulsi
and its various fractions (10-50 g /mL) add to 2.5 mL of 0.2 M
sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium
ferricyanide [K3Fe(CN)6] solution. Vortexed the reaction mixture well
and then incubate at 50C for 20 min using vortex shaker. At the end
of the incubation, add 2.5 mL of 10% trichloroacetic acid to the
mixture and centrifuge at 3,000 rpm for 10 min. Then mix supernatant
with 2.5 mL of deionised water and 0.5 mL of 0.1% ferric chloride.
The colour solution reading to be taken at 700 nm against the blank
with reference to standard using UV Spectrophotometer. Here,
ascorbic acid can be use as a reference standard.

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v. Preparation of sanitizer:

1. Lemon sanitizer: Add 44ml of 70% Isopropyl alcohol in flask. In that

add 2ml Glycerol and 4ml Rose water. Then add 50ml Lemon juice.

2. Neem-Lemon sanitizer: Add 44ml 70% Isopropyl alcohol in flask. In

that add 2ml Glycerol and 4ml Rose water. Then add 25ml Neem
extract and 25ml lemon juice.

3. Tulsi-Lemon sanitizer: Add 44ml 70% Isopropyl alcohol in a flask.

In that add 2ml Glycerol and 4ml Rose water. Then add 25ml Tulsi
extract and 25ml lemon juices.

vi. Antimicrobial and Antifungal activity of sanitizer:

Antibiotic sensitivity test (AST) of selected pathogens: 3 skin

pathogens Pseudomonas aeruginosa, Staphylococcus aureus,
Methicillin-resistant Staphylococcus aureus (MRSA) and yeast
Candida albican is selected for testing, to study the susceptibility of
bacteria to various antibiotics. Antibiotic was selected by referring
croHmans chart.

Agar well-diffusion method: (Abbas, S.Z. et al, 2016) For this

method, Mueller-Hinton agar plates to be use. Agar surface of each
plate is to be streaked by a sterile cotton swab with the reference
bacterial strain. Then punch agar plate with a sterile cork borer of 4
mm size and then pour 100 L of each sample (sanitizer) with
micropipette in the bore. Allow plates to stand for 30 min. Then
incubate the plates at 37C for 48 h. Zone of inhibition (mm) of the
bacterial strains against three Herbal sanitizers by Agar well diffusion

19
 method (AWDM) by standard protocol. A standard method of
National Committee for Clinical Laboratory Standards NCCLS.

vii. Effect of sanitizer on biofilm: (Hassan, A., et al., 2011)

Tube method: This is a qualitative method for biofilm detection.

Inoculate a loopful of test organisms in 10 mL of trypticase soy broth
with 1% glucose in test tubes. Incubate tubes at 37C for 24 h. After
incubation, decant tubes and wash with phosphate buffer saline (pH
7.3) and dry it. Tubes then stained with crystal violet (0.1%). Excess
stain is washed with deionized water. Dry the tubes in inverted position.
The scoring for tube method is to be carried according to the results of
the control strains. Biofilm formation considered positive when a
visible film lined the wall and the bottom of the tube. The amount of
biofilm formed is score as 1-weak/none, 2-moderate and 3-high/strong.

10. EXPECTED RESULTS:

1. Expected that all three herbal sanitizers should show an anti-microbial

activity against skin pathogens. As test for phytochemicals is to perform. It
is expected that

2. They should show an anti-biofilm activity.

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11. REFERENCES:

1. Hassan, A., Usman, J., Kaleem, F., Omair, M., Khalid, A., Iqbal,M.
(2011) Evaluation of different detection methods of biofilm formation in
the clinical isolates Brazilian Journal of Infectious Diseases volume 15,
issue 4, pg. 305-311.
2. Bernabe, Desierto, N.C., Maliclic, G.A., Manglangit, C. A.,
Marasigan, T. M., Austin. (2013) Citrus fruits as a bacterial hand
sanitizer.
3. Bolon, M.K. (2016) Hand Hygiene: An Update Infectious disease clinics
of North America, volume 30, issue 3, pg. 591607.
4. Kolhapure, S.A., Mondal, S. (2004) Evaluation of the antimicrobial
efficacy and safety of PureHands herbal hand sanitizer in hand hygiene
and on inanimate objects volume 101, issue 2, pg. 55-57.
5. Larson, E.L., Hughes, C.A., Pyrek, J.D., Sparks, S.M., Cagatay, E.U.,
Bartkus, J.M. (1998) Changes in bacterial flora associated with skin
damage on hands of health care personnel American Journal of infection
control volume 26, issue 5, pg. 513-521.
6. Yamani, H.A., Pang, E.C.,Deighton,M.A. (2016) Antimicrobial Activity
of Tulsi (Ocimum tenuiflorum) Essential Oil and Their Major
Constituents against Three Species of Bacteria volume 7.
7. Londhe, J., Jagtap,S.D., Doshi, C., Jagade, D. (2015) Formulations of
Herbal Hand Wash with Potential Antibacterial Activity International
Journal of Research in Advent Technology.
8. Lauharanta,J., Ojajarvi, J., Sarna, S., Makela, P. (1991) Prevention of
dryness and eczema of the hands of hospital staff by emulsion cleansing
instead of washing with soap Journal of hospital infection volume 17,

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 issue 3, pg. 207-215.
9. Maruti, J., Dhanavade, Chidamber, B., Jalkute, Jai, S., Ghosh and
Sonawane, K. D., (2011) Study Antimicrobial Activity of Lemon (Citrus
lemon L.) Peel Extract British Journal of Pharmacology and Toxicology,
volume 2, issue 3 pg. 119-12.
10. Maruthamuthu., and Kandasamy., (2016) Ferric reducing anti-
oxidant power assay in plant extract Bangladesh Journal of
Pharmacology volume 11, pg. 570-572.
11. Thombare,M.A., Udugade,B.V., Hol,T.P., Mulik,M.B, Pawade, D.A.
(2015) Formulation and evaluation of novel herbal hand sanitizer Indo
American Journal of Pharmaceutical Research, volume 5, issue 01, pg.
483-488.
12. Wani, N.S., Bhalerao, A.K., Ranaware, V.P., Zanje, R. (2013)
Formulation and Evaluation of Herbal Sanitizer International Journal of
PharmTech Research Volume 5, issue 1, pg. 40-43.
13. Mathur, P. (2011) Hand hygiene: Back to the basics of infection control
The Indian journal of Medical research, volume 134, Issue 5, pg. 611
620.
14. Raut, R. R., Sawant, A. R., Jamge, B. B. (2014) Antimicrobial activity
of Azadirachta indica (Neem) against Pathogenic Microorganisms
Journal of Academia and Industrial Research (JAIR),Volume 3, Issue 7,
pg. 327-329.
15. Babeluk, R., Jutz, S., Mertlitz, S., Matiasek, J., Klaus, C. (2014)
Hand Hygiene Evaluation of Three Disinfectant Hand Sanitizers in a
Community Setting Journal.pone.0111969 Volume 9, Issue 11.
16. Choudhari,S., Sutar,M., Chavan, M. (2016) Formulation, evaluation
and antibacterial efficiency of herbal hand wash Indo American Journal

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 of Pharmaceutical Research volume 6, issue 4, pg. 5202-5209.
17. Abbas, S.Z., Hussain, K., Hussain, Z., Ali, R., Abbas, T. (2016) Anti-
Bacterial Activity of Different Soaps Available in Local Market of
Rawalpindi (Pakistan) against Daily Encountered Bacteria Volume 7,
Issue 11.
18. Mishra,U.S., Murthy, P.N., Pasa,G., Nayak, R. K. (2011)
Formulation & Evaluation of Herbal Gel Containing Methanolic Extract
of Ziziphus Xylopyrus Asian Journal of Biochemical and Pharmaceutical
Research, Volume 1, Issue 4 , pg. 207-218.
19. Jain, V.M., Karibasappa, G.N., Mali, G.V. (2016) Comparative
assessment of antimicrobial efficacy of different hand sanitizers: An in
vitro study Dental research journal volume 13, issue 5, pg. 424-431.
20. Afsar, Z., Khanam, S. (2014) Antimicrobial activity of the extracts of
cassia fistula and milletia pinnata against ampicillin resistant strains of
clinical origin Asian Journal of Phytomedicine and Clinical Research
volume 2, issue 1, pg. 22-29.
21. Afsar, Z., Khanam, S. (2016) Formulation and evaluation of
Polyherbal soap and hand sanitizer International research journal of
pharmacy volume 7, issue 8.
22. Abelson,P.H. (1990) Medicine from plants volume 247, Issue 4942, Pg.
513.
23.Kawaii,S., Yasuhiko,T., Eriko,K., Kazunori,O., Masamichi,Y.,
Meisaku, K., Chihiroito, Hiroshi,F. (2000) Quantitative study of
flavonoids in leaves of Citrus plants Journal of Agricultural and Food
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24. Sherwood, C. (2017) How Does Hand Sanitizer Kill Bacteria?
http://www.livestrong.com/article/177415-facts-about-hand-sanitizers

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APPENDIX-I

Composition of media

1. Luria Bertani Agar

Casein enzymic hydrolysate 1gm

Yeast extract 0.5gm

Sodium chloride 1gm

Agar 2.5gm

D/W 100ml

pH 7.50.2

2. Mueller-Hinton Agar

Beef extract 0.2gm

Casein acid hydrolysate 1.75gm

Starch 0.15gm

Agar 2.5gm

D/W 100ml

pH 7.30.1

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3. Trypticase Soy Broth with 1% Glucose

Pancreatic Digest of Casein 1.7gm

Sodium Chloride 0.5gm

Papaic Digest of Soybean Meal 0.3gm

Glucose 1gm

Dipotassium Phosphate 0.25gm

D/W 100ml

Ph 7.3 +/- 0.2

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APPENDIX-II

Composition of Reagents

1. 90% Methanol:
Mix 10ml of distilled water with 90ml of Methanol.
2. Fehlings A Solution:
Dissolve 34.64gm of crystalline copper sulphate pentahydrate in water
and dilute up to 500ml volume.
3. Fehlings B solution:
Dissolve 173gm of potassium sodium tartrate tetra hydrate and 50gm
sodium hydroxide in water and dilute to 500ml volume.
4. 95% Ethanol:
Mix 5ml of distilled water with 95ml of Ethanol.
5. 0.2M Sodium phosphate buffer (pH 6.6):
Take 3.12gm of NaH2PO4.2H2O in 100ml D/W and 7.17gm of
Na2HPO4 in 100ml D/W.

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