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Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
139
ISSN 0975-234X (Print)
0975-4377 (Online)
DOI: 10.5958/0975-4377.2020.00024.5
Vol. 12 | Issue-03|
July-September| 2020
Available online at
www.anvpublication.org
Research Journal of Pharmaceutical Dosage
Forms and Technology
Home page www.rjpdft.com
RESEARCH ARTICLE
Formulation, Phytochemical, Physical, Biological Evaluation of Polyherbal
Vanishing Cream, and Facewash
Vani Mamillapalli1*, Mounika Katamaneni1, Vamsi Meghana Tiyyagura1, Praneetha Kanajam1,
Aslesha Padmavathi Namagiri1, Haripriya Thondepu1, Bhavani Appikatla1,
Bhavana Devangam1, Padmalatha Khantamneni2
1Department of Pharmacognosy and Phytochemistry, Vijaya Institute of Pharmaceutical Sciences for Women,
Enikepadu-521108, Vijayawada, Krishna (Dt.), Andhra Pradesh, India.
2Department of Pharmacology, Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu-521108,
Vijayawada, Krishna (Dt.), Andhra Pradesh, India.
*Corresponding Author E-mail: vanimamillapalli@yahoo.co.in
ABSTRACT:
The present study aims at formulation of polyherbal vanishing cream and face wash, determination of total
flavonoid content of herbs used, evaluation of formulations for various physical parameters followed by
antibacterial and antifungal activities. The problems of premature ageing, damage due to irradiation, loss of
pigmentation, moisture, nourishment, and acne can be solved by supplementation of health benefits provided by
selected multiple herbs in the formulations. The use of polyherbal vanishing cream and face wash promotes skin
health and beauty. The selected nine herbs in vanishing cream and eleven herbs in face wash formulations
contain good amount of flavonoids capable of protecting skin against damage. The total flavonoid content of
herbs used was determined, followed by evaluation of various physical parameters such as pH, viscosity,
spreadability and compared with marketed formulations. Further, the formulations were tested against gram
positive S.aureus, gram negative E.coli, and fungus C.albicans which are common associates of acne and
cosmetic appliances. The results indicated that both the formulations displayed better antibacterial and antifungal
activities than marketed formulations. Therefore, they can be tested further for their performance and quality
control parameters.
KEYWORDS: Polyherbal vanishing cream, facewash, skin problems.
Received on 17.04.2020 Modified on 08.05.2020
Accepted on 25.05.2020 ©AandV Publications All right reserved
Res. J. Pharma. Dosage Forms and Tech.2020; 12(3): 139-149.
DOI: 10.5958/0975-4377.2020.00024.5
INTRODUCTION:
Skin, the largest human organ is the first line of defense
for external exposure to UV radiations, dust,
environmental bacteria, fungi etc., which cause ageing
and other infections. Skin has been reported to reflect the
general inner-health status and aging [1].
UV exposure is known to negatively affect immune
system functions, cause erythema, changes in skin
elasticity, structure (roughness, scaling, volume, and
wrinkles), trans epidermal water loss, and skin cancers.
The adverse effects of ultraviolet radiation on the skin,
are caused by excessive generation of reactive oxygen
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
140
species [2]. Prevention is the best and most effective way
to work against skin aging effects [3,4]. The best
prevention strategy against the harmful action of free
radicals is a balanced nutritional diet containing rich
anti-oxidative flavonoids [5]. Acne vulgaris is an
extremely common disorder of skin which includes
follicular hyper proliferation, increased sebum secretion
and colonization of organism. Acne is not infectious
disease, generally caused by various factors such as
environmental pollution, harmful chemicals, bacteria,
fungi and treated by different mechanisms such as
controlling the sebum production, use of antibiotics
against bacteria and fungi that are responsible and
sometimes anti-inflammatory agents [6]. Natural or
synthetic sponges used in daily hygiene and in removing
make-up act as reservoirs and vehicles in the
transmission of bacterial species such as Staphylococcus
epidermidis, Staphylococcus aureus and Escherichia coli
[7]. Some strains of E. coli are pathogenic, which means
that they can cause infection that leads to illnesses, such
as diarrhea, urinary tract infections, respiratory illness,
and pneumonia [8]. S. aureus is the most dangerous of
many common staphylococcal bacteria species. These
gram-positive, sphere-shaped (Coccal) bacteria often
cause skin infections but can also cause pneumonia,
heart valve infections, and bone infections [9]. Candida
albicans, a type of yeast fungus found in moist areas of
human body such as mouth, skin and genitals. Yeast
infections on the skin are called cutaneous candidiasis
which may result due to lack of hygiene, excessive
sweating, and use of harsh facial products, rough
scrubbing, and facial tissue irritation. The skin rash due
to candidiasis leads to itching, ulcers, dry skin patches,
burning, and pimples [10]. Facial skin is delicate, regular
use of ordinary soaps can cause loss of moisture in the
skin. A low fat moisturizer that disperses into the skin is
called as a vanishing cream [11]. The cream acts as a
moisturizer, anti-bacterial, anti-fungal agent, and fairness
expert, removes ageing signs, and provides required
nutrients to the skin. A face wash is a mild cleanser that
does the vital job of cleansing the skin, prevents acne,
anti-wrinkle, anticancer, makes germ free, smooth, fresh,
moisturizes the horny layer without causing any
harshness to the skin, and makes to look younger [12].
Frequently researched antioxidants such as flavonoids,
have been referred as agents capable of promoting skin
health and beauty The use of flavonoids in cosmetic
formulation provides both medicinal and cosmetic
benefits [13]. They have been reported to possess potent
anti-oxidant properties and have been widely used in the
skin care industry either as topically applied agents or
oral supplements in an attempt to prolong youthful skin
appearance. Several thousand molecules having a
polyphenol or flavonoid structure have been identified in
plants being generally involved in defense against UV
radiation, UV-induced skin inflammation, oxidative
stress, DNA damage, and risk of skin cancers or
aggression by pathogens [14]. These polyphenolic
compounds or flavonoids have proven to improve
skin microcirculation, protection of the blood vessels,
[15] inhibit the synthesis of melanin [16], inhibit the
release of inflammatory mediator - histamine [17],
reduce erythema, inhibit the platelet aggregation [18]
and thus produce beautifying effects on the skin surface.
The contribution of the traditional preparations, which
are normally polyherbal, is increasing day by day
because of the general impression that these products are
safe with multiple applications; while the single-
molecule based modern drugs used in allopathic system
can cause severe adverse effects [19]. Topical polyherbal
formulations, containing flavonoids are used against
acne, skin tan, and other skin problems due to their anti-
microbial, anti-inflammatory and anti-oxidant activities.
Skin, the organ of beauty has been an interesting
research fields but also a common field of interest for
humans throughout the years, from ancient times to
nowadays. With this view the medicinal plants for the
study were selected based on literature review. The
current research has been planned to carryout in vitro
flavonoid content of the selected plant species,
formulate, and evaluate the polyherbal vanishing cream
and face wash for pharmaceutical parameters followed
by antibacterial and antifungal activities.
MATERIALS AND METHODS:
The plant materials were collected collected in the month
of December during afternoon from local grounds of
Prasadampadu and Enikepadu, Vijaya Institute of
Pharmaceutical Sciences for Women, Enikepadu,
coordinates 16°32′45″N 80°34′12″E of Vijayawada rural
region, Krishna district, Andhra Pradesh, India. Dr. P.
Satya Narayana Raju, Plant Taxonomist, Department of
Botany and Microbiology, Acharya Nagarjuna
University, Guntur, Andhra Pradesh, India, identified
and authenticated the plant specimens namely jungle
geranium Ixora coccinea, negundo Vitex nigundo,
kalmegh Andrographis paniculata, pomegranate Punica
granatum, bryophyllum Millettia pinnata, pongam
Kalanchoe pinnata, lemon grass Cymbopogon flexuosus
and kachnar Bauhinea variegate (Figure 1a-8b). The
plant materials sandal wood, honey, seeds of mustard,
green gram, fruits of myrobalan, amla, and lemon, were
purchased from the local market. The liquorice root
powder, camphor oil, lemon grass oil, and xanthan gum
were collected from the department of Pharmacognosy,
Vijaya Institute of Pharmaceutical Sciences for Women,
Enikepadu, Vijayawada. Marketed formulations used
were Himalaya kesar, alfalfa face cream and Patanjali
neem, tulsi facewash. The collected plant materials used
in the formulation of vanishing cream are jungle
geranium (Figure 1a and 1b), kalanchoe (Figure 2a and
2b), and bryophyllum (Figure 3a and 3b).The collected
plant materials used in the formulation of face wash are
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
141
nirgundi (Figure 4a and 4b), kalmegh (Figure 5a and 5b),
pomegranate (Figure 6a and 6b), lemon grass (Figure 7a
and 7b), and kachnar (Figure 8a and 8b).
Fig. 1a: Ixora coccinea plant
Fig. 1b: Ixora coccinea leaves
Fig. 2a: Kalanchoe pinnata plant
Fig. 2b: Kalanchoe pinnata leaves
Fig. 3a: Millettia pinnata plant
Fig. 3b: Millettia pinnata leaves
Fig. 4a: Vitex nigundo plant
Fig. 4b: Vitex nigundo leaves
Fig. 5a: Andrographis paniculata plant
Fig. 5b: Andrographis paniculata leaves
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
142
Fig. 6a: Punica granatum plant
Fig. 6b: Punica granatum leaves
Fig. 7a: Cymbopogon flexuosus plant
Fig. 7b: Cymbopogon flexuosus leaves
Fig. 8a: Bauhinea variegate plant
Fig. 8b: Bauhinea variegate leaves
Preparation of extracts and preliminary
phytochemical screening:
The leaves, seeds and fruits were cleaned of debris and
other vegetative parts, washed properly with water, dried
and powdered using an electric mixer, sieved and stored
in an air tight container for further use. All the plant
materials 5g each were soaked in alcohol separately for
seven days, filtered, filtrates were concentrated to get
residues, dried and kept in refrigerator. Further, all the
selected plant materials were subjected to preliminary
phytochemical screening using standard methods (Table
1) [20,21,22].
Table 1: Preliminary phytochemical screening of alcoholic extracts
S. No.
Plant part
Al
Ta and Fl
Sa
St
Tr
1.
Mustard seeds
+
+
-
-
-
2.
Ixora leaves
+
+
+
-
+
3.
Karanj leaves
+
+
-
-
-
4.
Green gram seeds
+
+
-
+
-
5.
Amla fruits
+
+
-
-
-
6.
Bryophyllum leaves
+
+
+
+
-
7.
Liquorice root
+
+
+
-
-
8.
Sandalwood
+
+
-
+
+
9.
Nirgundi leaves
-
+
-
+
+
10.
Kalmegh leaves
-
+
-
+
-
11.
Pomegranate leaves
+
+
+
+
+
12.
Lemon grass leaves
+
+
+
-
-
13.
Kachnar leaves
-
+
-
-
+
14.
Myrobalan fruits
+
+
-
+
-
Al-Alkaloid; Ta-tannin; Fl-Flavonoid;
Sa-Saponin; St-Steroid;Tr-Triterpene
Quantative determination of flavonoids:
The extract (1.5ml) was added to 1.5ml of 2%
methanolic AlCl3 (Finar). The mixture was vigorously
shaken on centrifuge (Lab India) for 5 minutes at 200
rpm and the absorbance was read at 367nm after 10
minutes of incubation (Bio-tech). Quercetin (Sigma-
Aldrich) was used as standard for the calibration curve
(Figure 9, Table 2). The assay was carried out in
triplicate [23].
C = c.V/m
C – Total phenolic compounds mg/g of plant extract
c – The concentration of standard established from the
calibration curve mg/ml
V – The volume of extract in ml
m - The weight of pure plant extract
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
143
Table 2: Total flavonoid content of ethanolic extracts
S. No.
Ethanolic extracts
Flavonoid Content (mg/g)
1
Mustard seeds
32±0.0047
2
Crateva leaves
34±0.0047
3
Karanj leaves
36±0.0047
4
Green gram seeds
34±0.0081
5
Amla fruit
32±0.0094
6
Bryohyllum leaves
33±0.0081
7
Liquorice root
33±0.0081
8
Sandal wood
32±0.0094
9
Nirgundi leaves
34±0.0047
10
Kalmegh leaves
33±0.0081
11
Pomegranate eaves
31±0.0047
12
Lemon grass leaves
35±0.0047
13
Kachnar leaves
33±0.0047
14
Myrobalan fruits
31±0.0047
Values represent mean ± S.D.of three parallel measurements
.
Fig. 9: Standard calibration curve of quercetin for flavonoids
Formulation of poly herbal vanishing cream:
The alcoholic extract was prepared by adding 100ml
ethanol to combined selected powdered crude drugs
(Table 3) 5g each, in to a conical flask, capped with
aluminum foil followed by maceration for 5 days. The
oil phase was prepared by adding stearic acid (17%),
potassium hydroxide (0.5%), sodium carbonate (0.5%)
were taken into a porcelain dish and melted at 70ºC. The
oil phase was prepared by adding alcoholic extract of
crude drugs (4.5%), glycerin (6%), and water (71%) into
another porcelain dish and heated at 70ºC. The aqueous
phase was added to the oil phase by constant stirring at
70ºC. It was allowed to cool to room temperature, by
constant stirring. Perfume (0.5%) was added at last just
before the finished product was transferred to suitable
container [24] (Table 3, Figure 10a).
Evaluation of vanishing cream for physical
parameters:
The vanishing cream was evaluated for various
parameters as per official guide lines [25,26,27] and
compared with standard marketed formulation. The
appearance of the cream was judged by its color,
pearlescence, and roughness [24]. The pH of the
suspension was determined by using digital pH meter
(India Mart) at 270C by dispersing 5g of the sample in 45
ml of water [19]. The formulation was tested for the
homogeneity by visual appearance and touch [24].
Spreadability may be expressed by the extent of the area
to which the topical application spreads when applied to
the affected parts on the skin. The spreadability value
also determines therapeutic efficiency of the
formulation. Lesser the time taken for separation of two
slides, better the spreadability [28]. Hence, it was found
necessary to determine the spreadability of the
formulation. The most widely used method for
determining the spreadability of semisolid preparations
is parallel plate method. A modified laboratory apparatus
was used to evaluate spreadability. The setup consists of
two glass slides placed on a tripod stand. Excess of
cream (3g) was placed in between glass slides. The
upper slide is movable and the lower slide was made
immovable, firmly fixed to the stand. 100g weight was
placed on the slides for a period of 5 min to compress the
cream to uniform thickness. The excess cream was
scrapped off. Then 50g weight was added to one side of
the slide and the slide was pulled till it covers a distance
of 10cm. The time required to separate two glass slides
by a distance of 10cm was taken as a measure of
spreadability and was noted in seconds. A shorter
interval indicates better spreadability. Spreadability was
calculated by using the formula given below. The
determinations were carried out in triplicate and the
average of three readings was recorded. The results were
given in the table 4, and figure 10a [29].
S=m.l/t
Where,
S=Spreadability, m=Weight tied to upper glass slide,
l=Length of glass slide, t= Time taken to separate them.
Wetness was determined by applying cream on skin
surface of human volunteer. Type of smear was
determined by applying the cream on the skin surface of
human volunteer. After application of cream, the type of
film or smear formed on the skin was checked [30].
Emolliency, and amount of residue left after the
application of fixed amounts of cream along with
slipperiness was checked. The viscosity determinations
were carried out at 25ºC using a Brookfield Viscometer
(DV II+ Pro model), spindle number S-64 at 20rpm
speed. The determinations were carried out in triplicate
and the average of three readings was recorded [19]. The
type of emulsion of the prepared vanishing cream was
determined by dilution test and dye solubility test. In the
dilution test the emulsion (cream) was diluted with water
to determine the type of emulsion. The emulsion on
dilution with water, was stable as water is the dispersion
medium indicating o/w type of emulsion. In the dye
solubility test the emulsion (cream) was mixed with a
water soluble dye (Amaranth) and observed under the
microscope. The continuous phase appeared red,
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
144
indicating o/w type emulsion as water is in the external
phase and the dye will dissolve in it to give color [19]
(Table 4).
Formulation of poly herbal face wash:
Desired quantities (5g) of herbal drugs (Table 5) were
weighed and each herb is separately macerated with rose
water (100ml) in conical flask with moderate shaking for
3 days [28]. After 3 days filtration of the extracts was
carried out two times. The extracts were evaporated on
water bath at 60ºC temperature to a desired consistency.
The required quantity of gelling agent i.e. xanthan gum
(Table 5) was weighed accurately and dissolved in hot
rose water (not more than 60ºC; 50% weight of the batch
size) with moderate stirring. Air entrapment was avoided
and allowed to soak overnight. Required quantity of
lemon juice was dissolved in desired amount of honey
by gentle stirring. The required amount of concentrated
herbal extracts were added to the remaining amount of
rose water and mixed with above honey mixture by
gentle stirring. This was finally mixed with previously
soaked xanthan gum formulation. Prepared formulation
was filled in a suitable container and labeled accordingly
(Table 5, and Figure 10b).
Table 3. Formulation of vanishing cream
S. No.
Ingredients
Qty %
Therapeutic, cosmetic uses
1.
Mustard seeds, Brassica nigra
0.54
Rubefacient, removes dead skin, Skin whitening facemask,
antioxidant, moisturizer, antifungal [31]
2.
Ixora leaves, Ixora coccinea
0.54
Balancing, stimulant, moisturizer, antitoxic, antioxidant,
antimicrobial, anti-inflammatory [32]
3.
Karanj leaves, Kalanchoe pinnata
0.54
Antiseptic, anti-inflammatory [33]
4.
Green gram seeds, Vigna radiata
0.54
Rubefacient, nutrient, immune booster, antiageing, improves skin
glow, removes tan, excess oil, blackheads [34]
5.
Amla fruits, Emblica officinalis
0.54
Antioxidant, brightens complexion, skin softener, tightener,
exfoliator, cleanser, antimicrobial, antiageing, anti acne [35]
6.
Brypphyllum leaves, Millettia pinnata
0.54
Anti-inflammatory, antiallergic, antiseptic, antimicrobial [36]
7.
Liquorice root, Glycyrrhiza glabra
0.54
Soothening, highly rejuvenating, nutritive [37]
8.
Sandal wood, Santalum album
0.54
Moisturiser, balancing, stimulating, antiseptic, astringent, anti-
inflammatory, antibacterial, disinfectant, brightener, reduces sun
burn, blemish marks, acne, dead skin, antiwrinkle [35]
9.
Camphor oil, Cinnamomum camphora
0.5
Antiseptic, cooling, analgesic, anticancer, perfume [38]
10.
Glycerin
6%
Humectant [39]
11.
Stearic acid
20
Waxy substance, thickening agent [39]
12.
KOH
0.5
Prevents harshness of cream on skin [40]
13.
Na2CO3
0.5
Prevents harshness of cream on skin [40]
14.
Purified water
71
Stability of cream [40]
15.
Propyl paraben
0.10
Preservative [41;39]
16.
Methyl paraben
0.06
Preservative [41;39]
Table 4. Evaluation parameters of herbal vanishing cream
Physical parameters
Test formulation
Marketed formulation
Appearance
Smooth
Smooth
Colour
Creamish white
Pinkish white
Odour
Pleasant
Pleasant
pH
6.3
6.1
Homogeneity ; By visual and touch
Elegant
Elegant
Spread ability
8sec
6 Sec.
Type of Smear
non-greasy
non-greasy
Emolliency
No residue left
No residue left
Viscosity
27025cps
25623cps
Dilution test
o/w type
o/w type
Dye solubility Test
o/w type
o/w type
Table 5. Formulation of face wash
S. No.
Ingredients
Qty for 50ml
Therapeutic and cosmetic uses
1.
Nirgundi leaves, Vitex nigundo
2.5 ml
Antioxidant, anti-inflammatory, antibacterial, antifungal [42]
2.
Kalmegh plant, Andrographis paniculata
1.25 ml
Anti-inflammatory, antibacterial, antiviral, antifungal,
immunostimulant [43]
3.
Pomegranate leaves, Punica granatum
1.25 ml
Antioxidant, antibacterial, antiwrinkle, skin tightener, improves
blood flow, improves skin glow [44]
4.
Liquorice, Glycyrrhiza glabra
1.25 ml
Soothing, highly rejuvenating and nutritive qualities [37].
5.
Honey, Apis mellifera
2.5 ml
Light humectants, nutrient, thickening agent [45]
6.
Lemon grass oil, Cymbopogon flexuosus
0.5 ml
Anti-inflammatory, antifungal, antibacterial, antiallergic,
antistress [46]
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
145
7.
Kachnar leaves, Bauhinia variegata
1.25 ml
Antidermatosic, improves blood flow, antibacterial, restores pH
balance, improves skin glow. [47]
8.
Lemon Juice, Citrus limonis
0.5 ml
To lighten skin, reduce blemish marks on the skin, quite
effective for treating acne, pimples and as a natural pH adjuster
in cosmetics [48]
9.
Sandalwood, Santalum album
1.25 ml
Moisturiser, balancing, stimulating, antiseptic, astringent, anti-
inflammatory, disinfectant, antibacterial, reduces sun burn,
anti-wrinkle, reduces blemish marks, acne, improves skin
brightness, removes dead skin [35]
10.
Myrobalan fruit , Terminalia chebula
1.25 ml
Antioxiant, antibacterial, antiviral, antifungal, balancing, anti
acne, detoxifier [49]
11.
Xanthan gum, Xanthomonas compestris
3 gm
It is used as a non toxic thickener and stabilizer [50]
12.
Rose water, Rosa indica
q.s.
Solvent, antibacterial and antiseptic, eventually cure acne [51].
13.
Propyl paraben
0.0.5 mg
Preservative [41; 39]
14.
Methyl paraben
0.03 mg
Preservative [41; 39]
15.
Sodium lauryl sulfate
1.05 g
Surfactant, foaming agent [52]
Qty-Quantity
Evaluation of face wash for physical parameters:
The prepared face wash was evaluated for various
parameters as per official guide lines [25,26, and 27] and
compared with standard marketed face wash [53].
Physical parameters such as colour, appearance and
consistency were checked visually. Formulations were
applied on the skin then washed with water and visually
checked for its washability nature [54]. pH of 1 %
aqueous solution of the formulation was measured by
using a calibrated digital pH meter at constant
temperature [55]. The viscosity of face wash was
determined by using Brookfield Viscometer. The Values
Obtained for sample is noted and compared with
marketed formulation. Spreadability was determined by
parallel plate method [28,29]. The face wash was applied
on left hand dorsal surface of 1 sq. cm and observed in
time interval 1 to 2 h for irritant reactions if any [56]
(Table 6).
Table 6. Evaluation parameters of face wash
Parameter
Formulated face wash
Marketed face wash
Color
Greenish-brown
Green
Consistency
Good
Good
Washability
Good
Good
pH
5.2
4.7
Viscosity
1520 cps
1690 cps
Spreadability
Good
Good
Irritation
No
No
Fig. 10a: Prepared vanishing
cream
Fig. 10b: Prepared face
wash
Evaluation of anti-bacterial activity of prepared
formulations:
The micro-organisms gram-positive bacteria
Staphylococcus aureus (NCIM 2794) and gram-negative
bacteria Escherichia coli (NCIM 2256) were selected to
test the anti-bacterial activity i.e. ability to inhibit the
growth of microbes in the formulated vanishing cream
(Table 7 and Figure 11) and face wash (Table 8 and
Figure 12). Prior to testing, each of the bacterial strain
was cultured in nutrient agar plates and incubated for 18
to 24 hrs at 37ºC to obtain colonies. After overnight
incubation, colonies were selected with a sterile
disposable inoculating loop and transferred to a glass
tube of sterile physiological saline and mixed
thoroughly. Each bacterial suspension turbidity was
compared with 0.5 Mc Farland standard solution which
produces turbidity equivalent to 1.5×108 CFU/ml
(Colony forming units). Antimicrobial susceptibility was
tested by using well-diffusion method (National
Committee for Clinical Laboratory Standards). The
formulations were tested on Mueller Hinton plates to
detect the presence of bacterial growth. All plates were
inoculated with the test bacteria which have been
previously adjusted to the 0.5 Mc Farland standard
solution turbidity by the pour plate method.
Table 7: Antibacterial and antifungal activity of vanishing cream
S.
No.
Concentration
(mg/10ml)
Extent of inhibition (mm)
S. aureus
E. coli
C.albicans
Test
Mktd
Test
Mktd
Test
Mktd
1
0.1
6
4
8
5
8
7
2
0.2
8
6
10
7
10
9
3
0.3
9
8
11
9
12
10
Mktd- Marketed
The plates were allowed to dry for 3-5 min to remove the
excess moisture in laminar air flow chamber. 50ul
aliquots of each test and standard marketed cream and
facewash formulations with different concentrations (0.1
mg/10ml, 0.2mg/10ml, 0.3mg/10ml) were dispensed into
each well after the inoculation of the plates with bacteria.
The plates were sealed, labelled, and placed in an
incubator set at 37ºC. After 24h of incubation, each plate
was examined for inhibition zones. Inhibition zone
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
146
reader was used to measure the inhibition zones in
millimeters [57] (Figure 11 and Figure 12).
Evaluation of anti-fungal activity of prepared
formulations:
The fungal organism Candida albicans (ACTM 2091)
was selected for the study of anti-fungal activity of
prepared vanishing cream and face wash formulations.
The fungal organism was cultured in subouraud dextrose
agar (SDA) medium at 22ºC. Colonies were observed
after 5 days, and were diluted to 0.5 Mc Farland which is
equal to 1.5×108 CFU/ml. All plates were inoculated
with the test fungi which have been previously adjusted
to the 0.5 Mc Farland standard solution by pour plate
method in SDA medium. Anti-fungal susceptibility was
tested by using well-diffusion method (National
Committee for Clinical Laboratory Standards). 50µl
aliquots of each test and standard marketed cream and
facewash formulations with different concentrations (0.1
mg/10ml, 0.2mg/10ml, and 0.3mg/10ml) were dispensed
into each well after the inoculation of the plates with
fungi. The plates were sealed, labelled, and placed in an
incubator set at 22°C. After 5 days of incubation, each
plate was examined for inhibition zones. Inhibition zone
reader was used to measure the inhibition zones in
millimeters [57] (Figure 11 and figure 12).
Table 8: Antibacterial and antifungal activity of face wash
S.
No.
Concentration
(mg/10 ml)
Extent of inhibition (mm)
S. aureus
E. coli
C.albicans
Test
Mktd
Test
Mktd
Test
Mktd
1
0.1
13
12
11
12
8
7
2
0.2
15
13
13
13.5
10
9
3
0.3
17
15
15
15
12
10
Mktd- Marketed
RESULTS AND DISCUSSION:
The results of the preliminary phytochemical screening
study indicate that tannins and flavonoids were found to
be present in all the selected plant extracts (Table 1). The
ethanolic extract of karanj was found to contain highest
amount of total flavonoids 36mg/g followed by lemon
grass 35mg/g equivalent to quercetin among the plant
extracts. The plant extracts of jungle geranium (Ixora),
green gram, nirgundi contain total flavonoids 34mg/g
equivalent to quercetin. The plant extracts of
bryophyllum, liquorice, kalmegh, kachnar contain total
flavonoids 33mg/g equivalent to quercetin. The plant
extracts mustard, amla, sandalwood contain total
flavonoids 32mg/g equivalent to quercetin. Pomegranate
and myrobalan were found to contain less amount of
flavonoids 31mg/g equivalent to quercetin when
compared to other plant extracts (Table 2).
Flavonoids are indispensable components in a variety of
pharmaceutical, medicinal, neutraceutical, and cosmetic
applications. Studies have indicated that quercetin helps
to to inhibit the production of superoxide radicals [58,
59], suppress lipid peroxidation, [60], a process
responsible for inflammation, along with ageing [61,62].
Serum flavonoid content of flavonoids improves
capillary blood flow to the dermis. Dermal
microcirculation causes increased cutaneous blood flow
which may contribute to enhanced delivery of oxygen
and nutrients to the skin, a proposed impact of
flavonoids on skin health [63]. This effect leads to
improved skin structure, texture, and water homeostasis.
Flavonoids reduce the UV-induced DNA damage, by
promoting DNA repair. Topical application of
flavonoids may protect skin by absorbing UV rays
before they can interact with and damage cellular
components, thereby providing a sunscreen effect [64].
The prepared formulations comply with parameters as
per official guidelines [25,26,27] (Table 4 and table 6).
The formulations were compared with marketed
formulations (Himalaya kesar, alfalfa face cream). The
appearance of formulated and marketed vanishing
creams was smooth (Table 4 and figure 10a). The
formulated cream was in creamish white, the marketed
cream was in pinkish white colour. The odour of both
the creams was found to be pleasant. The pH value of
formulated cream was 6.3 whereas of the standard cream
was 6.1. Both the formulations were elegant by visual
and touch examination. The spreadability values were 8
sec and 6 sec for formulated and marketed creams. They
were found to be non-greasy and emollient. The
viscosity measurements were 27025cps and 25623cps
(centipoise). They were both o/w type formulations.
The results indicate that the formulated cream passes the
physical parameter tests as per official guidelines [25,
26,27] and further it was compared with marketed
formulation (Table 4). The polyherbal face wash (50ml)
was formulated (Table 5 and figure 10b) as per the
prescribed procedure and evaluated for various physical
parameters according to official guidelines [25, 26, 27].
The formulated polyherbal face wash was evaluated for
physical parameters (Table 6) and compared with
marketed formulation (Patanjali neem and tulsi face
wash). The results indicate that the formulated face wash
was in greenish brown colour. The marketed formulation
was in green colour. The formulations exhibited good
consistency and washability. The pH values measured
were 5.2 for formulated face wash and 4.7 for marketed
face wash.
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
147
Fig. 11 Evaluation of Antibacterial and antifungal activity of vanishing cream
Fig. 12: Antibacterial and antifungal activity of face wash
Research Journal of Pharmaceutical Dosage Forms and Technology. 12(3): July - September, 2020
148
The viscosity values measured were 1520 cps and 1690
cps for formulated and marketed formulations. Both
the formulations exhibited good spreadability and no
irritation. The formulated face wash obeys the physical
parameter tests as per Pharmaceutical guidelines
prescribed, the results were compared to marketed
formulation (Table 6). The formulated polyherbal
vanishing cream was evaluated for antibacterial activity
against gram positive S. aureus, and gram negative E.
coli bacteria. The antifungal activity was tested against
C. albicans. The results were compared with marketed
formulation (Table 7 and Figure 11). The results indicate
that prepared formulation of polyherbal vanishing cream
showed better antibacterial and antifungal activities than
marketed formulation. It showed maximum growth of
inhibition at 0.3mg/10ml concentration. The prepared
cream exhibited better antibacterial activity against gram
negative E. coli (11mm) than marketed formulation (9
mm). It also exhibited better antifungal activity (12mm)
than marketed formulation (10mm). The results of
antibacterial and antifungal activities of the prepared
cream formulation are in line with the findings of Eichie
et al., 2011 [65]. Therefore, it can be quoted that the
prepared formulation acts as an effective antibacterial,
antifungal vanishing cream (Table 5; Figure 6). The
formulated polyherbal face wash was evaluated for
antibacterial activity against gram positive S. aureus, and
gram negative E.coli bacteria. The antifungal activity
was tested against C. albicans. The results were
compared with marketed formulation (Table 8 and figure
12). The prepared formulation showed maximum
antibacterial and antifungal activities at 0.3mg/10ml. The
formulation exhibited better antibacterial and antifungal
activity than marketed formulation at 0.3mg/10ml. The
formulation showed better results towards gram positive
S.aureus. It exhibited activity equivalent to marketed
formulation against E.coli. Therefore, the results form an
evidence to quote that the formulation acted as an
effective antibacterial, antifungal facewash compared to
marketed face wash. The results of antibacterial [66] and
antifungal activities [65] of prepared polyherbal
facewash formulation are comparable to the studies of
Amgad et al., 2015 and Eichie et al., 2011 [66,65]. The
exhibited antibacterial and antifungal activities may be
attributed to the presence of flavonoids in the prepared
formulations.
CONCLUSION:
In the current study polyherbal vanishing cream and face
wash were formulated, evaluated for total flavonoid
content, various physical parameters, antibacterial and
antifungal activities. The results indicated that the
formulations passed the tests for pharmaceutical physical
parameters and exhibited better antimicrobial activities.
The prepared polyherbal formulations nourish,
moisturize, protect the skin against premature ageing,
irradiation, and acne may be due to the presence of
flavonoids. The prepared formulations may be further
studied their performance, quality control tests followed
by isolation, characterisation of responsible flavonoid,
ACKNOWLEDGEMENT:
The authors are thankful to Vijaya Institute of
Pharmaceutical Sciences for Women for providing
infrastructural facilities, encouragement and support.
CONFLICT OF INTEREST:
The authors declared no conflict of interest.
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