Project Complete5884

Extraction Of Oil From Tulsi,Neem And Aloe Vera Gel Formulation Of Multipurpose Herbal Cream
Chapter-1
Introduction
1.1 General
Cream is defined as semisolid emulsions which are oil in water (o/w) or water in oil (w/o) type and
these semisolid emulsions are intended for external application. Cream is classified as oil in water
and water in oil emulsion. It is applied on outer part or superficial part of the skin and its main
ability is to remain for a longer period of time at the site of application. The function of a skin
cream is to protect the skin against different environmental condition, weather and gives soothing
effect to the skin. There are different types of creams like cleansing, cold, foundation, vanishing,
night, massage, hand and body creams. The main aim of our work is to develop a herbal cream
which can give multipurpose effect, like moisturizer, reduce acne and skin irritation, reduce skin
diseases like eczema, psoriasis, dry skin, wrinkles, rashes etc. and also adding glow to the face.
We have used three herbal ingredients in our preparation which are Aloe Vera gel, Neem, Tulsi.
Aloe Vera gel is used as a moisturizer, to reduce pimples and acne and also used for treatment of
burn wounds. Neem is used as an antifungal and anti-inflammatory and it is also used to reduce
scar, pigmentation, redness and itching of the skin. Tulsi is used to add glow to the skin and to
promote wound healing. Herb is a plant that is valued for flavour, scent, medicinal or other
qualities. Herbs are used in cooking, as medicines, and for spiritual purposes. Herbs have a variety
of uses including culinary, medicinal, or in some cases even spiritual usage. General usagediffers
between culinary herbs and medicinal herbs Neem (Azadirachta Indica A. Juss) is a plant in the
Meliaceae family. benefits in pharmaceutical field, A. indica A. Juss has been widely used as
pesticide for various species of pests.
B.Tech Project,Department Of Chemical Engineering ,2022 Page 1

1.2 Extraction Of oil fromTulsi

1.2.1 Collection of plant material: Leaves of Ocimum sanctum L. (tulsi) were collected from
different sites of Bareilly District, Uttar pradesh, washed with sterile water and dried in shades.
Then the samples were powered in mechanical grinder.
Plate 1.1: leaves of Tulsi
Plate 1.2: Tulsi powder

1.2.2 Aqueous, methanol and ethanol extract: The dried tulsi (50g) powder was placed in the
thimble of Soxhlet apparatus and 200- 400 ml of distilled water, methanol and ethanol was used
for extraction procedure and the experiment was done separately for all the two solvents and
distilled water. The extraction was continued till clear solvent or water was seen in the thimble.
The extract was concentrated using rotary evaporator. Then the extract was dried in a digital water
bath till a dark green residue was obtained.
Plate 1.3: sohxlet extraction of tulsi leaves
1.3 Extraction of oil from neem

1.3.1 Collection of plant material
The medicinal plants used for the evaluation of antimicrobial study were Neem (Azadirachta
indica) . Fresh bark and leaves of Neem a leaves were collected in the month of december 2021
from the BDA hostel.

Fig 1.1: neem leaves

Source(https://www.wildturmeric.net/neem-leaves-health-benefits-medicinal-uses-for-skin-hair)
1.3.2 Preparation of the plant material

Leaves and barks were shade - dried and converted into coarse powder by grinder and then stored
at room temperature and were subjected to the following extraction protocols. Preparation of the
Extracts
Fig 1.2: Neem powder

Source (https://www.google.com/search?q=neem+powder&tbm=isch&ved=2ahUKEwjAkIX)

Powdered sample were subjected to successive extraction with water, and 50% ethanol by Soxhlet
method The extracts were collected and distilled off on a water bath at atmospheric pressure.
Extracts were stored in refrigerator for antimicrobial studies.
Plate 1.4: sohxlet extraction of neem leaves
1.4 Extraction of Aloe Vera gel

Mature, healthy and fresh aloe Vera leaves were collected and washed with distilled water. Then
after proper drying of leaves in hot air oven, the outer part of the leaf was dissectedlongitudinally
using a sterile knife. Then the aloe Vera gel that is the colorless parenchymatous tissue was

removed using the sterile knife. Then it is filtered using muslin cloth to remove the fibers and
impurities. Then the filtrate or the filter product which is a clear aloe Vera gel was used in the
preparation.
Fig 1.3: aloe vera leaf

Source(https://www.google.com/search?q=aloe+vera+pulp&tbm=isch&ved=2ahUKEwjittG)
Fig 1.4 Aloe vera pulp
Source(https://www.google.com/search?q=aloe+vera+pulp&tbm=isch&ved=2ahUKEwjitt G)

1.5 Research Gap of Study

There are various studies are done for the formation of herbal cream. But they had only done with
single herbal plant like either they had use Tulsi or Aloe vera or neem none the studied are made on all the
three together to make the herbal cream which is multipurpose latest study was made on the extraction of
oil from the seed of neem in the year 2019. We had done a study on the all the three herbal plants together.
1.6 Scope of the Study

Earlier there were no cream introduce which is completely herbal. There are creams which are only
for single purpose. But this cream is herbal and can be used for multiple purpose like Moisturizer,
Antiseptic, Glowing, and more purposes as well.
1.7 Objectives
Objective of this study is to help people from having different creams for different purposes.
However, by using this cream they can get rid of using multiple creams as the single cream can be used
for multiple skincare purposes thus avoiding the hassles of buying and carrying different skincare
ointments.
 Evaluation of the components we are using independently and analysing the usage.
 Evaluation of the components we are using all together and analysing their effect on human body.
 Formulating the Multipurpose herbal cream and evaluating it.

Chapter-2
Literature review
2.1 General
Extracted samples were analysed by gas chromatography technique (GC) with TCD as detector at
2000 C. Nitrogen were used as carrier gas.
S. V. Taralkar et al. (2014) Many reserchers used various models to describe solid-liquid
extraction process shows, in the study of the mechanisms and kinetics in the extraction process of
water soluble compounds from tilia sapwood, that a model based on a second-order extraction
process was the most suitable model for a solid– liquid extraction process. It was then possible to
build the kinetic models of a solid–liquid extraction and the extraction order and rate constant
remained to be determined by experiments. According to a second-order rate law described the
rate of dissolution for the solute contained in the solid from plant cells to solution by Eq.
dct/dt = k(cs-c1)2
Where k is the second-order extraction rate constant (L g−1 min−1), Cs the extraction capacity
(concentration of solute at saturation in mg L−1) and Ct is the concentration of solute in the
suspension at any time t (min). By considering the initial and boundary conditions, t=0 to t and Ct
=0 to Ct, the integrated rate law for a second order extraction was obtained.
C2.t = Cs K.t / 1+ k. t.c2
By transforming Eq., a linear form shown in Eq. can be obtained and the extraction rat can be
written as
t/ct - 1/kc s2 + t/cs
Ct/t = 1/[1/K+Cs2]+[t/Cs]
The initial extraction rate, h, as Ct/t when t approaches 0, can be defined as

K=K+Cs

and, the concentration of solute at any time can be expressed after rearrange
Ct = t/[1/h]+[t/Cs]
The initial extraction rate, h, the extraction capacity, Cs, and the second-order extraction rate
constant, k, can be determined experimentally from the slope and intercept by plotting t/Ct versus
t.
Sampath Kumar et al. (2010), has reported that Tulsi has got the great medicinal value. Tulsi is
considered to be a ubiquitous plant in India. Ocimum tenuiflorum (tulsi) is an aromatic plant in the
family Lamiaceae. It is an erect, much branched sub shrub 30-60 cm tall with hairy stems and
simple opposite green leaves that are strongly scented. tulsi plays a vital role in our everyday life
and is said to be the queen of herbal plants. it is the most common household plant in india and it
is sacred in hindu tradition. Many Hindu epics explain the importance, properties and uses of tulsi.
Tulsi is an erect sweet scented shrub which grows upto a height of 3 -5 feet. it is commonly grown
in gardens and in the periphery of temples. it has got a pungent taste and fragrant smell. it is the
only plant that can absorb carbon di oxide through-out its life. it releases the oxygen in the early
morning which is beneficial for the people in breathing dis-orders. Tulsi plant has a lot of
significance for mankind, due to the manifold medicinal benefits it provides. Tulsi leaves are
widely used in the preparation of Ayurvedic medicines. It is known to promote the longevity of
life.
Tulsi has got many medicinal properties out of which some are like, Antispasmodic, appetizer,
carminative, galactagogue, stomachic. Basil is antispasmodic, appetizer, carminative,
galactagogue, and stomachic. It is used for stomach cramps, gastric catarrh, vomiting, intestinal
catarrh, constipation, and enteritis. It had been sometimes used for whooping cough as an
antispasmodic.
1. Tulsi has antioxidant properties and reduces blood glucose levels. Thus it is useful for diabetics.
2. Tulsi reduces total cholesterol levels. Thus it is useful for heart disease patients..
3. Tulsi reduces blood pressure.
4. Tulsi is also used to prepare herbal tea. It helps in building up stamina.

5. It has been used for gastric disorders, cough, common colds, malaria, and headaches.
6. It is used as mouth wash for reducing tooth ache
7.Tulsi oil shows larvicidal activity against malarial larva.
8. It has immuno-modulatory properties.
The description of the tulsi leaves and their family genus is:
Tulsi Order: Lamiales
Family: Lamiaceae
Genus: Ocimum
Species: O. tenuiflorum
Binomial name Ocimum tenuiflorum L.
Synonyms Ocimum sanctum
Perperation of the medicine from the tulsi had a procedure like first we take Tulsi seed flour, 125
grams; black pepper seeds, 10 grames; bhang (cannabis),5 grams; Saffron, 2 grammes; almond
seeds, 125 grams; khova (khoya), 125 grams; Bengal gram flour, 125 grams crystal sugar, 250
grams; ghee (clarified butter), 250 grams.
Mix a major part of the ghee with the gram flour. Sprinkle a little milk over the flour. Take the
remaining portion of ghee in an iron or brass pan and put it on the stove. When the ghee is fairly
hot, add the gram flour and let it cook the ghee over a low flame. When the flour is nearly
halfcooked, break up the khova into small lumps, mix it with the gram flour, and continue heating
till both the khova and the flour have been cooked completely, and have begun to turn brown. Now
add the almond seeds, cut into small pieces, and let them cook for some time. Now add the Tulsi
seed flour, immediately after that add the cannabis powder, cardamom and pepper powder
according to your taste, mix well, and take the pan off the fire.
Meanwhile, prepare thick syrup from the sugar, add the saffron to it. The thickness of the syrup
should be adjusted to suit the weather conditions. The thickness (or consistency) of the syrup
should preferably be a little greater in monsoon, otherwise the sweet is liable to spoil easily, and
become soft due to moisture in the air; a thicker syrup gives better result. In the winter, on the
contrary, the syrup should not be made so thick and viscous; thicker syrup results in greater
hardness of the sweet. If it is desired to increase the proportion of ghee in the sweet so as to increase
its nutritive value, somewhat thinner syrup will be needed. Once syrup of the proper consistency

has been prepared, and the saffron added, mix the roasted flours into the syrup with constant
stirring. Now the sweet is ready.
All these medicinal ingredients makes Tulsi a must have for longer and peaceful life. This small
plant is certainly a very good source of medicinal properties. After in depth and rigorous research
it has been proved and certified that it is safe to consume Tulsi in any form. All these remedial
properties are well accepted and honored by modern science. Tulsi is the herb that cures the
mankind from all odds naturally in today’s superficial not-so good lifestyle. It is considered as
India’s Queen of herbs.
Kurniati et al. (2018), explained that Neem (Azadirachta Indica A. Juss) is a plant in the Meliaceae
family. A. indica A. Juss can easily grow in the tropical area with rainfall of 450 - 750 mm/year
above the sea level. It is mainly found in India and its leaf, flower, fruit, seed and root have been
used as medicine. The plant of this family contains secondary metabolites including alkaloids,
flavonoids, quinones, and terpenoids. A. indica A. Juss pharmaceutically carries high anti-oxidant,
anti-diabetes, anti-inflammatory, anti-malaria, antibacterial, and anti-fungal activity. Based onthe
previous research, beside its benefits in pharmaceutical field, A. indica A. Juss has been widely
used as pesticide for various species of pests. There are four important components contained in
neem, those are azadirachtin, salanine, meliantriol and nimbin which are tetranortriterpenoid
beneficial for insect barrier, repellent, and antifeedant. A study on antifeedant activity reported that
the neem seed extract yields antifeedant index (AI) of 98.19 % against plutella xylostella
bioindicator. Its extract against hymenia recurvalis bioindicator gave higher AI of 52.70 %
compared to the result against psara basali bio-indicator which gives AI of 49.85%
In this we researched about the antifeedant activity of neem leaf (A. indica A. Juss) has been
identified by against Tenebrio molitor bioindicator. The highest activity was obtained on ethyl
acetate extract at 0.5% concentration having Antifeedant Index (AI) of 51.53% and most active at
10% concentration of 82.05%. The method used to test the antifeedant activity is the no choice leaf
disk method. Secondary metabolites contained in neem leaf extract (A. indica A. juss) include
terpenoids, steroids, flavonoids, saponins and phenolics.
In this research, we used laboratory glassware, analytical balance of brand CPA 6235
SARTORIUS, tube rack, vacuum rotary evaporator of Hidolph Laborota 4004 Contro brand,
distillation apparatus, drop pipette, micro pipette. The materials which were needed in this research

are n-hexane technical, ethyl acetate technical, methanol technical, reagent Liberman-Bourchard
(acetate acid glacialH2SO4(P)), reagent Mayer (potassium tetraiodomercurate), reagent
Dragendorf (Bi(NO3)3) and reagent Wagner (I2 in KI), silica gel 60 (0.2 – 0.5 mm). T. molitor
bio-indicator and spinach leaf as test media. The plant material in this research is neem leaf
(Azadirachta indica A Juss) from Darussalam, Syiah Kuala district, Banda Aceh city. Sample
voucher was identified by Saida Rasnovi and stored in herbarium taxonomy in Biology
Department, Faculty of Mathematics and Natural Sciences, Syiah Kuala University.
Antifeedant activity was tested by employing no choice leaf disk method. The rough extract of A.
indica A. Juss leaf was made into four concentrations which were 0.5, 1, 5 and 10%. Fresh spinach
leaves were used as the test media. The spinach leaves were dipped in to the extracts for 1 min,
dried for 15 min, and weighed to record their masses (X1). The leaves were placed in test containers
and added in 5T. molitor bio-indicators. The containers then were covered with gauze. Observation
was conducted after 1 x 24 h of the application. The spinach leaves were weighed to record their
shrunk masses. The difference of X2 and X1 indicated the leaves’ masses scraped by the bio-
indicators (treatment) while the fresh spinach leaves dipped in the solvents were the controls (Eq.).
−
%AI= *100%
+
The result of, Preparing the A. indica A. Juss leaf sample by drying it was done to blot out the
containing water in the sample. The dried sample was refined to widen its surface such that the solvent
can penetrate through the cell wall and accelerate the extraction process of the secondary metabolite
compound. Maceration in the solvent could draw out the compound contained in the plant. n-hexane
extract drew out non-polar compound, ethyl acetate drew out semi-polar compound, while methanol
drew out polar compound. The results of A. indica A. Juss leaf extraction are presented in Table
Extract Sample Dried Extract(g) Yield(%)
n-Hexane 18.83 1.25
Ethyl acetate 82.10 5.47
Methanol 93.09 6.21
Table 2.1: Result of Neem Leaf Extract

Antifeedant’s work mechanism happens by producing feeding inhibition stimulant such that it disturbs
the stimulation perception for eating and affects the growth and the development of hormonal system
(neuroendokrin). Moreover, it also acts as ecdysone bloker that insects fail to moult their skin (moulting
inhibition) such that it causes anatomical abnormality and death. The difference in the antifeedant
indices is caused by the different concentrations of the chemical compounds contained in the each
extract, which affects the behavior of the insects. The higher the concentration is, the higher the
antifeedant activity is.
The present study shows that neem leaf (A. indica A. Juss) extract contains antifeedant activity to the
bio-indicator T. molitor. Ethyl acetate extract of neem (A. indica A. Juss) leaf is active antifeedanr at
the concentration of 0.05% which gives AI for 51.53% and its most active antifeedant is at the
concentration of 10% which gives AI of 82.05%. Secondary metabolite contained in the neem (A.
indica A. Juss) leaf are terpenoid, steroid, flavonoid, saponin and phenolic.
Mostofa et al. (2013), expressed that the, poultry production systems have led to marked increase in
the production of poultry meat and eggs throughout the world (Armstrong, 1986). It has triggered the
discovery and widespread use of a number of “feed additives”. The term feed additive is applied in a
broad sense, to all products other than those commonly called feedstuffs, which could be added to the
ration with the purpose of obtaining some special effects. The main objective of adding feed additives
is to boost animal performance by increasing their growth rate, better-feed conversion efficiency,
greater livability and lowered mortality in poultry birds. These feed additives are termed as “growth
promoters” and often called as non-nutrient feed additives (Singh and Panda, 1992). Many synthetic
drugs and growth promoters are supplemented to the broilers to effect rapid growth, but their use have
shown many disadvantages like high cost, adverse side-effect on health of birds and long residual
properties etc. Growth promoters are chemical and biological substances, which are added to livestock
food with the aim to improve the growth of chickens in fattening, improve the utilization of food and
in this way realize better production and financial results.
The efficacy of tulsi (Ocimum sactum) and neem (Azadirachta indica) leaves extract as a growth
promoter were studied in broiler. A total of 40 day-old broiler chicks were purchased from Kazi
hatchery and after three days of acclimatization the chicks were randomly divided into four groups
(n=10). No vaccination schedule was practiced and no antibiotic was added in ration of group A, B, C,
and D respectively. Group A served control without any supplements while group B, C and D were

supplemented with combination of tulsi and neem extract @ 1 ml, 2ml and 3 ml/liter of drinking water.
Live body weight gain was recorded weekly up to 6th weeks and hematological studies were performed
at 21st and 42nd day of experiments. At the end of 42nd day of experiment final body weight of group
A, B, C and D were 1561± 12.10 g, 1698± 12.87 g, 1608± 12.04 g and 1763± 13.28 g, respectively.
Tulsi and neem leave were selected for effectiveness as growth promoter on poultry. Mature and
disease free tulsi and neem leave were collected from BAU campus. For the preparation of dust, the
leaves were dried in sun for 10 days and followed by oven at 55-60°C for 2 days. The dried leaves
were pulverized with a blender. A 25 (unit) mesh diameter sieve was used to obtain the fine dust, the
dust was preserved in airtight plastic container until they were directly used for screening and
preparation of water extract. Ten (10) gram each leave powder was added to 80ml of distilled water
and was shaking overnight at room temperature, filtered and distilled water was added up to 100ml to
make 10% extract. (Mollah et al., 2012)
Table 2.2: Effect of tulsi and neem leaves extract on hematological parameter of broiler

From the findings of the present study it can be concluded that supplementation with neem and tulsi
leave extract @ 1-3 ml/L drinking water causes significant increase in live body weight and
improvement in weekly weight gain and feed efficiency as compared to that of control group of broiler.
Thus, neem and tulsi leave extract supplementation in the broiler rations may be useful for the safe,
economical and efficient production of broiler and this formulation could be used as an alternative to
commercial growth promoters.
Hossain et al. (2019), in this we had studied about the, neem tree (Azadirachta indica) is an evergreen
tropical plant originating in South Asia but increasingly encountered in Africa, America and Australia.
It belongs to the Meliceae family, grows rapidly in tropic and semitropic climate with an extended dry
season, and is used in many countries for afforestation, fuelwood production as well as an avenue or
shade tree.1-3 All parts of the neem plant such as leaves, seeds, bark, flowers, fruit and root are useful
with applications in toiletries, pharmaceuticals, furniture manufacturing, cattle and poultry feeds,
nitrification of soils for various agricultural crops, and pest control.2,4 Of all plant components
however, neem seed oil and its constituents have attracted by far the greatest interest over the past few
decades.
Conventional extraction of oil and azadirachtin, a botanical insecticide, from Azadirachta indica
involves defatting the seeds and leaves using hexane followed by azadirachtin extraction with a polar
solvent. In order to simplify the process while maintaining the yield we explored a binary extraction
approach using Soxhlet extraction device and hexane and ethanol as non-polar and polar solvents at
various ratios and extraction times. The highest oil and azadirachtin yields were obtained at 6 h
extraction time using a 50:50 solvent mixture for both neem leaves (44.7 wt%, 720 mgAza/kgleaves) and
seeds (53.5 wt%, 1045 mgAza/kgseeds), respectively.
Table 3 summarizes the extraction of neem oil and azadirachtin from neem seeds and leaves using
Soxhlet extraction under various experimental conditions. Soxhlet extraction is a solid-liquid process
commonly used to obtain oil from seeds and other plant components.11 Solvent selection is important
to provide a good yield from the extraction. The solvent typically used for oil extraction is n-hexane
due to its high stability, boiling point, non-polarity, low corrosiveness and high oil yield.2,3,12 Oil
yield further depends on moisture content, extraction time, size of particle and solvent:solid ratio. In
the case of azadirachtin, a combination of n-hexane extraction to remove oil followed by ethanol

extraction of azadirachtin from defatted neem seed cake has been reported (Table 3). The authors
obtained an oil yield of 52.5% (v/w), while ethanol extraction and subsequent purification steps
resulted in recovery of 5 g of azadirachtin from about 950 g of defatted neem seed cake.
Table 2.3: Oil and azadirachtin yield from neem seeds and leaves using solvent extraction under various
process conditions
The seeds were cleaned to remove any sticks, unwanted leaves, bad seeds, sand and dirt to ensure oil
produced is not contaminated and of high quality. The cleaned neem seeds were dried at 55 o C for 72
h until constant weight, and the moisture content determined by following the Eq. The dried clean neem
seeds were dehulled by hand followed by roasting for about 5 min to enhance oil extraction. The roasted
neem seeds were crushed in a blender and sieved to obtain particles ranging from 425 and 710 µm in
size. The sieved neem powder was then stored under vacuum in an airtight container at 4 o C prior to
use.

Fig 2.1: Neem oil yields (a) and azadirachtin yields (b) from neem leaves at various solvent ratios
and extraction times (n=3)
Soxhlet extraction of oil and azadirachtin from neem leaves was carried out using pure n-hexane
and ethanol as well as various binary solvent mixtures as shown in Fig. 1. It was observed that
binary solvent had a higher affinity for neem oil and azadirachtin from neem leaves at the studied
solvent ratios and extraction times. The highest oil and azadirachtin yields were about 45% and
720 mg/kg, respectively, at a solvent ratio of 50:50 for 6 h extraction time. In the case of single
solvent using ethanol (ratio 0:100) and n-hexane (100:0), the pure n-hexane had a higher oil yield
(41.0%) compared to the pure ethanol (36.7%). Conversely, the extracted azadirachtin yield from
neem leaves was higher using ethanol as single solvent. The extraction using the single solventn-

hexane and ethanol resulted in 11 ± 1 wt% and 24 ± 2 wt% lower oil yields compared to the binary
solvent ratio of 50:50 at extraction time of 6 h.
Fig 2.2: Neem oil yield (a) and azadirachtin yields (b) from neem seeds at various solvent ratios
and extraction times (n=3)
The presence of interaction between solvent types and extraction time means that the way oil yield
and azadirachtin concentration changes for different solvents depends on the extraction time and
vice versa. Thus, both solvent types and extraction time is needed, as well as their interaction, to
generate yield for oil and azadirachtin. Two way ANOVA of all extraction experiments has shown

that the R2 and R2 adj were at least 0.94 and 0.87, respectively. It is generally recommended that
the R2 value should be ≥ 0.80 for a good fitted model.23 Nonetheless, a large R2 value does not
necessarily indicate an adequate model. Therefore, it is equally important to predict the R2 adj value
for a model, which gives a more appropriate evaluation of model adequacy.
Mossini et al. (2009), has expressed that, The presence and growth of fungi in food and feeds may
cause spoilage and result in a reduction in quality and quantity. Fungi produce a variety of
secondary metabolites as products of their metabolism; mycotoxins are metabolites that have
deleterious effects on other organisms. Ochratoxin OPEN ACCESS. A (OTA), a potent mycotoxin
produced by certain species of Aspergillus and Penicillium, originally associated with mouldy
legumes, fruits, meat and cereal products, is at present receiving increasing attention for its
nephrotoxic effects and its potential carcinogenic activity [1–3]. Cereal products are the major
group of food commodities in which the above toxin is of high impact. Control techniques that are
cheap, ecologically sound and environmentally safe to eliminate or reduce the incidence of
economically important pathogens are of great importance, so the search for substances meeting
these needs is an important research topic.
In vitro trials were conducted to evaluate the effect of Azadirachta indica (neem) extracts on
mycelial growth, sporulation, morphology and ochratoxin A production by P. verrucosum and P.
brevicompactum. The effect of neem oil extract from seeds and leaf was evaluated at 0.125; 0.25
and 0.5% and 6.25 and 12.5 mg/mL, respectively, in Yeast Extract Sucrose (YES) medium.
Ochratoxin A production was evaluated by a thin-layer chromatography technique. Oil extracts
exhibited significant (p ≤ 0.05) reduction of growth and sporulation of the fungi. No inhibition of
ochratoxin A production was observed. Given its accessibility and low cost, neem oil could be
implemented as part of a sustainable integrated pest management strategy for plant disease, as it
has been shown to be fungitoxic by inhibition of growth and sporulation.
Azadirachta indica A. Juss (Meliaceae) (neem plant) extract is one of the most important plant
products which inhibit mycotoxin production. It comprises several parts, such as fruit, seed, leaf
and oil, with several active compounds. The main chemical fractions of neem oil with antifungal
activities are a mixture of triterpenoidal and tetranortriterpenoid compounds. Azadirachtin, 6-
deacetyl-nimbin, azadiradione, nimbin, salannim and epoxyazadiradione were the major
compounds obtained from chemical fractions of neem oil. When tested alone they did not have

any appreciable activity, but when mixed they showed antifungal activity, indicating possible
additive/synergistic effects. The ability to cause retardation of fungus growth is the basic mode of
action of a number of classical antifungal agents. However, neem leaf constituents are known to
potentially inhibit aflatoxin production in Aspergillus parasiticus without affecting fungal growth.
According to, the main feature of neem extracts, particularly those derived from leaves, is that they
do not retard fungal growth, but appear to interfere with aflatoxin production.
They had told that the Neem seed oil (NO) and neem leaf extracts (NL) did not show the same
effects on fungi. Direct contact of fungus with NO and NL on YES medium resulted in differences
between the macroscopic features of the colonies but not in the microscopic ones. Colonies of
fungi grown on YES-NO had practically the same size and appearance as those of controls (YES):
white color, radially sulcate, moderately deep and produced exudates (Figure).
Fig 2.3: P. verrucosum (isolates K11, K13) and P. brevicompactum (isolate K20) on YES and on
YES-Neem Oil (YES-NO) as described in ‘Materials and Methods’
Fungi colonies grown on YES-NL were not only bigger in size than control (YES), but their
appearance differed too: green color, radially sulcate, lightly deep and produced more exudates
(Figure).

Fig 2.4: . P. verrucosum (isolates K11, K13) and P. brevicompactum (isolate K20) on YES and on
YES-Neem Leaf Extracts (YES-NL) as described in “Materials and Methods”.
The extracts’ different effects on radial growth, sporulation and mycotoxin production may be
either due to the solubility of the active compounds or inherent to fungal metabolism. NO extracts
showed higher inhibitory effects than NL, probably owing to the presence of azadirachtin at its
highest concentration in the mature seeds. Toxin production generally decreases as mycelium
formation decreases, but research has shown that antifungal potentiality against growth may not
coincide with the inhibitory potential of toxin production. Current analyses revealed inhibitory
potential of NO extracts on sporulation and mycelium growth of fungi, but not on OTA production
(Figure).

Fig 2.5: Thin Layer Chromatography of OTA production by Penicilium isolates (K11, K13 and
K20) without neem extracts (Control) and with neem leaf (NL) and neem Oil (NO) extracts.
Ultraviolet light visualizationThin Layer Chromatography of OTA production by Penicilium
isolates (K11, K13 and K20) without neem extracts (Control) and with neem leaf (NL) and neem
Oil (NO) extracts. Ultraviolet light visualization
In this we study the effects of neem extracts on growth, sporulation, morphology and OTA
production by Penicillium verrucosum and P. brevicompactum were investigated. Thin layer
chromatography failed to show any inhibition of OTA production; however, neem extracts
affected the growth rate and sporulation of isolates. Assays show that neem extracts have
fungitoxic activity, even though their mode of action is still not fully understood. It is also quite
possible that different chemicals or different ratios of chemicals found in neem trees have varied
effects on fungi. Evidence from the current and other studies shows that fungal species react
differently to compounds from the neem tree. Additional research is needed to determine the
potential usefulness of neem products in fungal control programs.
Panchal et al. (2019), has described that the, Ocimum Sanctum also known as Tulsi family of the
ocimum sanctum is laminaceae. Ocimum sanctum are produced in India and Southeast Asia, India
is the largest sources of medicinal plant in whole world. Herbs have been provided therapeutic
potential to the health of individual. The demand of this plant are increasing day by day for
medicinal purpose. There are approximately 35,000 medicinal plants which are used for the

therapeutic effect according to Ayurveda and siddha and unani and other traditional system. In
which ocimum sanctum is one of the most important for medicinal purpose. It is employed in the
treatment of various disease such as antimicrobial infection, antifungal, anticancer, arthritis,
chronic fever, antifertility, eye disease, hepatoprotective, antispasmodic, and analgesic,
antiemetic. Cardio protective. This medicinal herb have also been shown to reduce blood glucose
levels, making it an effective treatment of diabetes. There are many chemical constituent present
in ocimum sanctum such as, oleanolic acid, rosmarinic acid, ursolic acid eugenol, , linalool,
carvacrol,  elemene,  caryophyllene, germacrene. Ocimum sanctum is considered to have
diuretic, stimulant property. Volatile oil, fixed oil also obtained from the leaves of medicinal herbs.
Monoterpene are obtained from the the volatile oils such as, camphene, myrcene, sabinene, in
which some mono terpene produced oxygen such as linalool, borneo.
The materials required for the formation. Chemicals- Mayer reagent, Wagner’s reagent, Lead
ethanoate, Alkaline reagent, Ferric chloride, Molisch’s reagent, Alkaline reagent, Barford’s
reagent, Iodine solution, Ninhydrin solution, sodium hydroxide, all chemicals were used to fi nd
out the presence of phytochemical constituents which were obtained from the research lab of
Galgotias university. Plant Material- Fresh Leaves of selected medicinal herb Ocimum sanctum
(Tulsi) was harvested from the herbal garden of Galgotias university, Greater Noida in the month
of December, 2018. The collected leaves were thoroughly washed with tap water to avoid dusts
and other unwanted materials accumulated on the leaves from their natural environment. The dust
free leaves were shade, dried at room temperature. After 4-5 days for obtaining aqueous extract,
the properly dried leaves were then grinding into the fi ne powder by using the grinding machine
than the powder material of tulsi leaves were weighed properly. The fine powder of tulsi leaves
was stored in a clean and tightly closed container for extraction.
Aqueous extract of ocimum sanctum leaves which is used in the various treatment. Fresh juice of
Tulsi leaves is employ in karma. This technique helps to ease headache and diseases of head and
neck. Tulsi leaves act as nerving tonic. Tulsi leaves extract reduces pimples, acne and scars
effectively (Figure).

Fig 2.6: Ocimum sanctum leaves
Fig 2.7: Aqueous extract of Ocimum sanctum leaves
They had some test for the alkaloids they are:

Mayer’s test- 5 mg extract of Ocimum Sanctum (Tulsi) was transferred in the test tube and then
added 1% hydrochloric acid HCl, the obtained solution was gently heated. Red colour indicate the
presence of alkaloids because Potassium mercuric iodine are present in Mayer’s reagent.

Wagner’s test- In this test 5 mg extract of ocimum sanctum was taken in a test tube than 0.5 of
wagner reagent was added in a solution shaked well. Apperance of reddish brown colour showing
the alkaloids are present. Reddish brown colour beacause of iodine forms a complax is insoluble
and has the colour brown redish.
Dragendorff test-5 mg extract of ocimum sanctum tulsi was taken in tube. And then one drop of
dragendroff reagent was addedin the test tube. orange-red colour, showing the presence of
alkaloids. Dragendroff reagent was prepared using Bismuth nitrate, Nitric acid, iodine and water
because of these chemicals it gives orange red colur in the preseance of alkaloids.
The obtained result from whole study confirm the validity of the use of Ocimum sanctum plantas
medicine in ancient medicinal traditions and suggest that some of the plant extracts possess
compounds with antimicrobial properties. cirsilineol, circimaritin, isothymusin, apigenin and
rosameric acid, are present in isolated aqueous extract of ocimum sanctum which may be useful
against fever, syphilitic, ulcer, inflammatory disease wounds, such as antimicrobial infection,
analgesic, antifungal, arthritis, anticancer, eye disease, antifertility, hepatoprotective, chronic
fever, antispasmodic, antiemetic, cardio protective etc. In protective antioxidant supplement
ocimum sanctum leaf extract may be used after the analysis of certain tests.
Borah R et al. (2018), has expressed that, The plant kingdom is an excellent source of potential
drugs and in the recent years there has been an increasing awareness about the importance of
medicinal plants. Medicinal plants are rich source of different types of medicines and produce
various bioactive molecules. Herbal plant extracts are very useful and are the major sources of
medicine which play vital role in controlling various types of pathogens (Doss, 2009) and as
growth promoters. These are the cheaper source for therapeutics and viable solution for various
pathogens.The medicinal plants extract have now emerged as a good alternative as they are rich in
a wide variety of secondary metabolites such as tannins, phenolics, alkaloids and flavonoids etc
which enhances growth, innate immune response and disease resistance against pathogenic
bacteria in human as well as in different organisms (Edoga et al., 2005).
Collection of plant material: Leaves of Ocimum sanctum L. (tulsi) were collected from different
sites of Dibrugarh District, Assam, washed with sterile water and dried in shades. Then the samples
were powered in mechanical grinder.

Aqueous, methanol and ethanol extract: The dried tulsi (50g) powder was placed in the thimble
of Soxhlet apparatus and 500- 700 ml of distilled water, methanol and ethanol was used for
extraction procedure and the experiment was done separately for all the two solvents anddistilled
water. The extraction was continued till clear solvent or water was seen in the thimble. The extract
was concentrated using rotary evaporator.
Percentage yield = Final weight of the dried extract *100

Initial weight of the powder
The yield of residue after Soxhlet extraction and evaporation of 50 gm dried plant leaves in
methanol, ethanol and water were as follows:
Extract Yield amount (%)
⁄
Aqueous 5%
Methanol 8%
Ethanol 7%
Table 2.4: Amount of plant extracts yield percentage in different solvents
The phytochemicals analysis in Ocimum sanctum (Tulsi) leave extracts in the two solvents and
aqueous conditions were summarized in Table. Various bioactive molecules were found in Tulsi
leaf extract from the phytochemical screening. The amount of extraction is more in case of organic
solvent then that of water. From the quantitative analysis it was found that high amount of phenols
are present in Tulsi leaf ranging from 1.6 to 7.6 percentages. Consequently the amount of alkaloid
and flavonoids ranged from 0.91 to 1.28 and 1.56 to 2.24 percentages respectively.
Extract Phenolic Alkaloid Flavanoid

Aqueous 1.61±0.56 0.91±0.66 1.56±0.64
Methanol 7.61±0.55 1.28±0.03 2.24±1.02
Ethanol 4.61±0.56 0.94±0.58 1.91±0.56
Table 2.5: Percentage of total phenolic, alkaloid and flavonoid contents in plant extract

Fig 2.8: GC- MS chromatogram of the methanolic extract of the leaves of Ocimum sanctum
Ocimum sanctumhas various properties such as antistress, antiseptic, analgesic, anti-

inflammatory, antimicrobial, immunomodulatory, hypoglycemic, hypotensive, cardioprotective
and antioxidant (Williamson, 2002, Tanwar et al., 2015). Eugenol (1-hydroxy-2-methoxy-4-
allylbenzene), the active constituents present in O. sanctum have been found to be largely
responsible for the therapeutic potentials (Sailaja et al., 2010). This plant has various properties
such as antistress, antiseptic, analgesic, anti-inflammatory, antimicrobial, immunomodulatory,
hypoglycemic, hypotensive, cardioprotective and antioxidant.
The presence of various bioactive compounds in the tulsi leaves justifies the uses for various
ailments by living population. The results confirm the use of Ocimum sanctum plant as traditional
medicinal properties andsuggest that some of the plant extracts possess compounds with
antimicrobial properties that can be used asantimicrobial agents in new drugs for the therapy of
infectious diseases caused by various pathogens. It is more beneficial to use tulsi asan herbal
medicine as compare to chemically synthesized drug.
Orafidiyaa et al. (2004), this study was designed to investigate possible synergistic effect ofaloe
vera gel on the anti-acne properties of Ocimum gratissimum oil and to compare the activities of
both agents singly and in combinations with the anti-acne agent Dalacintm– a 1% Clindamycin

phosphate solution. The gel and extracts of aloe vera (Aloe barbadensis Miller) are widely used
unofficially in topical and internal folk medicines, in beverages and in cosmetic products (Grindlay
and Reynolds, 1986). The gel is said to possess immunomodulatory properties, antidiabetic,
anticancer and antibiotic activities (Reynolds and Dweck, 1999), and has been further suggested
to enhance intended therapeutic and/or protective effects when included in topical preparations or
devices for the management of certain pathologic skin conditions in man (Olsen et al., 2001;
Visuthikosol et al., 1995). Chemical analysis has shown the gel to contain various carbohydrate
polymers, particularly glucomannans and/or pectic acid, along with a range of other organic and
inorganic components (Grindlay and Reynolds, 1986). Aloe gel and extracts have been clinically
investigated (Olsen et al., 2001; Syed et al., 1996; Visuthikosol et al., 1995); they promote general
health and provide skin protection and healing, showing only mild side effects. A 0.5% aloe vera
extract in a hydrophilic cream, for instance, was found more effective than placebo, eliciting
neither toxicity nor side effects in a 60-patient randomized controlled clinical study that
investigated the management of Psoriasis vulgaris with aloe extract (Syed et al., 1996).
The research followed the tenets of the Declaration of Helsinki promulgated in 1964 and approved
by the Institutional Human Experimentation Committee. A careful explanation of the aims of the
tests was given to the subjects. Each subject freely signed an informed consent form and filled the
questionnaire provided. The study is a pilot for a subsequent larger trial. Approval was obtained
from the Research and Ethical Committee of the Obafemi Awolowo University Teaching
Hospitals Complex, Ile-Ife, Nigeria. Eighty-four subjects consisting mainly of undergraduate
students of the Obafemi Awolowo University, Ile-Ife, Nigeria, presenting with clinically
significant Acne vulgaris participated in the study. Power calculation following the methods of
Kirkwood (1988) indicated a minimum of 11 subjects per group for statistical significance (power
¼ 90% at 5% significance level). The subjects were randomly 16 L.O. Orafidiya et al. assigned to
seven groups each of 12 candidates. One of the seven test preparations was administered to one
group of the subjects, respectively. Subjects who presented with alcohol or drug abuse were not
included in the study. Those on long-term antibiotic medication were excluded. Pregnant and
nursing mothers as well as subjects who claimed to have sensitive skin were also excluded.
Concomitant medications considered to be vital to the general health of the subjects were permitted
and noted in the subjects’ report sheets. Such medications included analgesics, non-steroidal anti-
inflammatory drugs and antispasmodics.

Fig 2.9: Mean number of papulopustules counted on subjects’ faces relative to baseline counts (%)
on Days 3 and 7 of treatment with test preparations, placebo and controls.
Ocimum lotion preparations containing 2% oil and graded quantities of aloe vera gel have
generally shown increasing effectiveness in acne lesion treatment with increase in the aloe gel
contents. Favorable effect of aloe vera plant product(s) in treatment of acne had been earlier
suggested (Mantle et al., 2001). The results of the present study have confirmed this, in that the
neat aloe dispersion test product (negative control preparation) exhibited some anti-acne activity
(Tables) but this was not significantly different from the placebo preparation; on the other hand
compliments of results reveal that greater potency of Ocimum oil preparations against acne
condition is obtained when aloe gel is incorporated into the Ocimum oil products. This
combination hastened and more thoroughly accomplished resolution of the clinical lesions (Table,
Fig.). Other instances of rapid and improved dermal restoration when aloe gel or extract is included

or combined with auxiliary therapeutic agents are also known (Heggers et al., 1997; Heggers et
al., 1996).
Muñoz OM et al. (2015), has expressed that, Aloe vera (Aloe barbadensis Miller) is a perennial
monocot plant with turgid green leaves joined to the stem in a rosette pattern. Aloe leaves consist
of a thick epidermis (skin) covered by a cuticle surrounding the mesophyll that includes
chlorenchyma cells and thinner walled cells that form the parenchyma (filet). The mesophyll cells
contain a transparent mucilaginous jelly called Aloe vera gel. Aloe vera is native to western
meridional Africa and belongs to the group of crassulacean acid metabolism plants (CAM plants)
with nocturnal CO2 assimilation, which prevents water loss in hours when there is less evaporative
demand. The species is therefore adapted to arid and semi-arid regions such as northern Chile
(from the II to IV Regions). These regions have an arid Mediterranean climate with mean annual
precipitation of 170 mm in rainy years and 40 mm in dry years. The mean maximum temperature
is 32°C at midday in summer, with occasional peaks of up to 45°C. The average minimum night
time temperature in winter and spring is 6°C.
Aloe vera (Aloe barbadensis Miller) leaves were gathered in February, 2010 in Coquimbo in the
IV Region of Chile, at a latitude of 31°S. Specimens were delivered to the Facultad de Ciencias,
Universidad de Chile and plants were identified by Dr. Jose San Martin (University of Talca, Plant
Biology and Biotechnology Institute) as Aloe barbadensis Miller. Samples were kept frozen (-
40ºC) and protected from light until the analytical procedure took place. The leaves used for
extractions measured between 40 and 60 cm in length and were taken from 3-year old plants.
Whole leaves were cleaned by washing them individually with distilled water and water with 0.5%
chlorine. The spikes and margins were removed before slicing the leaf. The cortex was carefully
separated from the parenchyma using a scalpel-shaped knife. Filets were washed thoroughly with
distilled water to remove the exudate from surfaces. Fresh aloe filets were stored for no longer
than 1 h at -18ºC prior to lyophilization.
Gel production yield was calculated considering the average weight of aloe vera leaves to be 100%.
The leaf cortex represented 34.23% of the total, while the gel filet was 64.03%, with 1.74% loss
due to handling. This yield does not differ significantly from previous reports of 35.8% cortex,
61.3% gel filet and 2.9% loss. The yield from the manual pressing method for gel and cake was
45.58% and 52.02% of fresh filet, respectively, with 2.4% loss in the pressure filter. The

percentage yield obtained for fresh gel was less compared with other reports in the literature. The
pressure cake yield was greater than that found by, who reported 13.1% cake production with
retention of 4.6% in the pressure filter. Lyophilized gel yield was 0.90% while pressed cake yield
was 0.92%, confirming that most gel and cake is water. Water may take two forms in plant and
animal tissues; “free” and “bound.” Free water is the common form of absorbed water and is easily
released, while bound water is linked to molecules.
Fig 2.10: Shows the amount of Mannan in the gel and cake.
The mannan content was still higher than in another report from the literature; where the authors
found it to be only 25%. Therefore, plants grown in semi-arid conditions in Chile have a higher
mannan content.
Our results indicate that aloin is present in the lyophilized gel, cake and cortex in very small
amounts of 0.0228 %, 0.0117% and 0.0525% respectively. The higher amount of aloin in the
cortex is due to the presence of cells adjacent to vascular bundles that synthesize aloin. Zambrano
et al. Reported that aloin fluctuates from 0% to 0.107% in pure gel. In our results, aloin was less
than 11% of 0.107. In the lyophilized cake, aloin was about 50% of that found in the gel
(approximately 11.4 mg per L of gel).

Barbara Joanna Bałan et al. (2014), has described that the, Aloe vera (L.) Burm. f. (Aloe
barbadensis Mill) Liliaceae, succulent plant native to northern Africa, is presently cultivated in
many regions of the world. Traditionally, its inner part of parenchyma, which contains aloe gel,
was used for the treatment of minor wounds, inflammatory skin disorders, thermal and radiation
burns and to alleviate chronic osteoarthritis pain. It also possesses some antimicrobial activity.
Now, aloe gel is also increasingly consumed as a dietary supplement. Some data suggest its
immunomodulatory properties. Recently, it has been reported that Aloe vera possess, both in vitro
and in vivo, antimicrobial properties. Its in vitro inhibitory activity on some clinically isolated
cariogenic and periodontopathic bacteria was described. In other study, the bacteriostatic effect on
Listeria monocytogenes, a bacteria responsible for foodborne diseases was observed. Aloe gel and
its extracts also exert antimicrobial activity against multidrug-resistant bacteria (MDR) from
clinical isolates. In the study of Kwon et al., the antimicrobial activity of Aloe vera peel extract in
distilled water against Staphylococcus aureus, Bacillus spp., Enterococcus spp., Escherichia coli,
Salmonella typhimurium, Pseudomonas aeruginosa and Vibrio spp.
The study was performed on 91 female inbred Balb/c mice 6-8 weeks old, weighing about 20 g,
delivered from the Polish Academy of Sciences breeding colony. For all performed experiments
animals were handled according to the Polish regulations concerning the wellness of laboratory
animals (Polish National Institute of Health) standards. All experiments were accepted and
conducted according to ethical guidance of the Local Bioethical Committee. Mice were housed 4-
5 per cage and maintained under conventional conditions (room temperature 22.5-23.0°C, relative
humidity 50-70%, 12 h day/night cycle) with free access to standard rodent diet and water.

Fig 2.11: The effect of aloe gel mice feeding for 21 days in 50 µl or 150 µl daily dose on the
proliferation of their splenocytes in in vitro culture with mitogen PHA
Fig 2.12: The effect of aloe gel mice feeding for 14 or 21 days on the ex vivo chemokinetic
(spontaneous migratory) activity of their splenic cells in 24 h tissue culture
The results of splenocytes proliferation in cell cultures established with mitogen PHA are
presented in Fig. 2.11. Cells collected from aloe-fed animals responded more vigorously to
mitogen than cells isolated from the spleens of control mice. Feeding mice with a lower dose of

aloe gel resulted in better splenocytes response to a lower dose of mitogen than feeding with a
higher one. Response to a higher dose of PHA was the same in both aloe-fed groups. The results
of experiments performed for evaluation of the influence of in vivo aloe gel administration to mice
on the in vitro spontaneous migration of their splenocytes in tissue culture, are presented in Fig.
2.12 In this type of experiment, a stimulatory effect was observed only in mice fed with a higher
(150 µl) daily dose of aloe gel, disregarding whether they belong to the group fed for 14 or 21
days.
Fig 2.13: Stimulatory effect of aloe gel feeding on anti-SRBC antibody production in mice
These data demonstrate that aloe gel administered orally to mice behaves as a stimulator of cellular
and humoral immunity, increasing their splenic cells mobility and response to mitogen PHA, and
anti-SRBC antibody production.

Chapter-3
Material And Methods
3.1 General
The cream was prepared by using the cream base that is bee’s wax, liquid paraffin, borax,
methylparaben, distilled water, rose oil, Aloe Vera gel, dimethyl sulphoxide extracts of Neem and
Tulsi. The cream was prepared by using the trituration technique/extemporaneous method for
geometric and homogenous mixing of all the excipients and the herbal extracts. By using slab
technique, we have developed three batches of our herbal cream, namely F1H, F2H, and F3H.
F4H All Four batches were evaluated for different parameters like appearance, PH Results.
3.2 Material
• Neeem oil
• Tulsi oil
• Aloe vera gel
• Rose water
• Distilled water
• Bees wax
• Methylparabean
• Liquid paraffin
• Borax
3.3 Instrument
• Round bottom flask
• Heating mental
• Condenser
• Conical flask
• Beaker
• Soxhlet apparatus
• Stir rod

3.4 Role of material/ingredients
Sr.No. Ingredient Role

1 Aloe vera gel Anti-ageing,anti-inflammatory,moisturizer reduce acne
and pimples.
2 Tulsi Antibacterial, adds glow to the face
3 Neem Promote wound healing, relieves skin dryness, itching and
redness.
4 Bees wax Emulsifying agent, stabilizer and gives thickness to the
cream
5 Liquid paraffin Lubricating agent
6 Borax Alkaline agent which reacts with emulsifying
7 Methyle paraben Preservative
8 Rose water Fragrance
3.5 Formulation of cream

3.5.1General
Heat liquid paraffin and beeswax in a borosilicate glass beaker at 75 ℃ and maintain that heating
temperature.(Oilphase). Inanotherbeaker, dissolveborax, methylparabenindistilledwater and
heatthisbeakerto 75 ℃todissolveboraxandmethylparabenand to getaclearsolution.(Aqueous
phase). Thenslowly add this aqueous phaseto heated oilyphase. Then add ameasured amount of
aloe Vera gel, Neem extract, and Tulsi extract and stir vigorously until it forms a smooth cream.
Then add few drops of rose oil as a fragrance. Put this cream on the slab and add few drops of
distilled water if necessary and mix the cream in a geometric manner on the slab to give a smooth
texturetothecreamandtomixalltheingredientsproperly. Thismethod is called as slabtechnique
or extemporaneous method of preparation ofcream.

S.No. Ingredient Formula Formula Formula Formula Formula

F1H F2H F3H F4H F5H
1 Neem oil 1.5ml 1ml 1ml 1ml 0.3ml
2 Tulsi oil 0.5ml 0.2ml 0.4ml 1.5ml 1.2ml
3 Aloevera 1.5ml 1ml 1ml 1ml 0.5ml

pulp

4 Beeswax 3g 3.5g 3.2g 3.5g 2g
5 Liquid 10ml 15ml 12ml 7ml 3ml

paraffin
6 Borax 0.2g 0.4g 0.3g 0.2g 0.2g
7 Methyl 0.02g 0.04g 0.03g 0.04g 0.02g

parabean
8 Rose water 4ml 5ml 3ml 3ml 2ml
9 Distilled Q.S Q.S Q.S Q.S Q.S

water
Table- 3.1 Formulation
(a) (b) (c)
(d) (e)
Plate 3.1: Formulation (a) F1H (b) F2H (c) F3H (d) F4H (e) F5H

3.6 Evaluation of cream

 Physical evaluation:- In this test, the cream was observed for color, odor, texture, state
 Irritancy :- Mark the area (1 cm2) on the left-hand dorsal surface. Then the cream was
applied to that area and the time was noted. Then it is checked for irritancy, erythema, and
edemaif any for an interval up to 24 h and reported (table 4).
 Wash ability:- A small amount of cream was applied on the hand and it is then washed
with tap water .
 PH :- 0.5 g cream was taken and dispersed in 50 ml distilled water and then PH was measured
by using digital PH meter
 Viscosity:-Viscosityofcream wasdonebyusingBrookefieldviscometer at atemperature
of 25 ℃ using spindle No. 63 at 2.5 RPM.
 Phase separation :-Prepared cream was kept in a closed container at a temperature of 25-
100 ℃ away from light. Then phase separation was checked for 24 h for 30 d. Any change
in the phase separation was observed/checked .
 Spread ability :-The spreadability was expressed in terms of time in seconds taken by two
slides to slip off from the cream, placed in between the slides, under certain load. Lesser
the time taken for separation of the two slides better the spreadability. Two sets of glass
slides of standard dimension were taken. Then one slide of suitable dimension was taken and the
cream formulation was placed on that slide. Then other slide was placed on the top of the
formulation. Then a weight or certain load was placed on the upper slide so that the cream between
the two slides was pressed uniformly to form a thin layer. Then the weight was removed and excess
of formulation adhering to the slides was scrapped off. The upper slide was allowed to slip off freely
by the force of weight tied to it. The time taken by the upper slide to slip off was noted.
Spread ability= m × l/t
Where, m= Standard weight which is tied to or placed over the upper slide (30g)
l= length of a glass slide (5 cm)
t= time taken in seconds.
 Greasiness:-Here the cream was applied on the skin surface in the form of smear and
checked if the smear was oily or grease-like.

Chapter-4
Result and Discussion
4.1 General
4.2 Result
Out of the several prepared formulations, we chose the two formulations that best suited the quality
parameters and were cost effective as well. The evaluation of the two selected formulations was
performed and the results are discussed here.
Physical Evaluation: In this test colour, odour, texture and state of the three formulations were
checked.
Sr NO Parameters Formulation(F4H) Formulation(F5H)

1 Colour Creamish Creamish
2 Odour Pleasent Pleasent
3 Texture Smooth Smooth
4 State Semisolid Semisolid
Table 4.1: Physical Evaluation Observation
Irritancy: Mark the area (1 cm2) on left hand dorsal surface. Then the cream was applied to that area
and the time was noted. Then it is checked for irritancy, erythema, and edema if any for an interval up
to 24 h and reported. According to the results all the three formulations that is F4H and F5H showed
no sign of irritancy, erythema and edema.
Sr No. Formulation Irritant effect Erythema Edema
1 F4H Nil Nil Nil
2 F5H Nil Nil Nil
Table 4.2: Irritancy study observation

Washability: Washability test was carried out by applying a small amount of cream on the hand and
then washing it with tap water. Both the formulations were easily washable.
Sr No. Formulation Washability
1 F4H Washable
2 F5H Washable
Table 4.3: Washability Observation
pH: According to the results, the PH of both the formulations that is F4H and F5H were found to be
nearer to skin PH so it can be safely used on the skin.
Sr No. Formulation pH
1 F4H 5.8
2 F5H 6.1
Table 4.4: pH Observation
Viscosity: Viscosity of cream was done by using Brooke field viscometer at a temperature of 25 ℃
using spindle No. 63 at 2.5 RPM. According to the results both the formulations showed adequate
viscosity.
Sr No. Formulation Viscosity(cps)
1 F4H 21020
2 F5H 18820
Table 4.5: Viscosity Observation

Phase Separation: Prepared cream was kept in a closed container at a temperature of 15-30 ℃ away
from light. Then phase separation was checked for 24 h for 7 d. Any change in the phase separation
was observed/checked. According to the results no phase separation was observed in the formulations.
Sr No. Formulation Phase separtion
1 F4H No phase separation
2 F5H No phase separation
Table 4.6: Phase Separation Observation
Greasiness: Here the cream was applied on the skin surface in the form of smear and checked if the
smear was oily or grease-like. According to the results, we can say that both the formulations were
non-greasy.
Sr No. Formulation Greasiness
1 F4H Non greasy
2 F5H Non greasy
Table 4.7: Greasiness Observation

Chapter-5
Conclusion
By using Aloe Vera gel, Neem and Tulsi the cream showed a multipurpose effect and all these
herbal ingredients showed significant different activities. Based on results and discussion, the
formulations Formulation 4 and Formulation 5 were stable at room temperature and can be safely
used on the skin. For better result we should have the pH between 5.7 to 6.9. The cream
formulation we have done is having a good appearance, adequate viscosity and there is no phase
separation was observed. These formulation undergoes through all the necessary steps which a
cream should go. Its purely herbal cream.
The present study shows that neem leaf (A. indica A. Juss) extract contains antifeedant activity to the
Bio indicator T. molitor. Ethyl acetate extract of neem (A. indica A. Juss) leaf is active antifeedanr
at the concentration of 0.05% which gives AI for 51.53% and its most active antifeedant is
at the concentration of 10% which gives AI of 82.05%. Secondary metabolite contained in the neem
(A. indica A. Juss) leaf are terpenoid,steroid,flavonoid,saponin
The presence of various bioactive compounds in the tulsi leaves justifies the uses for various ailments
by living population. The results confirm the use of Ocimum sanctum plant as traditional
medicinal properties andsuggest that some of the plant extracts possess compounds with antimicr-
obial properties that can be used asantimicrobial agents in new drugs for the therapy of infectious
diseases caused by various pathogens. It is more beneficial to use tulsi asan herbal medicine as compare to
chemically,synthesized-drug.

Reference
1) KP Sampath Kumar, Traditional Indian Herbal Plants Tulsi and Its Medicinal Importance,
Research Journal of Pharmacognosy and Phytochemistry, 2010; 2(2): 103-108
2) Ridha Kurniati, Nurdin Saidi, Antifeedant Activity From Neem Leaf Extract, Jurnal
Natural, Vol. 18, (1) 2018 DOI 10.24815/jn.v18i1.8781
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Extraction Of Oil From Tulsi,Neem And Aloe Vera Gel Formulation Of Multipurpose Herbal Cream

B.Tech Project,Department Of Chemical Engineering ,2022 Page 1

1.2 Extraction Of oil fromTulsi

Plate 1.1: leaves of Tulsi

Plate 1.2: Tulsi powder

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Plate 1.3: sohxlet extraction of tulsi leaves

1.3 Extraction of oil from neem

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Fig 1.1: neem leaves

1.3.2 Preparation of the plant material

Fig 1.2: Neem powder

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Plate 1.4: sohxlet extraction of neem leaves

1.4 Extraction of Aloe Vera gel

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Fig 1.3: aloe vera leaf

Fig 1.4 Aloe vera pulp

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1.5 Research Gap of Study

1.6 Scope of the Study

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The initial extraction rate, h, as Ct/t when t approaches 0, can be defined as

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Extract Sample Dried Extract(g) Yield(%)

n-Hexane 18.83 1.25

Ethyl acetate 82.10 5.47

Methanol 93.09 6.21

Table 2.1: Result of Neem Leaf Extract

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Fig 2.6: Ocimum sanctum leaves

Fig 2.7: Aqueous extract of Ocimum sanctum leaves

They had some test for the alkaloids they are:

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Percentage yield = Final weight of the dried extract *100

Extract Phenolic Alkaloid Flavanoid

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Ocimum sanctumhas various properties such as antistress, antiseptic, analgesic, anti-

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