INTRODUCTION:

Plants are the oldest resources of pharmacologically active compounds and have provided humankind with many medicinal leaf valuable compounds from centuries. Hands are the primary mode of transmission of microbes and infections, hand hygiene is therefore the most important measure to avoid the transmission of harmful germs and prevent the nosocomial infections. To defend the screen from harmful microorganisms and to avoid spreading of numerous contagious diseases, hand washing is extremely significant precautions. Hand washing is the act of cleaning the hands with the purpose of removing soil, dust, and pathogenic microorganisms and avoid transmitting of transient microorganisms.

INFORMATION ABOUT PLANT

Nerium oleander

Nerium oleander L. (Apocynaceae), the only species currently classified in the genus Nerium, The plants are best adapted to the west coast, southern states, Florida, and Texas and will withstand dry conditions and wind, as well as salty, marshy soils, making them popular in coastal regions. Oleanders grow 6 to 12 feet tall and wide, and some varieties can be trained to grow into small trees up to 20 feet tall. The plant has a leaf like that of the almond, but smaller, and the flower is red like a rose. The plant itself (which loves hilly country) forms a large bush; the root is red and large, and, if this is dried, it gives off a fragrance like wine.

Classification:

Kingdom: Plantae

Clade: Angiosperm, Eudicots

Order: Gentianales

Family: Apocynaceae

Sub-Family: Apocynaceae

Tribe: Wrightieae

Genus: Nerium L.

Species: N. oleander

Synonyms

Nerium indicum mill

Nerion oleandrum

Kaneri

Oleander Medik

The whole plant is said to have anticancer properties. Nerium oleander has also been used in the treatment of cancer the flowers, leaves, leaf juice or latex, bark and roots have been used against corns, warts, cancerous ulcers, carcinoma, ulcerating or hard tumors.

Chemical Constituents of Nerium oleander leaves

Chemical name	Chemical structure
α-tocopherol	The word tocopherol comes from Tocos and pherein after the essential role vitamin E plays in animal reproduction. the ending -ol is standard chemical name ending for an alcohol. Tocopherol is used to in many Pharmaceutical product as a stabilizer. Some plastics technical oils and greases contain Alpha tocopherol as an oxidant.
Oleandrin	Oleandrin is a toxic cardiac glycoside. Ingestion of Oleander can cause both gastrointestinal and cardiac effects. The gastrointestinal effects can consist of nausea and vomiting, excess salivation, abdominal pain, diarrhea that may or may not contain blood, and especially in horses, colic.
Vitamin C	Vitamin C is an essential, water-soluble vitamin. It cannot be synthesized by our bodies and must be ingested regularly. It is known as a coenzyme and as an antioxidant. It can prevent the oxidation of other compounds by being oxidized instead.
Digitoxingenin	They are widely distributed in plants mainly has a glycoside sand or either toxic or insect deterrents. As potent cardio tonics, through their inhibition Na/K-ATPases, these steroids were largely studied (Digoxin and its derivative).
Ursolic acid	Ursolic acid has medicinal action, both topical and internally. Ursolic acid is used in many cosmetic preparations for its anti-inflammatory antitumor and antimicrobial properties. Ursolic Acid has antibacterial and antifungal activity.

Medicinal Importance of ursolic acid

Molar mass: 456.7 g/mol

Formula: C₃₀H₄₈O₃

Ursolic acid (3-hydroxy-urs-12-ene-28-oic acid), UA, is a pentacyclic tri terpenoid

Exhibiting a wide range of pharmaceutical properties. Ursolic acid is a secondary plant metabolite, usually present in the leaves of Nerium oleander. The health promoting activities of this compound have been unknowingly used for centuries as an ingredient of herb extracts employed in folk medicine. In recent years researchers, looking for natural biologically active substances, came back to this source of knowledge acquired over generations. Ursolic acid has medicinal action, both topical and internally. Ursolic acid can be found in plants that are used for cosmetics additives. Ursolic acid can serve as a starting material for synthesis of more potent bioactive derivatives, such as experimental antitumor agents, anti-inflammatory, antiviral, antibacterial, insecticidal, apoptotic, anti-aging, and antioxidant properties.

MATERIALS:

All the materials used in Herbal hand wash formulation was collected from our college pharmaceutics lab. Different Materials were used because of its properties in the making of handwash gel.

The Information of Materials used in formulation and its roles are given below.

1. Ursolic Acid:

The ursolic acid is an active ingredient in the formulation. The ursolic acid is a ethyl acetate extraction, extracted from the dried leaves of nerium oleander. It is added in formulation by dissolving it in Ethyl acetate. Ursolic acid has a medicinal action both topically and internally.

Ursolic acid is used in the formulation for its antibacterial, antimicrobial and antifungal activity.

2. Hydroxypropyl Methylcellulose:

Hydroxypropyl Methylcellulose is used in a formulation for its Gel formation ability. It has used in formulation because it provides a acceptable physical property to Gel and it will remain stable for a 3 to 4 months without any change upon storage.

3. Sodium Lauryl Sulphate:

Sodium lauryl sulphate is used in formulation as it has a property of foaming.

4. Methyl Paraben:

Methyl Paraben is generally used as a Preservative in the formulation. It provides a Good stability to the formulation during long term storage.

5. Glycerin:

Glycerin is generally used in the formulation to make the stable gel. As it maintain the viscosity of the handwash gel. Glycerin prevents the over gelling of formulation and stabilizes the gel for long time during storage.

6. Ethyl acetate;

As we are using a natural plant extract it is very important to dissolve the extract into the formulation. The ethyl acetate is used to prepare a solution of ursolic acid and dissolve in the formulation, so no particles of extract were seen in the gel.

7. Brilliant blue:

Brilliant blue is used in formulation to provide a lusture to the gel. It gives a Good appearance to the gel and also covers the colours of different ingredients and gives a single attractive colour to the handwash gel.

8. Distilled water:

Distilled water is used as a solvent in the formulation. It is also used to dissolve the methyl paraben completely by gentle heating.

METHOD OF EXTRACTION OF URSOLIC ACID:

The ursolic acid is one of the chemical constituent of the leaves of Nerium oleander which has the several pharmacological activities such as anti-inflammatory, anti-microbial, anti-viral, anti-bacterial, anti-tumour, Antifungal etc. hence, Ursolic acid is used in the formulation as a anti-bacterial agent as it is important to avoid bacterial infections.

Extraction of ursolic acid is carried out by using an apparatus called Soxhlet Extractor. Soxhlet extraction is used when the desired compound has a limited solubility in a solvent, and the other constituents are insoluble in that solvent. It allows for unmonitored and unmanaged operation while efficiently recycling a small amount of solvent to dissolve a larger amount of material.

Fig 1. Extraction Using Soxhlet Apparatus

Extraction:

The plant was collected from the Nursery named Ankur Ropvatika, Near Vijapur Road, Solapur. The leaves are separated from the plant and washed under tap water several time to remove dust. Then the leaves were shade dried for 5 days until the complete moisture is removed from the leaves. Then the dried leaves were grinded to make powder of it.

Now, the weighed quantity of 25gm of powder was placed in the extraction chamber and 200 ml of ethyl acetate is added in the extraction flask. The continues 7 cycles were carried out until the Ursolic acid is completely dissolved in the solvent. After this the solvent were concentrated by evaporation technique by using hot water bath. Then the dried powder of ursolic acid is collected and weighed.

% yield:

25gm of leaves powder = 5.03 gm’s of Ursolic acid

100gm of leaves powder = x

% yield = 20.12%

Fig 2. Recrystallized Product

Recrystallization:

Recrystallization of ursolic acid is carried out by dissolving the

Compound in ethyl acetate and gently warmed then filtered in

warm condition only and the recrystallized product is obtained.

EVALUATION OF URSOLIC ACID:

The evaluation of ursolic acid is carried out by three parameters i.e. Physical evaluation, Chemical evaluation and biological evaluation.

Physical Evaluation:

Colour- White creamy like

Odour- Characteristic

Melting point- 270°c

Solubility- Freely soluble in ethyl acetate, Insoluble in water.

Fig 3. Triterpenoid Test

Chemical Evaluation:

Carboxylic acid Test- Positive

Alcohol group Test- Positive

Triterpenoid Test- Positive

Antibacterial Test

The antibacterial test is carried out by using two different agar media's i.e. MacConkey agar and nutrient agar with two different bacteria’s i.e. Staphylococcus aureus and Bacillus subtilis. The test is carried out by using Well diffusion method, incubated at temperature 27°c and results were observed after 48 hrs.

Fig 4. Antibacterial test (MacConkey agar media)

1. MacConkey agar media

A) Staphylococcus aureus

Zone of inhibition - 17 mm

2. Nutrient agar media:

B) Bacillus subtilis

Zone of inhibition - 15 mm

INTRODUCTION TO HAND WASHING:

Keeping hands clean is one of the most important steps we can take to avoid getting sick and spreading germs to others. Many diseases and conditions are spread by not washing hands with soap and clean, running water. Not washing hands harms children, about 1.8 million children under the age of 5 die each year from diarrheal diseases and pneumonia, these are the top two killers of young children around the world.

Hand washing can also prevent people from getting sick with germs. Preventing sickness reduces the amount of antibiotics people use and the likelihood that antibiotic resistance will develop. Hand washing can prevent about 30% of diarrheal related sicknesses and about 20% of respiratory infections (e.g., colds). Antibiotics often are prescribed unnecessarily for these health issues. Reducing the number of these infections by washing hands frequently helps prevent the overuse of antibiotics. Hand washing is the single most important factor leading to antibiotic resistance around the world.

FORMULATION OF HERBAL HAND WASH GEL:

Various herbal hand wash gel formulation batches were prepared. The desired concentration of gelling agent, foaming agent, emollient were measured accurately and dispersed in purified water with moderate stirrer speed. The required quantity of methyl paraben was dissolved in remaining quantity of purified water by gentle heating. The required quantity of extract i.e. Ursolic acid is dissolved in sufficient quantity of ethyl acetate by gentle heating and mixed in above mixture with constant stirring until complete gel is formed. The hand wash gel is then transferred in suitable containers and stored in cool and dry place.

FORMULATION TABLE:

Table No.1 (For 50ml Of Hand wash gel)

Sr No.	Ingredients	F1 (gm)	F2 (gm)	F3 (gm)	F4 (gm)	F5 (gm)
1.	Ursolic Acid	0.5	0.5	0.5	0.5	0.5
2.	Hydroxypropyl Methyl Cellulose	1	1	1	1.25	1.3
3.	Glycerin	0.2	0.2	0.5	0.6	0.6
4.	Sodium Lauryl Sulphate	0.4	0.4	0.5	0.75	0.75
5.	Methyl Paraben	0.05	0.05	0.075	0.1	0.1
6.	Ethyl Acetate	-	5ml	8ml	8ml	8ml
7.	Brilliant Blue Solution	q.s	q.s	q.s	q.s	q.s
8.	Distilled Water	q.s	q.s	q.s	q.s	q.s
9.	Perfume	q.s	q.s	q.s	q.s	q.s

*q.s- quantity sufficient

CHARACTERZATION OF HARBAL HAND WASH GEL:

Ph:

The pH was determined by using digital pH meter (Equiptronics EQ-610). The pH was determined by taking 50 ml of gel into the beaker of 80 ml capacity and the pH electrode is dipped into the gel and the pH has recorded.

Viscosity:

The viscosity of herbal hand was determined by using digital Brookfield viscometer. Measured quantity of herbal hand was taken into a beaker and the tip of viscometer was immersed into the hand wash gel and the viscosity was measured in triplicate.

Spreadabilty Test:

The spreadabilty test for formulated Hand wash gel was carried out by placing a amount of gel in between two clean slides. Above the slides 1 kg weight is placed for better results. After 2 min the weight is removed and the diameter of gel spreaded was measured.

Stability Studies:

The stability of herbal hand wash gel was carried out by storing a measured amount of gel at different temperature i.e. 25°c, 37°c, 40°c, for 1 week. During stability studies no change in colour and no phase separation were observed in the formulated hand wash. Also the formulation withstands its activity.

Irritability Test:

The herbal hand wash gel was tested for irritability by applying it on left hand palm and washed off with water.

Antimicrobial Studies:

The screening of anti-microbial efficacy of the formulated herbal hand wash gel was aseptically performed on Bacillus subtilis and Staphylococcus aureus by using Dip well Agar Diffusion Technique. A well was prepared in the plates (containing 15ml of Nutrient and MacConkey agar medium respectively for both bacterias). 100μl of the test compound (herbal hand wash gel) was introduced into the well. The standard antibiotic discs like streptomycin were used as a standard. The plates were incubated overnight at 37°C. Efficiency of hand wash gel was determined by measuring the diameter of zone of inhibition.

Table no. 2

Sr No.	Evaluation Parameter	Observation
1.	pH	6.1±0.2
2.	Viscosity	53c Pascal’s
3.	Spreadability	2.45cm
4.	Stability	Stable
5.	Irritability	No irritancy
6.	Zone of inhibition	21mm & 20mm

RESULTS AND DISCUSSION:

According to the zone of inhibition formed against different bacterial isolates, showed that the hand wash prepared with Ursolic acid has great activity. It produced the widest zone of inhibition against S. aureus with diameter of 20 mm and B. subtilis with diameter of 18 mm. The zone of inhibition is due to the Active constituent Ursolic acid which is belongs to Triterpenoid class. As the antibacterial effect of herbal hand wash gel against gram positive bacterias shows greater activity so, as it has resulted that the prepared hand wash gel is has the good antibacterial activity and can be used with the patients suffering from bacterial infection.

CONCLUSION:

All the observations and results obtained from our research work, here by new conclude that the Ursolic acid extracted from the leaves of the plant Nerium oleander has the good antibacterial, antifungal, anti-microbial activity. The use of Ursolic acid in the preparation in order to prepare a herbal hand wash gel with a good antimicrobial activity to prevent the bacterial infection causes due to the improper cleansing of hands. The safety, efficacy and Antibacterial effect of the final formulation is tested and the results obtained were compared with a marketed product and it was observed that our formulation and the marketed product has comparatively similar activity.

ACKNOWLEDGEMENT:

It is a genuine pleasure to express a deep sense of thanks and gratitude to our guide our Principal Prof. R.Y. Patil Sir, D.S.T.S Mandal’s college of pharmacy, Solapur for his valuable guidance and inspiration. We are heartily thankful to all the teaching and non teaching staff for their help and cooperation.

REFERENCES:

1. National Center for Biotechnology Information. PubChem Database. Ursolic acid, CID=64945, https://pubchem.ncbi.nlm.nih.gov/compound

2. Journal of Chemical and Pharmaceutical Research, A Review on Chemistry and Pharmacological activity of Nerium oleander L. By Garima Zibbu and Amla Batra, Lab No: 5, Biotech lab, Department of Botany, University of Rajasthan, Jaipur

3. Antibacterial test, Practical manual of microbiology, second year B pharmacy, D.S.T.S. Mandal’s college of pharmacy, Solapur

4. International Journal of Pharmacy and Pharmaceutical Sciences nerium oleander: it’s application in basic and applied science: a review by saabiya farooqui, 2tulika tyagi P.G. Department of Zoology, Ranchi University, Ranchi-834002 Jharkhand India. 2Department of Botany Rajasthan University of Health Sciences Sector-18, Kumbha Marg, Pratap Nagar, Jaipur, Rajasthan.

5. Ursolic Acid," Examine.com, published on 7 November 2014, last updated on 14 June 2018, https://examine.com/supplements/ursolic-acid/

6. Hidayat Hussain, Ivan R. Green, Iftikhar Ali, Ikhlas A. Khan, Zulfiqar Ali, Abdullah M. Al-Sadi & Ishtiaq Ahmed (2017) Ursolic acid derivatives for pharmaceutical use: a patent review (2012-2016), Expert Opinion on Therapeutic Patents, 27:9, 1061-1072, DOI: 10.1080/13543776.2017.1344219

7. International Journal of Pharmaceutical Sciences and Research Phytochemical and Pharmacological Potential of nerium oleander: a review by Vikas Gupta, National Institute of Ayurvedic Pharmaceutical Research, Patiala, Punjab, India and Payal Mittal Akal College of Pharmacy and Technical Education, Mastuana (Sangrur) 2, Punjab, India

8. Review Ursolic Acid—A Pentacyclic Triterpenoid with a Wide Spectrum of Pharmacological Activities by Lukasz Wozniak, Sylwia Ska˛pska and Krystian Marszalek, Department of Fruit and Vegetable Product Technology, Institute of Agricultural and Food Biotechnology, 36 Rakowiecka Street, 02-532Warsaw, Poland

9. Solubilities of Ursolic Acid and Oleanolic Acid in Four Solvents from (283.2 to 329.7) Klie-Ping Fan, Tao Kong, Lu Zhang, Sheng Tong, Zhe-You Tian, Yong-Hua Duan, and Xue-Hong Zhang Journal of Chemical & Engineering Data 2011 56 (5), 2723-2725

10. https://en.wikipedia.org/wiki/Ursolic acid

11. https://www.ulprospector.com/en/na/PersonalCare/Detail/604/213576/Ursolic-Acid-60

12. http://www.smartskincare.com/forum/viewtopic.php?t=6339#top https://www.truthinaging.com/ingredients/ursolic-acid

Received on 03.04.2019 Accepted on 20.05.2019

Research J. Topical and Cosmetic Sci.10(1):01-06.

DOI: 10.5958/2321-5844.2019.00001.3