INTRODUCTION:

Traditional medicine system is one of the centuries-old practices and long-serving fellow human beings in fighting disease and leading a good life. The distinctive method of their traditional medicine scheme has been used by indigenous people for millennia and among the most famous are the Chinese, Indian, African medicine systems.

Traditional medicine relates to any ancient and cultural healthcare practices that differ from scientific medicine and are mainly orally transmitted by groups of distinct cultures¹.

The World Health Organization (WHO) notes that assigning one definition to the diverse range of traditional medicine features and components is challenging but that a working definition is crucial. It therefore concludes that the various health methods, methodologies, understanding and beliefs of traditional medicines incorporating medications based on plants, animals and minerals, spiritual therapies, manual methods and exercises applied singularly or in conjunction to preserve well-being as well as treating, diagnosing or preventing disease². Moreover, it has been stated that more than 70 percent of the population of the developing world still relies on complementary/ alternative medicine technologies, otherwise known as traditional medicine, for instance, up to 80 percent of the population in Africa, 71 percent in Chile, and 40 percent in Colombia, and others.^3,4However, the increase in population, insufficient drug supply, prohibitive treatment costs, side effects of several allopathic drugs and the growth of resistance to presently used infectious disease medicines have resulted in enhanced emphasis on the use of plant products as a source of medicine for a broad variety of human diseases^5,6. Approximately 121 pharmaceutical products have been developed in the last decade on the basis of traditional knowledge from different sources.^7,8 In human society from time medicinal plants have played an important role in prevention and control of diseases. Neem leaves has antibacterial properties and could be used for controlling air borne bacterial contamination. Azadirachta indica. A. Juss (neem) perhaps very useful traditional plant⁹. Every part of the tree has been used for traditional medicines remedies, most of the parts of the plants such as fruits, seeds, leaves, bark and roots contain compounds with proven antiseptic, antiviral, antipyretic, anti-inflammatory, antiulcer and antifungal properties. At present, synthetic drugs form a major line of treatment in the management of many diseases and few synthetic pharmacologically active substances can currently be administered through transdermal patches and production is technically demanding the development and optimization of dermatological delivery system which is a challenging task in respect to herbal drugs. A number of transdermal patches have been developed by various investigators to achieve controlled release for extended duration. Hence, turning to safe, effective and time-tested ayurvedic herbal drug formulation would be a preferable option and prime concerning issue of our study. So, aim of the studies to extract, prepare and evaluate herbal Transdermal Patches by using polymer for sustained action and for the treatment of Anti-bacterial activity.

MATERIALS AND METHODS:

Materials:

Azadirachta indica. A. Juss (neem) leaves were collected from the fields of Rangareddy district, Telangana, India. HPMC E15M was purchased from Oxford Pharma Labs. Hyderabad. All other chemicals and solvents used were of analytical grade.

Methods:

Extraction of leaves of Azadirachta indica¹⁰

Methanolic extract:

The shade dried leaves were subjected to size reduction and passed in to sieve no 20 and then 40. About 500g of the dried powder was extracted continuously in Soxhlet apparatus with methanol for 72 hours to obtain the crude extract. The extract was dried under vacuum oven.

Phytochemical Studies^11,12

The Methanolic extract was subjected to phytochemical studies to find out the presence and absence of constituents.

Table No.1: Phytochemical tests

Experiment

Observation

Inference

Test for Alkaloids

Dragendroff’s test: The extract was treated with Dragendroff’s reagent (potassium bismuth iodide solution)

Orange brown Precipitate was formed

Presence of alkaloids

Mayers’ reagent’s: The extract was treated with Mayer’s (potassium mercuric iodide solution) reagent

Precipitate formed

Presence of alkaloids

Wagner’s reagent: The extract was treated with Wagner’s reagent (iodide and potassium tri-iodide solution)

Reddish brown Precipitate was formed

Presence of alkaloids

Test for Glycosides

Brontragers test: To the extract add dilute H2SO4 and filtered. Filtrate was extract with little chloroform and layer was separated out and add equal volume of dilute NH3.

Red colour observed in ammonical layer

Presence of glycosides

Test for Saponins

Foam test: Shake the extract with water.

Foam was formed

Presence of saponin Glycosides

Test for Tanins

Ferric chloride test: To the extract few drops of ferric chloride solution were added

Dark black colour formed

Presence of tannins and phenolic compounds

Bromine water test: The extract was treated with bromine water

Discoloration of bromine Water

Presence of tannins and phenolic compounds

KMnO4 test: The extract was treated with dilute KMnO4

Discoloration of solution

Presence of tannins and phenolic compounds

Test for Reducing Sugar

Benedict’s test: 0.5ml of extract solution and 5 to 8 drops of Fehling’s solution was added.

No brick red precipitate

Absence of reducing sugar

Test for Amino Acids

Ninhydrin test: The extract was heated with 5% ninhydrin solution on boiling water bath for 10 min.

No purple colour formed

Presence of amino acids

The extract was treated with solution sodium hydroxide and lead acetate solution and boiled

No black precipitate is formed

Presence of amino acids

Test for Flavonoids

Shinoda test: The extract was treated potassium hydroxide solution and then 10% ammonia.	Yellow colour Precipitate formed.	Presence of flavonoids
To extract add few drops of Lead acetate solution.	Yellow colour Precipitate formed.	Presence of flavonoids

Test for Terpinoids

1.4gm of extract was treated with 0.5ml of acetic anhydride and 0.5ml of chloroform and added concentrated solution of sulphuric acid

No Red violet colour was obtained

Absence of terpenoids

Test for Steroids

Liebermann- Burchard Test: To extract add chloroform solution a few drops of acetic anhydride and 1ml of con. H₂SO₄ were added through the side of the test tube and set aside for a while.	Brown ring was formed at the junction.	Presence of steroids
Salkowski Test: To the extract add chloroform solution and few drops of con. H₂SO₄ was added, shaken and allowed to stand.	Greenish fluorescence was formed.	Presence of steroids
Liebermann’s Reaction: Mix 3ml of extract with 3ml of acetic anhydride, heat and cool. Add few drops of Con. H₂SO₄	Blue colour was formed	Presence of steroids

Compatibility studies of drug/extract and polymers^13,14

FTIR Studies:

The application of infrared spectroscopy lies more in the qualitative identification of substances either in pure form or in the mixture and as a tool in establishment of the structure. Since I.R. is related to covalent bonds, the spectra can provide detailed information about the structure of molecular compounds. In order to establish this point, comparisons were made between the spectrum of the substance and the pure compound. The infrared data is helpful to confirm the identity of the drug and to detect the interaction of the drug with the polymer. Infrared spectra of drug and polymer, alone and in physical mixtures were taken. Then it was investigated for any possible interaction between polymer and the drug.

Drug Permeability studies^15,16

The permeability study of the drug was carried out across the cellophane membrane using a Franz diffusion cell. The 40mg/ml extract/drug suspension was prepared in phosphate buffer pH 7.4 and sonicated to ensure uniform drug distribution. The one ml of the above suspension was taken in the donor compartment. The dialysis membrane was mounted between the donor and the receptor compartment. The receptor compartment contained phosphate buffer pH 7.4 as elution medium. The medium was stirred for uniform drug distribution and temperature was maintained at 37±.5⁰C. The samples were withdrawn at regular intervals and estimated spectrophotometrically at 310nm to determine amount of drug diffused.

Preparation of Azadirachta indica A. Juss Transdermal Patch¹⁷

The methanolic extract of leaves of Azadirachta indica A. Juss, the four (M1, M2,M3 and M4) transdermal patches were prepared using drug with polymer in different ratios (1:1,1:2,1:4 ,1:6). Weighed quantity of polymer was dissolved in calculated quantity of methanol and kept for stirring. Calculated amount of extract was added to the above mixture and stirred well until a homogenous mixture was formed. Then calculated amount of permeation enhancer and glycerine were added. The resultant mixture was poured into a Petri dish and air dried at room temperature for 24hr.The patches were then peeled off from the Petri dish with the help of a knife and kept in desiccators.

Table No.02: Formulation of transdermal patches of Azadirachta indica A. Juss

Formulation code
Ingredients	M1 (1:1)	M2 (1:2)	M3 (1:4)	M4 (1:6)
Drug (mg)	40	40	40	40
HPMC E15 (mg)	40	80	160	240
DMSO (%w/v)	30	30	30	30
Dibutyl phthalate (%w/v)	30	30	30	30
Methanol (ml)	q. s	q. s	q. s	q. s
Glycerine (ml)	q. s	q. s	q. s	q. s

M = methanolic extract

Evaluation of Azadirachta indica A. Juss Transdermal Patch^18,19

Physical Appearance:

All the transdermal patches were visually inspected for colour, clarity, flexibility, and smoothness.

Uniformity of weight:

This was done by weighing four different patches of individual batch taking the uniform size at random and calculating the average weight. The tests were performed on patch which was dried at 60˚C for 4 hr prior to testing.

Thickness of the Patch:

The thicknesses of the patches were assessed by using digital Vernier calliper at different points of the patch. From each formulation three randomly selected patches were used. The average value for thickness of a single patch was determined.

Drug content determination:

The patches (2×1 cm²) were taken and cut into small pieces, added to a beaker containing 100 ml of distilled water the medium was stirred magnetic bead for 5hrs. The solution was filtered and analysed for drug content with proper dilution at 310 nm U.V spectrophotometrically.

Folding Endurance:

This was determined by repeatedly folding one patch at the same place till it broke. The number of times the patch could be folded at the same place without breaking gave the value of folding endurance.

Percentage Moisture uptake:

The patches were weighed accurately and placed in desiccators containing aluminium chloride. After 24 hr, the patches were taken out and weighed. The percentage moisture uptake was calculated as the difference between final and initial weight. It was calculated by using following formula.

Percentage moisture uptake =

[Final weight -Initial weight/Initial weight] ×100

Determination of surface pH:

The patches were allowed to swell by keeping them in contact with 5ml of distilled water for 2 hr at room temperature and pH was noted down by bringing the electrode in contact with the surface of the patches, allowing it to equilibrate for 1 min.

Tensile Strength and Percentage Elongation:²⁰

Tensile strength of the patches was determined with Universal strength Testing Machine (Hounsfield, Slinford, Horshan, U. K). It consisted of two load cell grips. The lower one was fixed and upper one was movable. The patches of size (2×1cm²) were fixed between these cell grips and force was gradually applied till the patches broke. The tensile strength of the patches were taken directly from the dial range reading in kg.

In-vitro diffusion studies:²¹

In-vitro diffusion study was performed by using a Franz diffusion cell with a receptor compartment of 250ml. The cellophane membrane was mounted between the donor and the receptor compartment of the diffusion cell. The formulated patches were cut in size of (2×1cm²) and placed over the cellophane membrane and the receptor compartment of the diffusion cell was filled with phosphate buffer pH 7.4.The whole assembly was fixed on a magnetic stirrer and the solution in the receptor compartment was constantly and continuously stirred using magnetic beads at 100 rpm; The temperature was maintained at 37±0.5⁰c.Tha samples of 5ml were withdrawn at the time interval of 1hr up to 21 hr and analysed for drug content U.V spectrophotometrically at 310nm against blank. The receptor medium was replaced with an equal volume of phosphate buffer pH 7.4 at each time of sample withdrawal. The cumulative amounts of drug permitted were plotted against time.

Ex-vivo Permeation studies:²²

Ex-vivo permeation study was performed for optimized formulation by using a Goat abdomen skin membrane in Franz diffusion cell with a receptor compartment of 250ml. The Goat abdomen skin membrane was collected from the slaughter house washed with water twice and again washed with distilled water and was mounted between the donor and the receptor compartment of the diffusion cell. The formulated patch was cut in size of (2×1cm²) and placed over the goat abdomen skin membrane and the receptor compartment of the diffusion cell was filled with phosphate buffer pH 7.4. The whole assembly was fixed on a magnetic stirrer and the solution in the receptor compartment was constantly and continuously stirred using magnetic beads at 100 rpm; The temperature was maintained at 37±0.5⁰C. The samples of 5ml were withdrawn at the time interval of 1hr up to 21 hr and analysed for drug content spectrophotometrically at 310nm against blank. The receptor medium was replaced with an equal volume of phosphate buffer pH 7.4 at each time of sample withdrawal. The cumulative amounts of drug permitted were plotted against time.

Diffusion Kinetics:²³

To analyse the mechanism for the release and release rate kinetics of the dosage form, the data obtained was fitted in to Zero order, First, Higuchi matrix, Korsmeyer’s Peppa’s model. By comparing the r2-values obtained, the best model was selected.

SEM Characterization:

The surface morphology of samples was examined at OU Institute of technology, Hyderabad. The optimized formulation was examined by SEM (Scanning electron microscopy) with operating voltage ranging 5.00 k v. Electron microscopic study was conducted to visualize drug distribution in the patch. SEM study was carried out using SEM (JSM 6700JEOL, Tokyo, Japan) for examining the transdermal patch of herbal extract for optimized formulation.

Screening of Antimicrobial activity:^24-26

Organisms used: Bacillus subtilis (Gram⁺ve),

Pseudomonas aeruginosa (Gram^-ve)

Media used: Nutrient Agar

Test used: ME patch

Standard: Penicillin, Streptomycin (Ambistyrin)

Preparation of Nutrient Agar:

8.2gm of agar powder was dissolved in 25ml of water. The medium was steamed in boiler to precipitate any heat coagulable material. Then the medium was filtered. The filtrate was distributed in 5ml quantity into culture tubes. The tubes were plugged with non-absorbent cotton. The medium in the tubes were sterilized by autoclave not less than 15 minutes at 15 pounds per sq. inch at 121^oC.

Preparation of Paper Disc:

By using standard punching machine Whatman filter paper was cut and standard paper of 6.0mm diameter was prepared. The paper discs were sterilized in a hot air oven at 160^oC for 1hour. The paper discs were then impregnated with the test solution.

Inoculation of Organism to Petri dishes for the anti-bacterial activity:

The marketed standard drug solution (Penicillin, Streptomycin) paper disc (1mg/ml), extract solution paper disc (1mg/ml) (1:10) (1:20), test optimized formulation solution paper disc (40mg) were prepared by proper dilutions using sterilized distilled water. The sterilized boiling tubes, agar media, Petri plates, sterilized formulation paper disc’s, sterilized standard drug paper disc’s, sterilized extract paper discs were taken under laminar air flow chamber. The All the inoculation procedures should be performed only in laminar air flow chamber to aseptic conditions. The nutrient media was first poured into boiling tube and then test culture is inoculated to the tubes and mixed properly. After uniform distribution the nutrient culture media is poured in to the petri plates and set aside for uniform distribution. The prepared standard paper discs, formulation paper discs, extract paper discs were added slowly on to the settled agar petri plate with the help of forceps. Markings are made for identification of respective readings and after inoculation Petri plates were transferred to the incubator. The plates were incubated at 37⁰C for 24 hours, after that plates were observed for anti-bacterial activity by measuring zone of inhibition and reported.

RESULTS AND DISCUSSION:

Compatibility studies of drug and polymer by FTIR spectroscopy:

The IR spectrum of Neem extract sample revealed presence of major functional groups. The characteristic functional groups of Neem extract and formulation mixtures of extract and polymer showed the peaks at following wane number region given: C-H str-2854.24cm^-1, O-H str-3360.31cm^-1, COOH str-1374.02cm^-1, S=O str-1040.26cm^-1, -NO² str-1522.49cm-¹. There was no appearance of any characteristic peaks which shows that there was no chemical interaction between the drug and polymer used. Hence the IR spectrum showed no incompatibility between the polymer and extract (Drug).

Fig.1. FTIR spectra of optimized Formulation

Permeability studies:

The permeation studies of drug was performed by using a cellophane membrane. From the results the drug release was found to be 98.96±0.16% at the end of 10 hours. So, the drug was found to have high permeability.

Evaluation of Transdermal Patches of Neem:

Physical Appearance:

The patches were found to be smooth with elegant appearance. Colour: Green, Odour: Odourless.

Uniformity of weight:

The weights of the patches are in between 0.689±0.014gm to 0.869±0.084gm. The patches exhibited uniform weight and there was no deviation in the weight of any formulations and within the limits.

Thickness of the Patch:

The results of thickness of the patches are in between 0.102±0.007mm to 0.240±0.010mm, which indicates that low standard deviation in different patch thickness are relatively similar. The results indicated that there was no much difference in the thickness within formulations.

Drug content determination:

Homogenous uniform drug distribution is one of the important characteristics of a transdermal patches that ensures the uniform reproducible controlled release of the drug from the patches. The results revealed that the drug content was almost uniform in the range of 90.16±0.311% to 101.02±1.052% in all the patches with low SD values, it was clear that there was proper drug distribution in the formulated patches. Hence it was concluded that drug was uniformly distributed in all the formulations.

Folding Endurance:

The folding endurance measures the ability of patch to withstand rupture. The folding endurance was decreasing with increase in HPMC E15 concentration. The results were in between 62±2.9 to 199 ±1.8 and results indicated that the patches would not break and had good mechanical strength along flexibility and would maintain their integrity with general skin folding when used.

Percentage Moisture uptake:

It was found that the percentage moisture uptake was increased with increasing in HPMC E15 concentration. The percentage moisture uptake results were in between 1.45±0.058 to 0.88±0.062. The results show that moisture absorption of all patches is within limits. The percentage moisture uptake was determined and followed the order: M4˃M3˃M2˃ M1

Determination of surface pH:

The pH of all formulations was determined using digital pH meter. The pH is also an important characteristic of patches so as to match the pH of skin and should not irritate when applied. The results are shown ranging from 5.2±0.19 to 6.1±0.13.

Tensile Strength and Percentage Elongation:

The tensile strength of the patches was found to vary with the combination of HPMC E15 ratio. The tensile strength results were in between 0.424±0.009 to 0.228±0.013 kg/mm² and the elongations were in between 29.51±1.342 mm to17.23±1.109 mm. The tensile strength measures the ability of patches to withstand rupture. This is justified because dissolved neem strengthened the bonding of polymer chains posing high tensile strength. It reflects that more the solubility of polymer high will be the tensile strength.

The anti-bacterial activity of test formulation Zone of Inhibition (ZOI) was compared with standard antibiotic drugs like Penicillin and streptomycin. The results are shown in Table.No.14.

SEM Characterization:

The surface morphology of optimized formulation M2 was examined by SEM (Scanning electron microscopy) with operating voltage ranging 5.00 k v and with resolutions 8.7mm x100. From the SEM studies we found that the drug was uniformly distributed in the patches. The result is shown in Fig.4.

Fig.4: SEM picture of optimized patch M2

CONCLUSION:

The Transdermal patches of Neem (methanolic extract) (M1, M2, M3, M4) were successfully prepared by solvent casting method using HPMC E15 in 1:1.1:2, 1:4, 1:6, ratios. The FTIR studies did not reveal any significant drug interactions. The prepared patches showed good results for physicochemical evaluations, in-vitro diffusion studies, ex-vivo permeation studies and anti-bacterial screening studies. On comparing major in evaluation criteria, formulation M2 was selected as best formulation. The release kinetics of all the patches showed zero-order kinetics and followed non-Fickian diffusion mechanism. The anti-bacterial screening study showed good anti-bacterial activity against Bascillus subtilis and Pseudomonas aeruginosa and zone of Inhibition (ZOI) was compared against standard anti-biotic drugs.

ACKNOWLEDGEMENTS:

Authors wish to give thanks to Management and Principal, Malla Reddy College of Pharmacy, Telangana State, for providing suitable research laboratory to carry out this project work

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Received on 09.11.2019 Modified on 23.03.2020

Research J. Pharm. and Tech. 2021; 14(7):3709-3715.

DOI: 10.52711/0974-360X.2021.00642

S. No	Formulation	Weight Variation (gm)	Thickness (mm)	Drug content (%)	Folding Endurance	Tensile strength (kg/mm²)	Elongation (mm)	pH
1	M1	0.689±0.014	0.102±0.007	101.02±1.052	136 ±2.9	0.388±0.013	29.51±1.342	6.1±0.13
2	M2	0.837±0.036	0.110±0.041	96.84±0.684	199 ±1.8	0.424±0.009	26.18±0.623	5.2±0.19
3	M3	0.772±0.048	0.126±0.026	90.16±0.311	113 ±6.8	0.297±0.010	19.61±1.255	5.6±0.12
4	M4	0.869±0.084	0.240±0.010	92.56±0.912	62 ±2.9	0.228±0.013	17.23±1.109	5.6±0.16