CHAPTER V. DISCUSSIONS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/30969/12/12... ·...

Formulation and Evaluation of Piperine Incorporated Topical Preparations Intended for Vitiligo

Chapter V 166 | P a g e

CHAPTER V. DISCUSSIONS

Irrespective of rich, hygiene, sex and living conditions, vitiligo affects as many as 65

million people worldwide. Surprisingly India accommodates 0.46 - 8.8% vitiligo patients

against 0.5-1% of the world Vitiligo population. No wonder a great deal of attention has

gained recently by hosting two days (23rd

-24th

September, 2010) official conference titled

“The first world congress on Vitiligo” hosted by The San Raffalele Scientific Institute in

Milan, Italy[240].

While much emphasis is being given in the developed world [241], although India

bears a soaring vitiligo statistics, no Vitiligo societies or organizations exists in the

subcontinent to minimizes social stigma and facilitate public awareness, collaborations in

Vitiligo research and services.

Patients affected with Vitiligo have to bear social setbacks along with its pathological

exertion. In the light of the reported works on antivitiligo activity of piperine as mentioned

earlier, this project was aimed to bring a ray of hope to the “down casted” unfortunates.

Thus, the present work focuses on the process development of a suitable topical

formulation incorporated with an antivitiligo agent. As it was reported earlier, piperine was

found not only to stimulate the replication of melanocytes but also induces the formation of

melanocytes dendrites. The intention of the formulation was to retain the piperine in deep

dermal layers of the skin, where melanocytes are present which facilitates repigmentation by

stimulating the melanocytes. Transdermal delivery of drugs at many occasions gets thwarted

not only owing to blockage of permeation by stratum corneum but also transdermal flux of

drugs with molecular weight higher than 1 kDa at 1 mmol/ cm2 finds difficult in penetrating

[242,243].

The main challenge of the aim was that the drug must penetrate the tough Stratum

corneum, but rather without getting down into the blood stream and undergo sink conditions,

the drug must retain in the deep dermal layers. Thus the fundamental requirement was to

enhance the tissue bioavailability near the melanocytes at the target site of deep dermal layers

and minimize the trespassing into the blood stream by which the drug gets thwarted.

Objectives of the work in accordance to the aim was to prepare, characterize and evaluate

piperine incorporated topical formulations by which tailoring of the drug as near to the

melanocytes has to be achieved. The objectives were further alienated into formulation



development of preliminary conventional formulations, later novel drug developments and

further extension of nanotechnology products. Under preliminary conventional topical

formulations, ointment and cream was considered in the preliminary stage. Novel products

like phytosomes and vesicular systems like transfersomes, otherwise known as ultra

deformable vesicular drug delivery systems and liquid crystalline nanoparticles (cubosomes)

were taken into account.

Permeation of peptide-based compounds across cellular barriers can occur by several

routes. One needs to be mindful that each of these routes of permeation can apply to different

peptide-based compounds; however, the physicochemical properties of the compound and the

physiological significance of each route will determine the relative importance in controlling

the compound’s net absorption. In screening compound permeation across cellular barriers,

delineation of the key rate-determining steps for controlling absorption needs to be identified.

Methods to delineate the functional relevancy of each route are now being enhanced further

by focusing the kinetics on potential paracellular and transcellular events. It is a well known

phenomenon that demonstrated that the delineation of the paracellular and transcellular mass

transfer resistances from observed cell-based assay permeability studies was possible. Several

studies conducted by Burton and colleagues further demonstrated that hydrogen-bonding

potential may provide a better means for predicting the potential passive transcellular

diffusion of peptides .

From the literature survey, exiting information mechanisms of antivitiligo activity of

piperine a bioactive alkaloid from Piper nigrum was gathered. Thus standardization of both

pepper and piperine gained attention. While the former was standardized by total ash value,

moisture content, powdered microscopy the later was done by chemical tests: Dragendroff’s

test, Mayer’s test, Hager’s test, Wagner’s test; TLC, melting point, spectral analysis and

infrared spectroscopy. Extraneous matter should be < 5% and their results were 0.4% which

shows extra pure and possibly practiced good harvesting practice.

As seen in the results, the test results were complying with that of the standard.

Extracted piperine was standardized with that of commercialised procured piperine which

was kept as reference (table 7). As the extracted piperine yield is quite less, the best method

of extraction which offers maximum yield and purity has to be taken into account. Therefore

piperine was extracted from black pepper by employing dual methods; soxhlation and

refluxation paying way for a comparison. The percentage yield indicates that the former was

marginally given better yield than the later (Soxhlation and refluxation was found to be 4.6%



and 4.2% respectively), thus soxhlation method was selected for scale up extraction process.

It is a well known fact that piperine should appear as needle shaped (acicular) crystals and

our results too perfectly matched with the expectations. But the process was slow, laborious

and the experience was burning; piperine when in direct contact with the skin gives a burning

sensation! At a time five soxhlation apparatus were employed as a part of process

management but still it took couple of months only to get required initial quantity for

formulation.

Our next dual efforts were to enhance the tissue drug bioavailability and absolutely

free from burning sensation by a suitable formulation. For the analytical requirements ie,

diffusion studies and consistency modified diffusion cell and consistency tester was

fabricated. The advantage what we claim for the diffusion cells are each unit (three

chambers) are detachable, easy for cleaning and maintenance, uniform temperature

distribution even from sidewise, cost effectiveness and replacement of units possible without

affecting the integrity of the entire system.

As a part of preformulation studies FTIR studies, X-ray, partition coefficient and

solubility were performed. FTIR studies indicate the corresponding peaks of alkenes, CH2-

CH2 -CH3, amines and ketonic groups which comply with the chemical formula of piperine.

When structure of Piperine and Infrared spectra are compared, the corresponding bonds were

found to present. This showed that Piperine was pure. X-ray studies of piperine before and

after sonication was taken and the peak heights in the later were reduced, indicating that

crystallinity of the drug has reduced. Partition coefficient was done to find out whether the

drug can partition into the lipoprotein skin membrane. For this reason n-hexane (Hexane has

polarity zero) which represents the nonpolar solvent and buffer as polar solvent was selected.

Results clearly indicate major part of the drug was partitioned into the nonpolar. It is

presumed that partitioning into the non polar is because piperine being a lipophilic drug. This

is supported by the findings of solubility studies which show the solubility in the descending

order: chloroform> ethanol> acetone but insoluble in water.

(i) Comment on conventional cream and ointment

In the process development of a suitable formulation, ointment and cream was

considered as conventional and compared. Sonicated piperine was used throughout in all the

formulations to enhance the incorporation. Melting point and Rf values of test (cream) and

standard (Piperine) were comparable which indicates there is no change in the physical and



chemical nature of the Piperine. This also reflects that the drug is compactable with other

excepients like bees wax, lanolin etc. There was no extra spot under UV lamp which suggests

there are no fluorescent impurities. This is having significant importance when we consider

confocal microscopy were we use florescent dye for detection of formulation inside tissue.

By sonication it was found that the long needle shaped crystals of average size of 59.8 µm

was able to bring down to 13.77µm. SEM data clearly indicates the reduction in particle size

and unorganized morphology in the later is evident. More important, its crystalline nature

was drastically reduced as it was proved in the X ray diffractogram (figure 31). This truly

facilitated the drug incorporation in both ointments and creams. The optimized formulation

was considered for further evaluations. Optimization was based on the tissue bio availability.

For this ex-vivo diffusion studies were performed by using an indigenously fabricated 3

chambered modified diffusion cell. Pig skin was selected because of its anatomical and

physiological resemblance with human skin. Care was taken to avoid distracting the tissues.

Drug quantification was done by tape stripping method which was commonly practiced by

dermatological researchers. But the drug concentrations of the deep epidermal and dermal

layers were done by extracting from the homogenised tissue. Although organoleptic

characters of ointment was acceptable, diffusion results were not promising. When urea as a

permeation enhancer was used, the drug diffusion into the buffer was increased by 0.87%

which ironically reflected less tissue drug bioavailability. Drug tailoring to the dermis was

augmented by 0.6% which was negligible. This could be because mechanism of urea in

permeation is by pulling apart the cells paying way for the drug to pass. In the process we

presume that the drug was free to pass through the dermal layers too by the channels created

by the urea. The results suggested that permeation and tissue retention of piperine in the

cream was more when compared to ointment. Piperine tissue concentration when cream was

considered lodged maximum in the skin rather than in the buffer. This was the core idea and

expectation of the work. Therefore drug release of the cream and its site specificity was found

to be superior to ointment. Thus, 1% piperine cream was selected for further evaluation

parameters like stability assessment and pharmacological studies. Melting point and Rf values

of test (cream) and standard(Piperine) were comparable which indicates there is no change in

the physical and chemical nature of the Piperine. This also reflects that the drug was

compactable with other excepients like bees wax, lanolin etc. It could be postulated that in

addition to transcellular permeation, paracellular permeation also was significant through

tight junctions. The final preparation was found to be smooth texture and consistency and

free from gritty nature with light yellow colour and peppermint odour. The globules retained



its size and no coalescence was found. Moisture absorption studies showed no significant

absorption of moisture. TLC studies of cream and pure piperine was shown to have same

spots and under UV light no multiple spots were seen which suggests no drug interaction and

pharmaceutically piperine was compatible with excipients. pH of the cream was found to be

6.5 which was matching with that of the human skin pH. The pH of skin products can affects

the skin and hair condition and cause breakage.The high pH affects the cuticle of the hair

that protects the inner cortex. Alkaline products raise the hair cuticle and go inside the hair

shaft. Mild acid has an opposite effect to alkalies and flattens the cuticle such that for easier

to comb and look shiener.

Acid-base optimization is the part of human homeostasis concerning the proper

balance between acids and bases, in other words the pH [259]. The body is very sensitive to

its pH level. Outside the range of pH that is compatible with life, proteins are denatured and

digested, enzymes lose their ability to function, and the body is unable to sustain itself.

Alkalies open the hair cuticle and acids close the cuticle hair is its strongest when the cuticle

is closed.

Minimum, maximum and average globule size of cream was found to be 4.72µ

52.75µ and 28.75 µ respectively while the average size of piperine was in the range of 50-60

µm. Thus once can deduce why we have gone for sonication of piperine before incorporation.

The globule size in the cream was retained for 4 weeks and there was no phase separation.

Topology studies by SEM revealed almost smooth globules. In addition irritancy test was

also performed on rabbits and found no redness, oedema, Inflammation and irritation which

give a green signal for pharmacological studies.

In permeation studies, since in all formulations we are targeting the drug at the dermis

and not to be diffused, it was rational to expect comparatively negligible fraction will get into

the buffer solution. In this case to reduce the error we need to apply a large quantity of

formulation and to meet this requirement a modified diffusion cell was fabricated. Semi

permeable membrane used here was pig skin from ear pinna. Pig has been widely recognized

as a model for dermal or transdermal drug delivery systems due to similarity to human skin,

both physiologically and anatomically.

In antivitiligo pharmacological studies unlike other activities we need to consider

pigmented animals. For this we had considered buffalo in the beginning. Apart from



complications of getting ethical committee (buffalo being a higher mammal) the owners

reluctant to provide their animals stating that our experiments may result in decreased milk

production. Ultimately pigmented rabbits were taken into account. Colour- brown or black ?

Availability of rabbits to satisfy the number of the same strain for all groups at the time of the

study was only brown. Protocol in one line was depigmenting all the animals and find out the

repigmentation rate by the cream. Also UV radiation therapy was used in conjugation with

the piperine cream to check any augmentation of the pigmentation was there. For

depigmentation, 4-hydroxy N anisole was used and the mechanism of this chemical was

bleaching. Depigmentation was achieved in almost 35 days in all the groups. The next step

was repigmentation. Vaguely animals were only cream was applied was found to repigment

in 15 days and the repigmentation was significant by 59 days (figure 39). But the group were

UV radiation therapy was conjugated with the topical application, the repigmentation was

seen significant in shorter period. But the rate of hair growth was less and the repigmentation

area was homogenous. This could be because of the stimulation of melanocytes by UV

radiation was not achieved uniformly throughout, just because targeting the melanocytes at

the dermal layer was not achieved fully. Regarding hair growth, it will be easy to understand

if we know the three stages of hair growth ie, anagen (growth phase), catagen (transitional

phase), telogen (resting phase). Anagen phase is the first phase of hair growth cycle which is

also known as the growing phase. At any one time, 80 - 90 percent of hair follicles on scalp

are in the anagen phase. During this period hair grows continuously. The growing will

continue for 3 to 7 years at the rate of half an inch a month.The hair bulb produces hair

pigment .Blood supply provides nutrients and minerals to hair. After the anagen phase, hair

will turn into a transitional phase before going to rest. This short phase is known as the

catagen phase which last for 2 to 4 weeks. During this time, hair detaches from the blood

supply. The detached follicle will slowly shrink to about 1/6 its size. The hair bulb stops

producing the color pigment. The bulb will be pushed upwards towards the surface when the

new hair is formed. Approximately 2 - 3 % of hair will be in this phase in scalp. Telogen

phase is the final phase of your hair growth cycle. It is also known as the resting phase where

the hair follicles will slowly fall off and replaced by a new hair. Around 10 - 15 % of the hair

in the scalp will be in telogen phase. 50 - 100 hairs from this phase will shed daily. This

period lasts for 3 months before the hair falls out. The hair follicles become weak and thin

and can be easily pulled them out - new hair follicle will emerge once the hair the hair falls.

When the animals were subjected to radiation the formulation was not able to protect the hair

bulb and perhaps the anagen phase of the hair growth was not fulfilled. This discussion is



supported by the fact that hair growth promotion by herbal drugs could be either due to

increased blood flow by angeogenesis or skin irritation. Piperine known for its burning

sensation when applied on the skin in low concentration (1%) may produce slight

vasodilatation. But due to the effects of radiation, this property was arrested [244,245].

After all, if we correlate the ex-vivo diffusion studies with the pharmacological studies

only 69.06% could be tailored at the dermal layers which led to consider developing novel

topical drug delivery systems [246].

(ii) Comment on phytosomes

In the next step, phytosomes were considered because of its unique ability of bonding

together with the phyto- constituent as its integral part. Such a complex was obtained by

reaction of stoichiometric amounts of phospholipid and the substrate in an appropriate

solvent. When treated with water, phytosomes assume a micelle shape forming liposome-like

structures, thus giving provision for incorporating into cream base for application. FTIR

spectral analys was was done to rule out the possibility of drug-excipient

interaction.Vibrational spectroscopy (IR) describes same kind of molecular informaton and

can be used to supplement or complement each other. Middle IR (400-4000cm-1

; vibration-

rotation region) was used for anaytical purpose. The atoms held by a chemical bond are the

main participants in vibration. Vibrations depend on mass of two vibratiing atoms, force

constant of bond between two atoms and other atoms attached. Thus vibrations are

characterstic for a group. In FTIR spectrogram results, the functional region was important

for stretching and the fingerprint region for bending. Transmission peaks at 3000-3100 cm-

1indicated aromatic functional group and 3300-3700 cm

-1 indicated hydrogen bonding (O-H).

The C=O and C=N stretching and alkenes were significant. The peaks were intact in both the

physical mixture and as it appeared in the pure drug which indicated that the excipients

neither altered the configuration of the drug nor disturbed its bonds. As the concentrations of

phytosomes were close to the theoretical value, it can be confirmed that the formulations

maintain good drug content. To have a chance of comparative topological examination, SEM

of pure piperine, phosphatidyl choline and the complex (phytosomes) were taken. In the

complex it was clear that the piperine and the PC fused to a common complex. To know the

effect of PC on tissue retention, the polymer concentration was gradually increased through

three formulations. Phospholipids being amicable with the lipoprotein bilayers of the skin are

promising candidates for drug carriers. It doesn’t mean that simply when one increase the



concentration drug permeation will induce. Paradoxically in the present study, the optimum

concentration was when the drug-polymer ratio was 1:1. Further as the PC concentration was

increased, the drug permeation not only hampered, there was an occlusive outer layer which

blocked further permeation through the skin. That is the reason in this formulation (1:1)

although relatively maximum drug was diffused into the buffer (22.60 ±0.37), tissue

bioavailability too was found to augment (72.07 ±0.01). The rate of many drug degradation,

increases about two or three times with every 10°C rise in temperature. Accelerated stability

study of the phytosomal formulation was based on Arrhenius principle which explains the

effect of temperature on the rate of reaction. k= A e –EaRT

where k= specific rate constant; A=

frequency factor; Ea= energy of activation; R= ideal gas constant; T= absolute temperature.

The drug degradation stress was induced by elevated temperatures, 45°C and 55°C. Studies

showed that dispersion complex emulsion system if stored at optimum temperature (27 °C)

will be pharmaceutically stable for 17. 65 months. Thus phytosomes when compared with the

previous optimized formulation (cream having 69.06%) the tissue drug bioavailability was

augmented by 3.01%. This shows not much significant probably because the further drug

release once drug has leached into the cream was comparable with that of the previous

preparation. But phytosomes as such cannot be considered for topical application for practical

reasons. Thus the investifations were in pursuit of alternative NDDS.

(iii) Comment on transfersomes

The next step was towards preparation of ultradeformable vesicles (Transfersomes). A

Transfersome carrier was an artificial vesicle esigned to be like a “cellvesicle” or a cell

engaged in exocytosis and thus suitable for controlled and, potentially targeted, drug delivery.

Transfersomes overcome the skin penetration difficulty by squeezing themselves along the

intracellular sealing lipid of the stratum corneum. There was provision for this, because of the

high vesicle deformability, which permits the entry due to the mechanical stress of

surrounding, in a self-adapting manner. Flexibility of transfersomes membrane was achieved

by mixing suitable surface-active components in the proper ratios. The resulting flexibility of

transfersome membrane minimizes the risk of complete vesicle rupture in the skin and allows

transfersomes to follow the natural water gradient across the epidermis, when applied under

non-occlusive condition. Transfersomes can penetrate the intact stratum corneum

spontaneously along two routes in the intracellular lipid that differ in their bilayers properties.



The main objective in the preparation of TS loaded with the bioactive compound (Piperine)

was to target the melanosomes present in the deep dermal layers and also to enhance the

tissue bioavailability compared to the previous formulations. TS are capable of crossing the

pores in permeability barriers extremely efficient even if the transfersome radius (t r) was

much greater than the pore size (r pore) ie., tr/rpore ˃ 0.25 the driven flux rate depends on the

transdermal osmotic gradient [247].

Vesicular systems are preferred as drug carriers because they can be used for site

specific drug delivery and improving therapeutic index of the drug. TS were selected as they

are special type of liposomes having flexibility which penetrate through the skin efficiently

when compared with conventional vesicles. Results of FTIR spectrogram showed stretching

of functional groups N-H (3429.7 cm-1

), aromatic C-H (3088.2 cm-1

), alkenyl C-H (3008.2

cm-1

) and aliphatic C-H (2937.1 cm-1

) groups respectively. These peaks were not altered in

the physical mixture of drug with PC indicating that there were no interactions occurred

between the Piperine and PC. It was hypothesised that piperine being lipophilic will lodge in

the lipid lamellar region rather than the hydrophilic vesicular lumen. Size of tween 80 based

vesicles were found to be small when compared to its span 80 counterparts, the later being

less hydrophilic its chain length was less which was responsible for the decreases in

comparative size. PC concentration was found to be directly proportional to the size and

entrapment efficiency. Thus it was logic to hypothesis that this phenomena also points out the

lodging of lypophilic drug must be with the lamella rather than the core. Sample collection

for SEM studies during the real time filtration was very tricky and tedious. Sample

preparation and SEM were taken at several stages, out of which just one showed here was

satisfactory. Indeed the picture clearly describes elasto-mechanical character of vesicles

while penetrating through the pores (figure 49). The size of the vesicles were almost same

before (66.97) and after extrudation (and 68.92) through a membrane whose pore size

(12) was smaller than that of the vesicles. The proviso was that the vesicular membrane

elasticity was dynamically to the local stress by the external. Thus we claim that our

formulation can considerably easily permeate through skin pores (20). TS with minimum

concentration of PC was found to diffuse more profusely (TT1) perhaps because of more

flexible nature. Ironically, just reverse was observed when span which was less hydrophilic

compared to tween was used. This strongly suggests there was an influence of edge activator

as well PC concentration in skin penetration and dermal retention status. Thus propensity of



permeation was influenced by permeability, trans-barrier force (transdermal osmotic

gradient). PC was a polar lipid molecule having one of two OH- group on glycerol are

involved and 3rd

combined with a highly polar molecule. This was achieved by condensation

of fatty acids with glycerol. The TS suspension must left dry out at the skin surface which

facilitates the vesicular penetration by the transdermal osmotic gradient which was described

before. Only when the water in the suspension evaporates due to the body heat, TS get the

driving inertia for penetration. Thus there was no scope and not necessary for preparing yet

another base for such kind of TS preparations. Span 80 shows more entrapment efficiency

when compared with tween 80 (table 16), this was because span 80 was less hydrophilic

(lower HLB) than tween 80. As piperine was hydrophobic, span 80 shows more entrapment

efficiency. pH of the suspensions indicated compatibility with that of the skin.

For treatment of vitiligo, the drug should penetrate through the skin and released in a

controlled manner. The more drug should be within the skin, not in the systemic circulation.

Pig skin was used for drug diffusion studies, since it was widely recognized as a model for

dermal or transdermal drug delivery systems due to the physiological similarity with human

skin. Designed for collecting large samples and so to accommodate more buffer solution,

which conventional Franz diffusion cells fails, diffusion cell which can precisely measure 60

ml was fabricated. For the convenience of centrifugation, comparatively large sample

volume, 5 ml was collected; n-propanol (Triniton X-100, 0.1% w/v can be alternatively used)

was used for disrupting the vesicles so that the drug will be free for estimation. These add to

other advantages of this apparatus. Drug concentration retained within the skin was found to

increase with PC concentration. But when span was used, drug was found to retain more in

the skin due to comparatively less hydrophilicity. Kinetics of lipid vesicle penetration

through the skin can be affected by enhanced water flow towards the skin surface. This was a

continuous process, possibly due to constant intra dermal flow to keep the decisive water

concentration lower than the limiting water concentration. The best fitting kinetic model of

kinetic analysis of diffusion data with the higher correlation was found with the Korsmeyer-

peppas model for all formulations. This indicates that the release of drug from the hydrogel

follows non-Fickian diffusion depending on the ‘n’ value. In the Korsmeyer-Peppas model r2

value was found to be 0.7929 with the ‘n’ value was more than 1, signifying super case-II

transport mechanism of drug release, edifying that the drug release was affected by erosion of

PC.



Although the average particle size was slightly increased there was no sedimentation.

Lesser the time taken for separation of two slides, better the spreadability. Since spreadability

was indirectly proportional to consistency, it was found that consistency slightly decreased

with increase in the polar lipid concentration. It was also noticed that there was no much

difference in spreadability when span 80/ tween 80 was used. The results of stability testing

were almost same before and after the freeze thaw cycles. This suggests that the consistency

of the formulation remains unaltered. Crystal growth should be more cautious than the

sedimentation, since the former ruptures the lamellar structure. Sedimentation can be

overcome by simple physical agitation. Thus the optimised formulation (TS1) was showing

better tissue bioavailability (75.25%±1.72) than the previous formulation (phytosomes having

72.07 ±0.01) which clearly indicates TS are persuasive enough to be considered.

(iv) Comment on cubosomes

As an effort to see the further possibilities of enhancing tissue bioavailability, cubical

liquid crystal nanoparticles (cubosomes) were explored. Cubosomes were considered because

of their unique biodegradable nature, edibility, biological compatibility, and incorporation.

The initial gel was further breakdown into cubosomes of heterogeneous dimensions. Rupture

usually occur in a direction parallel to shear direction and the energy required was

proportional to the number of network branches that rupture as suggested by Warr and Chen

[248].

Later controlled sonication was employed and allowed to stand aside. This allowed

the colloids to aggregate together to form homogenous cubosomes, likely via membrane

fusion. Thus, it has to say that initial levels top-down-approach was employed and later

bottom-up-approach. It was suggested that coarse cubosomes on the micron scale possess D-

surface, but after homogenization, the P-surface dominates possibly due to the addition of

polymer [249].

FTIR studies revealed stretching vibrations in the range of 3088.2, 2937.1 and 3429.7

which correspond to aromatic/ aliphatic groups of CH, alkenes CH and NH groups. The

peaks of pure drug and in the physical mixture didn’t alter stating that the drug was

pharmaceutically compatible.

SEM was used for morphological studies while TEM was greately used for studying

inner structural integrity along with size and external shape. TEM was a useful tool to



identify the internal structure of the cubosomes. TEM results show the coexistence of vesicles

and particles characterized by cubic organization which was in full agreement with the drug

entrapment and loading. Uranyl acetate as stain was used to get a phase contrast of

cubosomes from the environment. The ordered structure suggests the formation of cubical

nature rather than crystalline. For nanoparticles of smaller diameter ordered structures were

observed. Piperine, being lipophilic molecule has a tendency to partition into the hydrophobic

domains in the cubic phase. It was assumed that the drug at low levels only affect the lipid

bilayers slightly, which could not disturb the whole cubic phase.

Loading and release properties from cubosomes are governed by the solubilised

active. Loading properties are governed by the partition of actives between existing phases.

Higher the affinity of the active for the liquid crystal leads to higher loading. The bilayers are

associated with an energy known as curvature elastic energy.

Entrapment of active as well as size as detected by TEM was found to be

proportionate with the increase of GMO and poloxamer upto 76.25%.Thus entrapment

efficiency and size was directionally proportional. Functionalization was to control the

loading and release properties of the active by changing the properties of the cubic phase by

adding amphiphile molecules into the liquid crystal. Hydrophobic portion of amphiphile

inserts into the bilayers of the cubic phase and hydrophylic portion extends into the water

channels (5µ). Positive charged ampihiphile increases the loading of negative charged active.

Surfactants and large amphiphillic polymers used in this capacity are called “anchors” and

“tethers” respectively. Poloxamer 407 which was considered here was a block copolymer

which was considered as tethers. Glyceryl mono oleate (GMO) which was having solubility

of 10-6

M when placed in water produces reversed micelle and three different types of liquid

crystalline phases ie, lamellar, reversed hexagonal and cubic phases. In the present work

piperine being a lipophilic drug was added to the liquid GMO (when heated GMO was found

to be in liquid for hours) rather than in the tether[250].

Incorporation of cubosomes into hydrogel allowed to act as a base to apply topically.

To know whether by hydrogels we can tailor the drug into dermis, tissue deposition of drug-

hydrogel and cubosomes-hydrogel was studied. Although by increasing the concentration of

carbopol, negligible drug (10.9%) was localized at dermis. At the same time, 88.03±0.02 %

tissue bioavailability was achieved when GMO: drug ratio was kept as 1:1 with polaxamer. It

has to be specified that piperine being lipophilic, partitioning into the aqueous channels



becomes the rate limiting step. Ethanol in addition to be a solvent for piperine also acted here

as a hydrotrope to create a liquid precursor perhaps by exhibiting a “salting in behaviour”.

Thus the optimised formulation (TS1) was showing better tissue bioavailability than the

previous formulation transfersomes (75.25%±1.72).

Freeze thaw stability testing between 4°C ± 1 and 27°C ±1 for 8 cycles revealed good

spreadability which was constant, no crystal growth and slight sedimentation only towards

the last cycle. More than sedimentation, crystal growth should be more concerned since this

depressingly affects the hardening and consistency. Thus it has to say that the final

formulation was stable.

In ova studies were conducted in an attempt to establish biocompatability of the

formulation. This modle gives an oppurtuniuty to reduce the number of animals in

pharmacological experiments of this type of study. Normal blood vessel proliferency, no

atypical growth and regular embryonic movements significantly indicated that the

formulation was accepted biologically by the developing chick embryo.

Pharmacological evaluation (adult New Zealand (OB) black rabbits) of the piperine-

cubosomal hydrogel was performed to prove the anti Vitiligo efficacy. The studies revealed

that depigmentation took more time compared to that of brown rabbits. Depigmentation

commenced only by 35 days and completed by 60th day against 35 days complete

depigmentation in brown rabbits. Repigmentation for G3 (treatment with cubosomes + UVR)

commenced from 15th

day while G2 (treatment with cubosomes alone) there was no change.

The delayed response in black rabbits compared to brown rabbits was because the density of

melanin in the former was more than the later. G1 remained the same. By the end of one

month, pigmentation for G3 was found to be more homogenous and repigmentation for G2

started which suggests melanocytes proliferency and dendrites formations in the hair bulb.

Cubosomes have a unique bioadhesive nature, Hoath and Norlen reported that cubosomes

provide a distinctive electrical interrogation of the body because of the unique cubic phase

behavious on the human skin and skin substitutes [251].

Perhaps this could be the reason why cubosomal therapy when in conjugate with

UVR showed more homogenous. Almost complete repigmentation was observed towards 90th

day and more important the distribution of repigmentation was more homogenous than the

previous work with piperine cream. When we compare the depigmentation: repigmentation

period of studies of piperine-cream and piperine-cubosomes, black rabbits took almost double



the period to depigment, but recovery comparatively was found to be quite faster. Thus it has

to be emphasised that when comparing with cream, piperine-cubosomes therapy with or

without conjugate with UVR was found to be more effective.

Skin irritation refer to localized toxic effects resulting from a topical exposure of the

skin to a substance. The multicompartment model of skin irritation test when compared with

the chart has proved no allergic reactions as complying with the inova studies and the

formulation can be considered for clinical trials.

Confocal microscopy of histological specimen[252]

Confocal microscopy (CFM) of tissue specimen was used to observe the permeation

of cubosomes through excised pig skin. Tissue was kept inside the specimen chamber which

can provided supply of air (95%) and carbon dioxide (5%) through distilled water to produce

moist air at 37° which gives a artificial homeostasis for the tissue. Transmitted bright light

(20X, 40X) and Incident light- fluorescence was used to excite the flurochrom at 360 λmax

and observed by inverted microscopy. Laser, photomultiplier tubes, mercury lamp were used

for excitation and software- LASAF was used for the purpose. By using CFM resolution upto

200-250 nm can be obtained. Resolution zoom gives the pixel information. Refractive index

should be more than objective RI. Basic no of lasers: 4.405 diode (nuclear staining), 543

helium neon, argon, helium neon 633 (for IR so no use) are used. Argon laser has 3 sub lasers

(458 CFT, 488GFP and FITC, 514 YFP) thus there are total of seven lasers. High power laser

bleaches the dye and may not get proper results for interpretation. Different cells take up a

particular wavelength, so according to the dye select PMT1/ PMT2/PMT3.XY imaging and

XYZ imaging, voltage around 900 was used.

The tissue was not treated with formalin in order to avoid any undesired alteration in

the cubosomal permeation. Thus the tissue was found to degrade after 6 h. As the time

proceeds the cubosomes concentration were found to be recede in the stratum corneum and

exceeds in the dermal region. This shows cubosomal technology achieved its aim in targeting

and tailoring the drug at the dermis were maximum melanocytes are lodged.

DSC investigation

The transition peaks in the thermogram of treated pig skin with optimised formulation

of cubosomes were compared with that of the untreated skin, endothermic peak difference in

the former case (treated skin) shown a decrease in transition by 16.1°C. This decline can be



explained by the presence of cubosomes. Clues for the presence of cubosomes derived from

glyceryl mono oleate could interact with the lipid barrier of stratum corneum and penetrate

into later, which resulted in an increase of the enthalpy related to the lipid components of the

stratum corneum. Thus it has to be emphasised that cubosomes can produce an enhancing

effect on the skin penetration related to the effect of the lipophilic drug entity which may

penetrate deep into the dermal layer and fuse with skin lipids to loosen their structure

[238,253].

(v) Comparative drug quantification

Summarizing the tissue drug bioavailability for each optimized formulations, drug

tailored in the dermal region was in the descending order: ointment (22.5%) ˃ cream

(69.06%) ˃ phytosomes (72.07%) ˃ transfersomes (75.25%) ˃ cubosomes (88.03%)

Drug quantification from each optimized formulations diffused into the buffer was

phytosomes (22.60%) ˃ transfersomes (20.75%) ˃ cream (18.62%) ˃ ointment (16.2%) ˃

cubosomes (11.01%)

Drug left over the dorsal skin surface of each optimized formulation in the descending

order was ointment (59.99%) ˃cream (8.02%) ˃phytosomes (5.32%) ˃cubosomes (0.79%)

˃transfersomes (0.38%).

Rather than drug diffused into buffer and left over the dorsal surface of the skin, we

have to give much attention for the drug capable of targeting the dermis were melanocytes are

present. But at the same time let us discuss about the reason why certain fraction of the drug

was not able to either penetrate through the skin or got diffused into the buffer. The results

clearly indicate in ointment the drug left over on the dorsal surface was maximum thus it was

a poor formulation when compared to other formulations. The reason was that after the initial

penetration of the drug, the fatty acid bases must have become occlusive at the surface which

prevents further penetration of the drug. There was little effect upon addition of urea.

Mechanism of urea as permeation enhancer was pulling apart the cells leaving more gap for

the medicament along with the drug to penetrate. But in our case even though the inner skin

gaps became more due to urea, the occlusive layer is on top which prevents the penetration.

But still certain channels might have opened depending on the degree of conclusiveness

which shows a slight permeation enhancement, though it was negligible.



In cream the drug being lipophilic might have partitioned into the oil phase and thus

leaving the medicament, the oil phase along with the drug could penetrate through the tough

stratum corneum. But there was no special mechanism by which the drug can retain within

the dermis that could be the reason why the drug diffused into the buffer was more than

ointment. Thus it has to be said that the drug in cream was able to penetrate but also a major

part got diffused which was undesirable here. Even though when compared to ointment,

cream was able to retain more drug at the target which may be appreciable.

In phytosomes the drug was an integral part of the structure rather than occupying the

core. The phosphatidyl choline which resembles chemically much similar to the lipoprotein

bilayers of the skin was able to interact much and the drug was expected to lodge much more

than the conventional formulations. Contradictory to the expectations, although there was

almost 3% increase in dermal drug bioavailability, this was not attractive enough when

compared to the investment of the excipients and the process complications. This could be

because of the reason that although the phytosomes can enhance permeation, the phytosomal

complex was within the cream base. This means the case of cream almost repeats here,

although the presence of the complex had some contribution towards dermal drug targeting.

Interestingly phytosomes when in cream could penetrate but because of the lipophilic nature

the permeation was more thus reflecting the maximum drug diffused compared to other

formulations. The difference from cream was that phytosomal cream although could

penetrate more diffusion too was high. It may be appreciated that when compared to either

ointment or cream, the drug left over the skin was negligible which supports the above

statement.

Transfersomes are much bigger (in our case 66.97) than the skin pores (12). The

proviso was that the vesicular membrane elasticity was dynamically to the local stress by the

external. The driving drag down effect comes when; the transfersomal suspension gets dried

up due to evaporation of the continuous media which was facilitated by the body temperature.

Thus we claim that our formulation can considerably easily permeate through skin pores

(20). The proviso was that the vesicular membrane elasticity was dynamically to the local

stress by the external environment. The moisture of the beneath skin layers and the dry

outside layer pulls the transfersomes down, more correctly squeezes down through the

narrow pores. From the results of the drug fraction left over it was very clear that it was

negligible (0.38%) which was fascinating. It has to be noted down that Span 80 shows more



entrapment efficiency when compared with tween 80, because span 80 was less hydrophilic

(lower HLB) than tween 80. Naturally the lipophilic drug preferres span rather than tween.

From the results it was very clear that cubosomes could penetrate, tailor the drug into

the dermis which was the target of our interest with minimal diffusion. The advantage we got

here was drug being liphophilic was able to accommodate more into lipid GMO. Then it

becomes the partitioning of the drug from the lipid portions of the cubical structure into the

5µ water channels. It was hypothesised that the drug comes out through these channels. It

was an interesting factor that when cubosomes for the optimized formulation were

considered, drug diffusion could be kept minimal at 11.01% and the drug retention on the top

was negligible (0.79%). Thus the cubosomes of drug: GMO was 1:1 and Poloxamer 407

when kept at 9% with respect to drug-GMO was able to tailor the drug maximum at the

dermal region when compared to other intra and inter formulation variables awarding this as

the optimised formulation.

(vi) Discussion on drug release and release kinetics

In this study tissue drug retention capacity was more significant than drug release to

the blood stream. But still to know the nature of the drug release pattern, we must know the

release kinetics. The quantitative interpretation of the drug release values was supported by

generic equations that translates drug release curve in function of some parameters

mathematically related with the respective dosage forms.

Drug release kinetics are greatly influenced by crystallinity, solubility, particle size

amount of API (Active Pharmaceutical Ingredients) etc. A Water soluble API homogenously

distributed in a matrix system was mainly released by diffusion. At the same time,

liphophilic drugs, the mechanism was based on self erosion. In this case the API was

liphophilic.

The various mathematical model for the drug release kinetics are Zero order, First

Order, Higuchi model, Korsemeyer Peppas model, Weibull model, Hixson Crowell’s model,

Baker- Lonsdale model, Hopfenberg model etc. Here mathematical expressions for different

dosage forms are done by using the respective equations and ‘n’ value was found out. The

highest ‘n’ value among all the models (Zero, First, Higuchi etc.) can be found out which is

considered as the optimized drug release model. This is called “Kinetic drug release model

fitting” and the graphical expressions are known as “Kinetic drug release model fitting



curves”. Out of all kinetic models, the first four are the most vital and widely used. Thus

here more emphasis was given for these models.

Zero order release kinetics is used for utilized for dosage form do not disintegrate

through out the dissolution. No equilibrium conditions are obtained. Graph of Ct vs t should

produce a straight line. First order20

was first proposed by ‘Gibaldi’ and ‘Feldman’(1967) and

later by ‘Wagner’(1969).This was based on Noyes-Whitney equation. Graph of Log Ct vs t

should produce a straight line.Applied for drug release from solid particles in a liquid media;

also for a describing absorption and elimination[254].

Higuchi release model is to study the release of water soluble and low soluble drugs

incorporated in semisolid, solid matrix and matrixes including TDDS. Here the matrix acts as

a planar diffusion homogenous media. Matrixes due to the porous nature will have channels.

These channels with in the matrix generates a first percolation threshold to increase its

mechanical stability and below the second percolation threshold to release all the drug

content. As the drug diffuses out through the surface of an erodible matrix dosage form into

the dissolution media, the surface of the device gets depleted. This becomes a depletion layer

and distance for the drug to travel ( diffusion ) from the matrix of the dosage form into the

media becomes more. Thus boundary of the drug shifts to left (dh). The equation used was Ct

= C0+ KH t1/2

were Ct = amount of drug released in time t, C0 = initial amount of drug KH =

Higuchi rate constant. Graph of Ct vs t1/2

( square root of time ) should produce a straight

line[255,256].

Korsemeyers- Peppas is a hybrid of two equations developed one in 1983 by

Korsemeyers-et-al and another in1985 by Peppas. . In the excel graph we get two

information. One on R2 and another on equation for the linearity (y). Log time vs log %drug

release gives the R2 and represents the equation: y = m x + c, were y was the equation for the

linearity, “m” was slope, “x” was constant. “m” was considered as “n” value which was

used to determine whether the mechanism was Fickian , non Fickian or anomalous. If the

drug release follows diffusion, it may follow fickian equation. (drug amount relased vs √time

graph will be straight line.) If the ‘n’value of a slab in the equation was 0.5 then it follows

‘Fickian diffusion’. Some times this deviates, representing a non-Fickian, i.e if we get the

‘n’values between 0.5 and 1(or) n=1 for mass transfer following a ‘non-fickian’model. If the

‘n’ value is almost 0, then it is anomalous diffusion. To use this equation it is also necessary

that release occurs in a one dimensional way and the system width-thickness or length



thickness relation be at least 10. This model is generally used to analyze the release of

pharmaceutical polymeric dosage forms, when the release mechanism is not well known or

when more than one type of release phenomena could be involved [257].

The mechanism of release for all the formulations was determined by finding the R2

value for each kinetic model viz. Zero-order, First-order, Higuchi, and Korsmeyer-Peppas

corresponding to the release data of formulations.



Table 22: Mathematical models and equations concerned which is utilised in model

fitting curves [258]

Ct = amount of drug released in time t, C0 = initial amount of drug, K0 = zero order release

constant, K1 = first order proportionality constant, KH = Higuchi rate constant, C∞ = total

amount of drug dissolved when the dosage form is exhausted off the drug, Kk = Korsmeyer

rate constant.

Drug release kinetic model Equation X axis vs Y axis

Zero order:

Ct = C0+ K0t

Time vs %drug release

First order:

Log Ct = logC0+ K1 t/ 2.303

Time vs log %drug released

Higuchi:

Ct = C0+ KH t1/2

√time vs %drug released

Korsemeyers- peppas:

Ct/ C∞ = Kk tn

Log time vs log %drug release.



Comprehensive discussion on drug release kinetics of all formulations

Table 23:Drug release kinetics of cream and ointment

Formulation Zero First Higuchi Peppas n

Cream 0.777322389 0.51691858 0.947135035 0.505880848 0.6286

Ointment 0.962658117 0.837180238 0.93231288 0.739151109 0.7695

Drug+urea 0.975245 0.800999799 0.960843795 0.721089635 0.7679

All the formulations except cream were good fit into the zero order with R2 value >

0.9626, then higuchi followed by first order. Cream was good fit into Higuchi model with R2

value 0.9471 then zero order followed by zero order. The data obtained were also put in

Korsemeyer –Peppas model in order to find out ‘n’ value, which describes the drug release

mechanism.

The n values of Korsmeyer-Peppas model of all the formulations are in between 0.55-

0.9. Therefore the most probable mechanism that the release patterns of the formulations

followed was non-Fickian diffusion or anomalous diffusion (table 23).

Table 24: Drug release kinetics of phytosomal formulation

Phytosome

code

Zero First Higuchi Peppas n

P1 0.993752 0.878752 0.899746 0.9949 1.2994

P2 0.991302 0.863181 0.949023 0.9692 0.9582

P3 0.951157 0.894835 0.84127 0.9252 0.992

Formulation P1 best fit into Korsmeyer-Peppas model with R2 value 0.9949, then zero

order followed by higuchi equation. Formulations P2 and P3 best fit into zero order with R2

value > 0.9511, then Korsmeyer-Peppas followed by higuchi equation.

The n value of Korsmeyer-Peppas model for formulation P1 is > 1. Therefore the

most probable mechanism that the release patterns of the formulation followed was super

case-II transport. The n values of Korsmeyer-Peppas model for formulation P2 and P3 are in



between 0.55- 0.9. Therefore the most probable mechanism that the release patterns of the

formulations followed was non-fickian diffusion or anomalous diffusion (table 24).

Table 25: Drug release kinetics of transfersomes

Transfersome

code


TT1 0.8702 0.6171 0.9851 0.805 1.1621

TT2 0.9726 0.8103 0.9437 0.9562 1.1933

TT3 0.9723 0.773 0.9591 0.924 1.2

TS1 0.902 0.6521 0.9955 0.8293 1.0801

TS2 0.8961 0.6228 0.9966 0.7998 1.1091

TS3 0.9122 0.6442 0.9954 0.815 1.1

All the formulations except TT2 were good fit into the H iguchi model with R2 value

> 0.9954, then zero followed by peppas. TT2 was good fit into peppas model with R2 value

0.9562 then higuchi followed by zero order. The data obtained were also put in Korsemeyer –

Peppas model in order to find out ‘n’ value, which describes the drug release mechanism.

The n values of all the formulations are > 1. Therefore the most probable mechanism

that the release patterns of the formulation followed was super case-II transport (table 25).

Table 26: Drug release kinetics of cubosomes

Cubosome

code


C1 0.959458 0.868249 0.940581 0.977114 0.9696

C2 0.963083 0.90649 0.845306 0.943318 1.117

C3 0.909273 0.902614 0.701077 0.76742 0.7942

Formulation C1 best fit into Korsmeyer-Peppas model with R2 value 0.9771, then

zero order followed by higuchi equation. Formulations C2 and C3 best fit into zero order with

R2 value > 0.9092, then Korsmeyer-Peppas followed by Higuchi equation.



The n value of Korsmeyer-Peppas model for formulations C1 and C3 is between 0.5-

0.9. Therefore the most probable mechanism that the release patterns of the formulations

followed was non-fickian diffusion or anomalous diffusion. The n value of Korsmeyer-

Peppas model for formulation C2 >1, therefore the most probable mechanism that the release

patterns of the formulation followed was super case-II transport (table 26).

In sum, it can be said when cubosomes at drug: GMO ratio of 1:1 and Poloxamer 407,

9% were fixed, the drug piperine was able to tailor to the dermis at its maxima, which was

obvious in the in vivo experimental data.

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