Introduction

Aging comprises the various changes that occur in the organisms with the passage of time, leading to increased system entropy, loss of homeostasis and eventually death [5]. According to free radical theory, a shift in the antioxidant/pro-oxidant balance increases the oxidative stress. This leads to dysregulation of cellular function and aging. The most important free radicals in the body are the radical derivatives of oxygen, known as reactive oxygen species (ROS). Excess free radicals react with cellular lipids, proteins and nucleic acids, leading to local injury and eventual organ dysfunction. Lipid biomolecules are probably the most susceptible to free radical attack. Cell membranes are a rich source of polyunsaturated fatty acids, which are readily attacked by oxidizing agents, a process that is called “lipid peroxidation” [15, 16, 22].

Wrinkling is a natural part of aging. As we grow older, the skin gets thinner, dryer, less elastic, and hence wrinkled. Weakening of the skin’s connective tissue (collagen and elastin) in its underlayer causes sagging and wrinkling. It has been proved that accumulated damage by free radicals produced by exposure to UV radiation results in extensive damage to the skin tissues [20, 25]. Also, electron spin resonance spectroscopy (ESR) has provided irrefutable evidence that UV exposure leads to the generation of a host of free radicals, producing an effect commonly known as “photoaging” [19]. Extensive photoaging is characterized by wrinkles, telangiectases (abnormal dilation of blood vessels), benign skin growth, coarsening of skin, dryness, mottled hyperpigmentation, heliodermatitis and hyperplasia of elastic fibres. Generation of free radicals as a result of exposure to UV radiation is considered to be a major pathway for skin aging. Repetitive exposure to UV radiation leads to the formation of peroxyl free radicals, which break down to form malondialdehyde (MDA). MDA subsequently cross-links and polymerizes collagen, leading to loss of skin elasticity and decreasing the capacity of the skin to hold water. The outcome of cross-linking of collagen is generation of wrinkles [21, 32].

Curcumin (CU), a natural polyphenol, found in the rhizomes of Curcuma longa (turmeric) is well known for its medicinal properties since ancient times. Curcumin shows antioxidant activity as seen by its ability to inhibit lipid peroxidation. The antioxidant activity of curcumin arises mainly from scavenging of several biologically relevant free radicals that are produced during physiological processes. Apart from antioxidant action, curcumin has been reported to have a variety of biological activities and pharmacological actions, such as antioxidant, anti-tumor, anti-inflammatory, antidiabetic, antirheumatic, wound healing, anti-viral, hepatoprotective and anti-HIV [2, 14, 30].

Chemically, curcumin (Fig. 1) is diferuloylmethane [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] [2]. It is unstable in basic pH, and degrades within 1 h to trans-6-(40-hydroxy-30-methoxyphenyl)-2,4-dioxo-5-hexanal, ferulic acid, feruloylmethane and vanillin. The presence of foetal calf serum or human blood, or addition of antioxidants such as ascorbic acid, N-acetylcysteine or glutathione, completely blocks this degradation in culture media or phosphate buffer above pH 7. Under acidic conditions, the degradation of curcumin is much slower, with less than 20% of total curcumin decomposed at 2 h [2, 30]. Cutaneous absorption of curcumin is very poor and traces of topically applied curcumin reaches up to the dermis. Considering the fact that free radicals react with cellular lipids and protein on skin, topical application of curcumin can offer the advantage of delivering the drug directly to the skin and producing its local effects.

Fig. 1
figure 1

Structure of curcumin

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Creams and gels containing curcumin are popularized worldwide and marketed (e.g. Vicco Turmeric cream, Emami Gold, etc.) all over the world, but even after incorporation of high amount of curcumin in topical formulations, significant antioxidant and anti-aging effect could not be achieved. The barrier properties of intact skin limit the permeability of curcumin through skin [28].

To improve the topical bioavailability of curcumin, numerous approaches have been investigated. These approaches include loading curcumin into liposomes or nanoparticles, forming self-microemulsifying drug delivery systems (SMEDDS), transdermal films and synthesizing structural analogues of curcumin [3, 9, 23]. To reduce the barrier properties of stratum corneum, various chemical enhancers have also been used that allow drug permeation through the skin. Use of the chemical enhancers produces percutaneous irritation and sometimes dermal allergy which is not acceptable cosmetically [27, 28].

Phospholipids are complex molecules that are used in the formation of cell membrane and known as building blocks. Complexing of the phospholipids with the standardized botanical extracts has provided dramatic bioavailability enhancement and faster and improved absorption through skin [7, 35]. In our lab, we developed and evaluated vesicles of boswellic acid and found dramatic increase in absorption of boswellic acid through skin by vesicular system [29]. Curcumin is poorly absorbed and therefore strategies to improve its topical bioavailability need to be worked. Complexation with phospholipid has been used in the present studies to meet this objective. One system (phyto-vesicles) was developed by us and its superiority over other delivery systems (viz. liposomes and niosomes) was compared. The phyto-vesicles were also compared with plain curcumin and physical mixture of curcumin with phosphatidylcholine, in terms of better antioxidant, anti-aging and antiwrinkle effect when applied topically.

Materials and methods

Materials

Curcumin and cholesterol were purchased from Sigma Chemical Co., USA. The soy-phosphatidylcholine (Lipoid S 100) was obtained as gift sample from Lipoid, Ludwigshafen, Germany. Thiobarbituric acid, l-hydroxyproline and glutathione were purchased from Hi Media, India. All other chemicals and solvents were of analytical grade.

Preparation of curcumin-phosphatidyl choline complex

Curcumin and soy-phosphatidyl choline (PC) in the molar ratio of 1:1 were taken in a round bottom flask and dissolved in 20 ml dichloromethane. This mixture was stirred for 2 h at room temperature on a magnetic stirrer. The solvent was then removed under reduced pressure in a rotary evaporator at 30°C. The resultant curcumin–phosphatidylcholine (CU–PC) complex was washed with n-hexane to remove traces of PC, dried under vacuum (yield 91.38%, w/w), kept in amber-coloured glass bottle and stored at room temperature (Fig. 2) [13, 29, 36].

Fig. 2
figure 2

Flow diagram explaining preparation of CU–PC complex

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Characterization of complex

Thin layer chromatography

Thin layer chromatography (precoated silica gel TLC plates, 60F254, E Merck Ltd, Mumbai, India) of the curcumin, PC, CU–PC complex and physical mixture of curcumin with PC was performed using chloroform:methanol (7:3) as solvent system. After development, the plates were sprayed with 10% methanolic sulphuric acid and heated at 110°C for 10 min.

Differential scanning calorimetry (DSC)

DSC thermograms were recorded with a METTLER STAR SW 9.00 DSC instrument. The samples were sealed in the aluminum crimp cell and heated from 20 to 200°C at 10°C/min. The onset, peak and endset temperatures of curcumin, PC and CU–PC complex were determined and compared.

Melting point

Melting point of curcumin, PC and CU–PC complex was determined by digital melting point apparatus (Scientific International, New Delhi, India).

Infrared spectroscopic analysis

FTIR spectra were obtained on the 8400 Shimadzu FTIR spectrometer with the wave number 500–4,000 cm−1 using KBr pellets. Briefly, 500 mg potassium bromide and 5 mg sample were taken into a mortar, mixed and ground to a fine powder. Homogeneous disc was prepared by compressing the powder and inserted into IR sample holder carefully. The spectrum was recorded between 500 and 4,000 cm−1.

Preparation of vesicular systems

Preparation of phyto-vesicles

The CU–PC complex and cholesterol in 7:3 ratio were dissolved in chloroform:methanol (3:1) and then introduced into 250 ml round bottom flask with round glass neck. The flask was attached to rotary evaporator (Model RE-52, SONAR, New Delhi, India) and rotated at 60 rpm. The solvent was evaporated at 35 ± 2°C under reduced pressure to get film on the wall of flask. The casted film was dispersed in phosphate buffer saline (PBS, pH 6.8) at 55 ± 2°C. Upon hydration for about 2 h, the lipid swells and is peeled off from the wall of round bottom flask and vesiculate forming vesicles [29].

Preparation of liposomes

Liposome formulation was prepared by lipid film formation technique. Phosphatidyl choline and cholesterol were taken in 7:3 ratio in a round bottom flask and dissolved in chloroform. Curcumin was incorporated in chloroform at the time of film formation. Film was formed by evaporation of chloroform in a rotary vacuum evaporator at 35 ± 2°C.

After 1 h the resulted film was hydrated with phosphate buffer saline (pH 6.8) solution for about 45 min. Temperature was maintained at 55 ± 2°C. The lipid swells and peals-off from the wall of round bottom flask and vesiculate forming multilayer vesicles [29].

Preparation of niosomes

Niosomes were prepared by reverse evaporation technique with slight modification. Span 60 and cholesterol in 7:3 ratio were dissolved in diethylether in a sonication tube to which curcumin was added and dissolved. Further, PBS pH 6.8 was added and the mixture was sonicated in a probe type sonicator (Soniweld, India) for 1 min.

The mixture was then placed in a rotary evaporator and the organic solvent was removed under vacuum to form a gel. The gel was then subjected to vigorous mechanical agitation on a vortex mixer to form the suspension. The niosomal suspension after suitable dilution was allowed to stand for further 2 h at room temperature to allow complete swelling of the vesicles [29].

Characterization of vesicular systems

Vesicle shape

Vesicular systems were visualized by Philips Morgani 268 Transmission Electron Microscope. A drop of each formulation (phyto-vesicles, liposomes and niosomes) was placed on different carbon coated copper grids to leave a thin film on the grids. Then, the film was negatively stained with 1% phosphotungstic acid by placing a drop of the staining solution on to the film and the excess of the solution was drained off with a filter paper. The grid was allowed to dry and formulations were viewed under the transmission electron microscope and photographs were taken at suitable magnification.

Vesicle size

The size and size distribution of vesicles (phyto-vesicles, liposomes and niosomes) were determined by Malvern zeta sizer after dilution of formulations with phosphate buffer.

Entrapment efficiency

The entrapped drug was estimated by taking 1 ml of dialyzed vesicular suspension and digesting it with 0.1 ml of Triton X-100 for 5 min. The resulted solution was centrifuged at 3,000 rpm for 5 min. The supernatant was decanted off and suitably diluted with methanol. The drug was estimated spectrophotometrically at λ max 425 nm against methanol containing Triton X-100 as blank. The whole procedure was repeated three times and data is reported as mean ± SEM.

Screening of anti-aging effect of vesicular systems (UV-induced aging in mice)

Animals

Swiss albino mice (male/female) weighing 20–25 g were used to evaluate the anti-aging activity of the prepared formulations. The animals were housed in polypropylene cages at standard animal house conditions (25 ± 2°C temperature, 60 ± 5% relative humidity and 12 h dark/light cycle). The mice were fed with standard laboratory diet and water ad libitum throughout the experiment. The animals were maintained as per CPCSEA regulations and studies were performed after due permission by Institutional Animal Ethics Committee (IAEC) of the university.

Animal grouping

Fifty-four animals were divided into nine groups consisting of six animals in each group. A demarcated area of approximately 2 cm2 on the dorsal surface of the mice was shaved using a soft hair removing cream. The mice were observed for 48 h to exclude mice showing abnormal hair growth or a reaction to the depilatory preparation. A hair removing cream was preferred over a shaving blade to minimize free radical production due to trauma from the blade [31]. The study pattern was designed according to the following manner:

Group I:

Control group (without UV and/or formulation treated)

Group II:

UV-treated group

Group III:

UV treated + plain carbopol gel

Group IV:

UV treated + curcumin gel

Group V:

UV treated + CU–PC complex gel

Group VI:

UV treated + CU–PC physical mixture gel

Group VII:

UV treated + phyto-vesicles gel

Group VIII:

UV treated + liposomes gel

Group IX:

UV treated + niosomes gel.

All the vesicular suspensions were incorporated into 1% carbopol 934 gel separately, so as to make feasible for topical application. In addition, gels containing curcumin, CU–PC complex and physical mixture of curcumin with phosphatidylcholine were also prepared separately. The plain carbopol gel (without CU and/or PC) was used for the animals of Group III. The formulation was applied 1 h before UV exposure, once daily to the animals of respective groups [4, 31].

UV exposure to animals

In the present experiment an ultraviolet lamp (200–400 nm) was used to induce photo stress on the skin of mice. The irradiance of 3.6 ± 0.4 mW/cm2 was exposed to achieve 5 J/cm2 intensity of exposed light radiation. The time of light exposure was calculated according to OECD guidelines by following formula [4, 31]:

$$ T(\min ) = {\frac{{{\text{Irradiation\,dose (J/cm}}^{ 2} )\times 1,000}}{{{\text{Irradiance (mW/cm}}^{ 2} )\times 60}}}. $$

The respective gel formulations were applied prior to UV irradiation on shaved dorsal area of mice.

Biochemical estimations

After 30 days of study, mice were sacrificed under light ether anaesthesia and their skins were surgically removed. The excess of fascia and adherent tissues were removed by washing with chilled normal saline solution. A part of skin tissues was weighed and minced on glass plate over ice bags and homogenized to make colloid. The homogenates were centrifuged at 3,000 rpm for 15 min and supernatant fractions were collected and stored in deepfreeze for further estimation of biochemical parameters.

To study the UV protecting effects of different formulations, biochemical parameters were estimated by well-known procedures, i.e., total protein [11], hydroxyproline [34], malondialdehyde (MDA) [26], superoxidedismutase (SOD) [18], reduced glutathione (GSH) [10], catalase (CAT) [1] in each set of experiment.

Moisture content

Moisture content of skin tissue was determined by IR moisture balance (model FD 240, Kett US, CA, USA) to evaluate the hydrating (moisturizing) effect of different formulations. Immediately after sacrificing the animals, a part of skin from each mouse was deprived of the subcutaneous tissue and fascia. The trimmed whole skin weighing approximately 1 g was put on the pan of moisture balance and heated at 160°C till complete drying and moisture content was calculated by the following formula [17]:

$$ \text{Moisture\, content\,(\%)} = \frac{{\text{Weight \,before\, drying - Weight\, after\, drying}}}{{\text{Weight\, before\, drying}}}\times 100. $$

Histology of skin

For histological studies, skin tissues were fixed in freshly prepared 10% chilled formalin for 24 h. After fixation, skin tissue was rinsed in distilled water several times and dehydrated in graded alcohol series, cleared in xylene and embedded in paraffin wax to make tissue blocks. The 5-μm-thick sections were cut by using rotary microtome. Sections were stained with hematoxylin and eosin stain for demonstration of ultra structural epidermal and dermal changes [24].

Statistical analysis

Data were expressed as mean ± standard error of mean (SEM). Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by Dunnett’s test using GraphPad Prism 3 (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Characterization of complex

Thin layer chromatography

The R f value of curcumin, PC and CU–PC complex was found to be 0.78, 0.42 and 0.65, respectively. The CU–PC physical mixture showed two spots at R f values 0.78 and 0.43. Difference in the R f value of curcumin and CU–PC complex indicates the formation of complex.

Differential scanning calorimetry (DSC)

In DSC, an interaction is concluded by elimination of endothermic peaks, appearance of new peaks, change in peak shape and its onset, peak temperature/melting point and relative peak area. DSC thermograms of curcumin, PC and CU–PC complex are shown in Figs. 3, 4, 5. The thermogram of curcumin showed a single peak with an onset of 170.14°C and maximum occurrence at 176.24°C. Thermogram of PC exhibits two different peaks at 113.29 and 185.37°C, which may be due to the hot movement of phospholipids polar head group and due to phase transition from gel to liquid crystalline state, respectively. In the thermogram of the complex a single peak with an onset at 68.50°C and maximum occurrence at 75.99°C appeared, which is different from the peaks of the individual components (CU and/or PC) of the complex.

Fig. 3
figure 3

DSC thermogram of curcumin

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Fig. 4
figure 4

DSC thermogram of PC

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Fig. 5
figure 5

DSC thermogram of CU–PC complex

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Melting point

Melting point of curcumin, PC and CU–PC complex was found to be 176–178, 108–112 and 74–76°C, respectively.

Infrared spectroscopic analysis

The IR spectrum of CU–PC complex was found to be significantly different from the IR spectrum of curcumin and the phosphatidylcholine which supports the reaction of -OH group of curcumin at choline part of PC and hence complex formation.

Characterization of vesicular systems

As shown in the Table 1, average vesicle size of the phyto-vesicles was found to be 547.19 ± 18.87 nm, whereas liposomes and niosomes were smaller in size and have average vesicle size of 331.94 ± 17.13 and 238.25 ± 11.49 nm, respectively. Entrapment efficiency of liposomes and niosomes was found to be 62.13 ± 3.81 and 68.52 ± 4.21%, respectively, whereas the lipid material of phyto-vesicles contains complex of PC and curcumin (1:1 molar ratio).

Table 1 Average vesicle size and entrapment efficiency of various vesicular systems
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As shown in the Figs. 6, 7, 8, the photographs of transmission electron microscopy (TEM) clearly reveal the formation of vesicles.

Fig. 6
figure 6

TEM photograph of phyto-vesicles of curcumin

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Fig. 7
figure 7

TEM photograph of liposomes of curcumin

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Fig. 8
figure 8

TEM photograph of niosomes of curcumin

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Anti-aging effects of vesicular systems

Moisture content

Moisture content of skin in normal mice was found to be 67.79 ± 2.12%, which was decreased to 27.00 ± 1.03% after UV exposure (Fig. 9). Topical application of gel containing CU–PC complex before the UV exposure restored the moisture content to 70.48 ± 3.84%, which is higher than the moisture content of normal mice. Application of gel containing phyto-vesicles of curcumin was found to be the most effective than any other formulation, as the skin moisture content of this group of mice was found to be 74.49 ± 3.28, which is highly significant when compared to control group (p < 0.01).

Fig. 9
figure 9

Moisture content (%) of mice skin

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Application of gels containing liposomes and niosomes of curcumin also restored the skin moisture content to 62.88 ± 3.11 and 59.35 ± 2.64%, respectively, which is also significant but these formulations were found to be less effective than phyto-vesicles. In case of application of gels containing CU–PC physical mixture and plain curcumin before UV exposure, the moisture content was found to be 53.77 ± 2.53 and 46.59 ± 2.17%, respectively. Application of plain carbopol gel before UV exposure was found ineffective in restoring the skin moisture, as the moisture content was only 28.67 ± 1.01%.

Biochemical parameters

As shown in Table 2, total protein and hydroxyproline content of normal mice were found to be 148.24 ± 6.21 and 55.37 ± 2.32 mg/g of skin tissue, respectively. After UV exposure the level was decreased to 103.54 ± 4.04 and 23.26 ± 1.85 mg/g of tissue, respectively. Treatment with gel containing CU–PC complex restored the total protein and hydroxyproline level to 158.39 ± 6.27 and 60.28 ± 2.85 mg/g of tissue, respectively, which is higher than the normal mice. Moreover, application of gel containing phyto-vesicles of curcumin increased the same to 166.73 ± 7.31 and 66.25 ± 2.64 mg/g, respectively, which is highly significant as compared to control group of animals (p < 0.01). Thus phyto-vesicles were found to be more effective than CU–PC complex in terms of restoring the total protein and hydroxyproline content of mice skin. Application of gels containing liposomes and niosomes also restored the total protein level to 144.28 ± 4.84 and 147.03 ± 4.33 mg/g tissue, respectively, and hydroxyproline content to 52.19 ± 1.95 and 54.14 ± 2.13 mg/g tissue, respectively. Application of gels containing CU–PC physical mixture and plain curcumin were found to be least effective, as the total protein level was found to be 139.27 ± 6.11 and 138.72 ± 5.52 mg/g, respectively, and hydroxyproline content 48.67 ± 2.74 and 49.02 ± 1.93, respectively. Treatment with plain carbopol gel was found ineffective in restoring the total protein (105.21 ± 3.63 mg/g tissue) and hydroxyproline content (23.02 ± 1.13 mg/g tissue).

Table 2 Effect of the various formulations on total protein, hydroxyproline and malondialdehyde (MDA) in UV-treated mice
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Malondialdehyde (MDA) level of normal mice was 1.13 ± 0.09 μmol/g tissue, which was increased to 2.09 ± 0.13 μmol/g tissue after UV exposure. On treatment with gel containing CU–PC complex and phyto-vesicles, it was decreased to 1.23 ± 0.06 and 1.11 ± 0.02 μmol/g tissue. In this case also phyto-vesicles were found to be the most effective in providing protection against lipid peroxidation of skin. Treatment with gels containing liposomes, niosomes, CU–PC physical mixture and plain curcumin also decreased the level of MDA to 1.38 ± 0.04, 1.41 ± 0.03, 1.58 ± 0.10 and 1.53 ± 0.08 μmol/g tissue, respectively, but these formulations were found to be less effective than phyto-vesicles and CU–PC complex containing gels in providing protection against lipid peroxidation. Plain carbopol gel was unable to reduce the increased MDA level and in this group the MDA level was found 2.11 ± 0.16 μmol/g tissue.

As shown in Table 3, the level of enzyme catalase (CAT) and superoxidedismutase (SOD) was found to be 71.53 ± 3.93 and 87.29 ± 4.98 U/g tissue, respectively, in normal mice. After UV exposure, the level of these enzymes was reduced to 32.29 ± 1.93 and 24.59 ± 1.31 U/g tissue, respectively. Topical treatment of skin with gels containing phyto-vesicles and CU–PC complex increased the level of CAT to 96.48 ± 5.19 and 78.19 ± 4.25 U/g tissue, respectively. SOD was also restored after treatment with these formulations and found to be 103.21 ± 5.74 and 94.84 ± 5.12 U/g tissue, respectively. Application of gels containing liposomes and niosomes also increased the level of CAT to 65.93 ± 2.41 and 69.34 ± 3.73 U/g tissue, respectively, and level of SOD to 81.97 ± 3.91 and 86.21 ± 4.11 U/g tissue, respectively. In case of gels containing CU–PC physical mixture and plain curcumin the level of CAT was found to be 61.27 ± 2.84 and 60.32 ± 2.33 U/g tissue, respectively, and level of SOD was found to be 74.32 ± 3.08 and 70.39 ± 3.19 U/g tissue, respectively. Treatment with plain carbopol gel was ineffective in restoring the CAT and SOD, as the level of these enzymes was found to be 35.12 ± 1.28 and 23.16 ± 1.08 U/g tissue, respectively. Thus in this case also phyto-vesicles were found to be most effective in restoring the antioxidant enzymes (CAT and SOD) in skin tissue of mice.

Table 3 Effect of the various formulations on catalase, superoxidedismutase (SOD) and reduced glutathione (GSH) in UV-treated mice
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The level of reduced glutathione (GSH) in the skin homogenate of normal mice was recorded as 47.19 ± 2.14 mg/g tissue. After UV exposure the level was decreased to 13.62 ± 0.83, which is an indication of oxidative stress. Treatment with gels containing phyto-vesicles and CU–PC complex increased the GSH level to 53.19 ± 2.72 and 48.28 ± 2.11 mg/g tissue, respectively. In case of treatment with gels containing liposomes, niosomes, CU–PC physical mixture and plain curcumin the level of GSH was found to be 44.87 ± 2.19, 42.89 ± 2.16, 37.13 ± 2.38 and 34.16 ± 2.09 mg/g tissue, respectively. The animals treated with plain carbopol gel showed GSH level of only 14.13 ± 0.62 mg/g tissue. Here too phyto-vesicles were most effective in restoring the level of GSH among various formulations tested.

Histopathological studies

As shown in Fig. 10, the normal skin showed normal arrangement of connective tissue (consisting of collagen and elastin), normal epidermis and adipose tissue. After chronic UV exposure epidermal hyperplasia, infiltration of the skin layers with neutrofils and destruction of the integrity of the connective tissue could be seen in comparison to section from normal mice (Fig. 11). Severe elastosis was observed which was not only confined to the upper dermis but was noted to be more dense in deeper dermis layer. Discontinuous and fibroblastic collagen bundles are also observed after chronic UV exposure.

Fig. 10
figure 10

Photomicrograph of normal mice skin (E epidermis, D = dermis, CT connective tissue, CF collagen fibres, EF elastin fibres)

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Fig. 11
figure 11

Photomicrograph of UV-treated mice skin (E epidermis, D dermis, CT connective tissue, CF collagen fibres, EF elastin fibres, N neutrofils, BV blood vessels, V vacuoles)

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Treatment of mice skin with gels containing phyto-vesicles or CU–PC complex showed regenerative changes in the connective tissues and integrity is maintained. Application of these gels also prevented the premature elastic degeneration of collagen and remarkable protection of collagen bundles and elastin fibres was observed. In case of mice treated with gel containing CU–PC complex some damage could be seen but the section of mice treated with gel containing phyto-vesicles is like normal mice (Figs. 12, 13).

Fig. 12
figure 12

Photomicrograph of mice skin exposed to UV and treated with gel containing CU–PC complex (E epidermis, D dermis, CT connective tissue, CF collagen fibres, EF elastin fibres)

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Fig. 13
figure 13

Photomicrograph of mice skin exposed to UV and treated with gel containing phyto-vesicles (E epidermis, D dermis, CT connective tissue, CF collagen fibres, EF elastin fibres)

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When gel containing liposomes or niosomes was applied on mice skin there is significant protection of connective tissue, collagen bundles and elastin fibres at the level of deeper dermis but degeneration could be seen in upper dermis and epidermis (Figs. 14, 15). Niosomes were found to be more effective than liposomes but both were found less effective than either phyto-vesicles or CU–PC complex.

Fig. 14
figure 14

Photomicrograph of mice skin exposed to UV and treated with gel containing liposomes (E epidermis, EH epidermal hyperplasia, CT connective tissue, CF collagen fibres, EF elastin fibres, D dermis)

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Fig. 15
figure 15

Photomicrograph of mice skin exposed to UV and treated with gel containing niosomes (E epidermis, EH epidermal hyperplasia, CT connective tissue, CF collagen fibres, EF elastin fibres, D dermis, V vacuoles)

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In case of treatment with gel containing CU–PC physical mixture or plain curcumin there is very little protection against UV exposure. In these sections destruction of integrity of connective tissues, infiltration of skin layers with neutrofils and damaged collagen bundles and elastin fibres were observed (Figs. 16, 17). Little protection could be seen when compared with UV-exposed mice but these formulations were found less effective than others. Treatment with plain carbopol gel was found ineffective to protect the skin damage by chronic UV exposure and the skin section is almost same as in the UV-exposed mice (Fig. 18).

Fig. 16
figure 16

Photomicrograph of mice skin exposed to UV and treated with gel containing curcumin (E epidermis, EH epidermal hyperplasia, CT connective tissue, CF collagen fibres, EF elastin fibres, N neutrofils, V vacuoles)

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Fig. 17
figure 17

Photomicrograph of mice skin exposed to UV and treated with gel containing CU–PC physical mixture (E epidermis, EH epidermal hyperplasia, CT connective tissue, CF collagen fibres, EF elastin fibres, N neutrofils, V vacuoles)

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Fig. 18
figure 18

Photomicrograph of mice skin exposed to UV and treated with plain carbopol gel (E epidermis, EH epidermal hyperplasia, CT connective tissue, CF collagen fibres, EF elastin fibres, N neutrofils, V vacuoles)

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Discussion and conclusion

Phytomedicines, complex chemical mixtures prepared from plants, have been used for health maintenance since ancient times. But many phytomedicines are limited in their effectiveness because they are poorly absorbed. The recent trend is to improve the therapeutic performance of the conventional drug by formulating them as new drug delivery system rather than going for cumbersome and costly research for a new entity [7, 29, 35].

The effectiveness of any herbal product is dependent upon delivering an effective level of the active compounds. The bioavailability of lipophilic drugs like curcumin is very poor when administered orally or applied topically. Curcumin has its widespread use in treatment of a number of disorders including various types of cancer [33]. Poor absorption of curcumin through intestine as well as skin reported in pharmacokinetic studies warrants high doses to reach therapeutic levels of curcumin, which is sometimes inconvenient. Phospholipids play a major role in drug delivery technology. There are numerous advantages of phospholipids in addition to solubilizing property while considering them for a carrier system. Improved topical absorption of plant constituents by complexation with phospholipids has been reported in a number of cases [6, 8, 12].

In the present experiment complex of curcumin was prepared with soy phosphatidyl choline and characterized by TLC, DSC, melting point and IR spectroscopic analysis. The complex was also converted into vesicles (phyto-vesicles) and other vesicular systems (liposomes and niosomes) were also prepared to study their topical anti-aging effect against UV-induced skin damage.

Among the various components of the dermis, connective tissue consisting of collagen, elastic fibres and amorphous ground substances are assumed to be the most important factors in the process of cutaneous aging. The ground substances are known to have a large capacity to hold water in tissue, and they can be responsible for wrinkling and laxity of the skin [5]. Therefore, water content in the skin is presumed to be a critical determinant in the cutaneous aging. In the present investigation it was observed that chronic UV exposure reduced the moisture content of skin and simultaneous treatment with various gel formulations the moisture content was restored. The formulation containing phyto-vesicles was found to be most effective in retaining the moisture of skin.

In the present study, the reduction in total protein and hydroxyproline level appears to correlate with the reduction in collagen matrix or degeneration of tissue bundles. Application of all the gel formulations restored the total protein and hydroxyproline level but in this case also phyto-vesicles were found to be most effective among all the formulations.

The antioxidant enzymes, such as SOD, catalase and glutathione peroxidase constitute a mutually supportive team of defence against reactive oxygen species (ROS). Endogenous antioxidant enzymes are responsible for preventing or neutralizing the free radical-induced damage of tissues. After UV exposure, the level of all these three enzymes was reduced but on application of gel formulations they were restored significantly. Here too the gels containing phyto-vesicles and CU–PC complex were found to be most effective against oxidative stress.

In the present investigation the elevated level of end products of lipid peroxidation in skin of mice exposed to UV radiations were also observed. The increase in MDA level in skin tissues suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with gel formulations significantly reversed these changes. Here too the gel containing phyto-vesicles was found to be most effective in comparison to other formulations.

After chronic UV exposure severe elastosis was observed till deep in the dermis as compared to the skin section of normal mice. Epidermal hyperplasia, infiltration of the skin layers with neutrofils and destruction of the integrity of the connective tissue could also be seen. Discontinuous and fibroblastic collagen bundles were also observed which may be due to reactive oxygen species (ROS). Excess free radicals react with cellular lipids, proteins and nucleic acids, leading to degeneration of skin [5]. Treatment with various formulations containing curcumin reversed these changes and maintained the integrity of skin. Here too phyto-vesicles were found to be best in terms of providing significant protection against UV-induced damage.

The increased effectiveness of CU–PC complex and phyto-vesicles may be due to the amphiphilic nature of the CU–PC complex, which greatly enhances the water and lipid miscibility of the curcumin. The overall pharmacokinetics of curcumin has been improved after complexation with phosphatidyl choline. The phyto-vesicles were found to be better than CU–PC complex; this may be due to their small size when prepared as vesicles. Phyto-vesicles were more effective than CU–PC complex in terms of providing better hydration and retaining skin moisture, which will definitely work as anti-aging and anti-wrinkle agent.

In the present study the phyto-vesicles were found to be most effective than all other formulations and plain curcumin. The activity order was found to be as follows: phyto-vesicles > CU–PC complex > niosomes > liposomes > CU–PC physical mixture > curcumin > plain carbopol gel

Thus, the present study clearly indicates the superiority of CU–PC complex and their phyto-vesicles over other vesicular systems, CU–PC physical mixture and plain curcumin, in terms of providing enhanced anti-aging, antioxidant and anti-wrinkle effect.