Melnikova Nina, et al. / International Journal of Advances in Pharmaceutical Research

IJAPR Research Paper Available Online through www.ijapronline.org ISSN: 2230 – 7583

PROPERTIES OF BETULIN AND PHYTOCOMPOSITION IN PUMPKIN SEED OIL

Vorobyova Olga 1, Zhiltsova Olga 1, Smirnov Vasilij 2, Smirnova Olga 2, Lapshin Roman 3, Mukhina Irina 3, Melnikova Nina 2*

1Department of Pharmaceutical Chemistry, Nizhny Novgorod State Medical Academy, Russian Federation, 603600, Nizhny Novgorod, Minin sq., 10/1, Tel.: +7(831)432-80-75 2 Lobachevsky State University of Nizhny Novgorod, Russian Federation, 603950, Nizhny Novgorod, CSPO-20, Gagarin avenue, 23 3 Scientific Research Institute of Applied and Fundamental Medicine, Russian Federation, 603600, Nizhny Novgorod, Minin sq., 10/1.

Received on 22 – 07 - 2015 Revised on 28 – 08- 2015 Accepted on 02– 09 – 2015

ABSTRACT Properties of pentacyclic triterpene alcohol - betulin and thymol as components of phytocomposition in pumpkin seed oil have been studied. Pumpkin (Cucurbita Pepo) Seed Oil as medium was chosen because it is rich in natural antioxidants such as α-tocopherol (6 mg%), mixture γ-tocopherol and γ-tocotrienol (near 50 mg%), carotinoids (0,6 – 1 mg%) and , among them β-sitosterol (100-150 mg%), which was estimated by UV-Vis – spectroscopy and by RP- HPLChromatography. The increase in betulin dissolving in oil was promoted by means of antioxidant –thymol, because thymol and betulin form complex in molar ratio 1:1. Moreover, thymol intensified radical scavenging activity against stable 2,2’-diphenyl-1-picrylhydrazyl radical in Pumpkin Seed Oil. The topical anti-inflammatory effect of the phytocomposition of betulin and thymol in Pumpkin Seed Oil was demonstrated on acute cutaneous inflammation experimental model in mice. Dermatological cream with the phytocomposition has been developed. Key words: betulin, thymol, pumpkin seed oil, dermatological phytocomposition.

INTRODUCTION The disorders of metabolism of organism One of the natural lipid metabolism regulators is a both on the whole systematic level and on the an group of phytosterols occurring essentially in the epidermis surface, which means the disorders of vegetable oils. The sea buckthorn oil, food oils such epidermal barrier functions, respectively, are one of as olive oil, corn oil, sunflower oil, pumpkin seed oil, the reasons of occurrence of such diseases as etc., containing β-sitosterol as a main , dermatitis and psoriasis.[1, 2, 3]. show the good result in the treatment of lipid- associated diseases. Pumpkin seed oil (PSO) is the Author for Correspondence: most rich in β-sitosterol and it contains other isomers Melnikova Nina among them brassicasterol and campesterol (Fig. 1a). Department of Pharmaceutical Chemistry, It is assumed, that the hypocholesterolinic effect of β- Nizhny Novgorod State Medical Academy, Russian sitosterol is due to the structure similarity with Federation, 603600, Nizhny Novgorod, Minin sq., , because β-sitosterol may displace 10/1Tel.: +7(831)432-80-75,e-mail: cholesterol from low density lipoproteins (LDL). [4, [email protected], 5]. The other mechanism of β-sitosterol action is [email protected] formation of sufficiently stable complexes with cholesterol that makes difficult removal of the essential from corneous layer. [4, 3] Common action by unsaturated acids (up to 80%) and by IJAPR /Sept. 2015/ Vol. 6/Issue.09/ 296 – 305 296

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phytosterols promotes disorder correction of lipid [6] The antioxidant protection of carotinoids in PSO metabolism, too. [2]. Moreover, PSO is enriched with is caused by α, β, ∆ and γ-isomers of tocopherols and carotinoids, general formula are presented in Fig. 1b. tocotrienols. [7] а)

Δ7-sterol Δ5-sterol β-sitosterol b)

c)

Figure 1. Basic biologically active substances of PSO: а) phytosterols, phytostanols; R1: derivatives of ∆7 – stigmasterol, R2: β-D-glucopyranosid; b) carotinoids R- hydroxy- and epoxy- derivatives; c) tocols (α R1=R2=R3=CH3; β R1=R3=CH3 R2=H; γ R2=R3=CH3 R1=H; δ R1=R2=H R3=CH3)

The first report in which PSO acts as a topical anti-inflammatory agent and PSO is effective against acute and chronic skin inflammatory processes in mice was represented by M.L.M.de Oliveira et al. in the paper. [8] The anti- inflammatory activity of PSO has been explained by promising proportions of ω-6 and ω-9 of unsaturated fatty acids which are present in it. The effect is due to either their individual activity or the synergistic effect of these bioactive molecules. At the same time, these findings suggest that PSO may be an important alternative therapy for the treatment of inflammatory skin diseases, such as psoriasis, contact dermatitis, and atopic dermatitis. [8] Betulin is pentacyclic triterpene alcohol present in bark in high concentration; it is similar to phytosterol giving rise to hypolipidemic effects, too. Betulin doesn’t have special direct effect in vitro, but that which has been proved by study with volunteers; betulin shows antiallergical, and anti-inflammatory effects in accordance with an inhibition mechanism of synthesis of prostaglandin when it interacts with glucocorticosteroid receptors.

Figure 2. The structure of betulin

One of the ways to increase dissolving of betulin is acoustic action in the presence of components able to form complexes with higher than initial betulin. It is very important to improve dissolving of betulin by developing complex betulin with thymol being antioxidant for carotinoids and tocols within oil medium. This natural composition may be used in cosmetic creams and ointments as a base. This paper deals with development of dermatological phytocomposition of betulin and thymol in PSO and topical dosage form such as cream which is able to regulate lipid metabolism and to inhibit inflammation in skin. For this purpose we have studied: 1) the estimation

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Melnikova Nina, et al. / International Journal of Advances in Pharmaceutical Research of PSO content; 2) the determination of physicochemical properties and of radical scavenging activity of phytocomposition components; 3) the optimization of formulation of phytocomposition and dermatological cream; 4) the study of anti-inflammatory activity of the phytocomposition.

MATERIALS AND METHODS -1 Materials and Reagents. Betulin (C30H50O2) was obtained by method [9], mp. 260 ° C; purity 99.5%, IR, ν, cm : 1 3470 st (OH), 1640 st (C = C); H -NMR δ, ppm: 4.67m (1H, = CH2), 4.57 m (1H, = CH2), 3.78 br. s (1H, 28- CH2OH), 3.31 m (1H, 28-CH2OH), 3.17 m (1H, 3-CHOH), 2.36 m (1H, 19-CH), 1.66 s (3H, CH3), 1.23 s (3H, 13 CH3), 0.96 s (3H, CH3), 0.94 s (3H, CH3), 0.80 s (3H, CH3), 0.74 s (3H, CH3). C -NMR, δ, ppm: 76.71 (C-3), 109.46 (C-29), 150.24 (C-20), 57.87 (C-28). EI-MS m / z (%): 442 (M +, 40), 411 (60), 203 (95), 189 (100), 95 (85). Reagents. Glycerol (state standard 6824-96), (state standard 51652-2000), acetonitrile for chromatography grade 0 (specifications 2636-040-44493179-00), dichloromethane (state standard 9968-86), methanol (HPLC grade, Merck), pumpkin seed oil (state standard 42-8110-06), thymol (specifications 6-09-37-36-64), stearic acid (state standard 6484-96), ascorbylpalmitate (state standard R 55517-2013), allantoin (CAS 97-59-6), cetylstearyl alcohol (CAS 67762-30-5), ceteareth-25 (CAS 68439-49-6), glyceryl stearate (CAS 31566-31-1), chloramphenicol were used without special purification. Deionized water (resistivity >18 MΩ cm, Simplicity, Millipore Inc.) with pH 5.5 at 20±10C. Devices. IR - spectra were made by Shimadzu IR-Prestige-21 instrument (KBr tablets). H1- and C13-NMR spectra were obtained on Bruker Advance DPX-200 and Bruker DRX SF-500 - solution spectrometers DMSO-d6, comprising TMS as the internal standard. UV-Vis spectra were obtained on Analytik Jena Specord S-100. Melting points were measured using a capillary method by the apparatus Electrothermal 9200. HPL - chromatograms were obtained on brand HPLC Shimadzu LC-10 Avp, column Discovery C18 (250 × 4.6 mm, 5μm) with UV-detector. The formulation: the phytocomposition (wt%): betulin– 2.1; thymol - 6.4, lecithin – 2.0, pumpkin seed oil (PSO) at 100.0; the cream: betulin - 0.2, thymol - 0.8, lecithin – 0.2, PSO - 12.0, the base: cetylstearyl alcohol, stearic acid, sodium ascorbate, ceteareth-25, glyceryl stearate, allantoin, glycerin, chloramphenicol, water. Preparation of model mixtures for determination of carotinoids by UV-Vis spectrometry. The standard (1 - 0.25 g, 2 - 0.5 g, 3 - 0.75 g) and test solution (0.5 g) of pumpkin seed oil were placed in a 50 mL volumetric flasks, then thymol (0.1 g) was added into all samples. The mixtures were heated by water bath at 50ºC up to the dissolving of thymol, then hexane was added into the all flasks up to mark 50 mL at stirring. content was performed with bands at 424 and 434 nm. Total content (in mg) was calculated by the formula: ,

where Aobs. – absorption of the sample solution of pumpkin seed oil in hexane; b - correction of the optical 1% density relative to the baseline; V - volume of the analyzed solution, mL; E 1cm –specific extinction coefficients equal to β, β-carotene-3,3`-hexanediol - 2480 and for the α, β-carotene-3,3`-hexanediol - 2550; Co – concentration of 10 mg / mL (1% solution). Tocols quantification. An external calibration was performed prior to analyses of pumpkin seed oil, by injecting different volumes (10 and 20 μL) of tocol working solutions (0.2-5mg/L) on column. Standard curves (concentration versus peak area) were calculated by linear regression analysis. Injections in triplicate were made at each concentration for both the standards and the samples. The calibration curves were plotted using standard solutions of tocols and used for quantification. The total tocol content is expressed as milligrams per 100 gram of oil (mg%). Sample preparation for tocols analysis.The samples were stored in the dark at (21±2)°C until the measurements were performed. Pure PSO samples were weighted (about 50 mg) and diluted 10 times in hexane. Thereafter, 50 μL of above solution was taken into a screw-capped tube and diluted with 1 mL of a mixture of (50:44:6 methanol/acetonitrile/dichloromethane v/v/v). The sample was vortexed for 5 min and centrifuged for 10 min at 5000 rpm. After that, the sample was filtered through a 0.45 μm pore size filter and an aliquot of the clear liquid was directly injected into HPLC column, UV detection (284 nm and 295 nm) at 30ºC. Sample preparation for phytosterol analysis. Saponification of probes for RP-HPLC. 0.5 g PSO, 0.02 g ascorbic acid, 96% ethanol up to 50 ml was heated at 40oC by water bath until homogenization. The mixture was heated at 70oC; then it was incubated in the presence of 30 mL of 60 wt. % aqueous KOH solution during 30 minutes. After that, the mixture was homogenized with 100 mL of water added into two-phase medium. Unsaponifiable components of the reaction mixture were extracted with two portions of hexane (100 mL each). The combined hexane fractions were treated by 100 mL of 1 % ascorbic acid solution, then twice by 100 mL of water, respectively; IJAPR /Sept. 2015/ Vol. 6/Issue.09/ 296 – 305 297

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then hexane solution was dried by Na2SO4. At the last stage hexane was removed under nitrogen. Analyses were performed rapidly to minimize possibility of phytosterol oxidation. The lipid residues containing sterols were dissolved in 1 mL of eluent (85:15 acetonitrile/ethanol 95% v/v). Analyses were obtained by UV detection (210 nm) at 40ºC. Analisys of betulin was carried out using the same test and standard probes as for analyses of β-sitosterol, but betulin standard was used. Mobile phase: 90:10 acetonitrile/water v/v, UV detection (206 nm and 210 nm) at 40ºC. Before analysis the sample solution was filtered. Flow rate of mobile phase was equal to 1 mL / min. Statistical analysis was performed using Statistica 7.0 programme. Antimicrobial activity of the compositions was studied in accordance with the requirements of the State Pharmacopea of Russia XI (Part 2, Dec.28, 1998) by agar diffusion well-variant method. [10] Antioxidant effect. 2, 2’-Diphenyl-1-picrylhydrazyl (DPPH) assay: The hydrogen atoms or the electron donation abilities of the corresponding samples were measured from the bleaching of a purple-colored methanol solution of DPPH. This assay was carried out following the same method as reported elsewhere. [11,12] Added to 5 mL of a 0.004% methanol solution of DPPH (2,2-diphenyl-1-picrylhydrazyl, free radicals) was 50 μL of various concentrations of the samples dissolved in methanol. After a 30-min incubation period at room temperature, the absorbance was recorded against a blank at 517 nm. The inhibition of free radical DPPH, in percentages as DPPH• radical remaining (I%), was calculated in the following way: I% = (Acontrol – Asample/Acontrol) × 100, where Acontrol is the absorbance of the control reaction, containing all reagents except the test compound, and Asample is the absorbance of the test compound. Values are presented as mean ones ± SD of 5 parallel measurements. [11] Anti-inflammatory activity. Animals. Female white outbred mice weighing 25-30 g were used for this study. Mice were housed in standard polypropylene cages under controlled conditions of temperature (23-25°C), relative humidity (40-45%) and 12 h light/dark cycle, with free access to commercial pellet diet and water. Xylene-induced acute ear edema. Ear edema was induced in mice (n=9/group) by topical application on the inner and outer surfaces of the right ear of the following phlogistic agent – xylene (20 µl/ear). Immediately after the application of phlogistic agent the right ears were topically treated with PSO at 100% (20 µl/ear), 6,4% thymol in mineral oil (20 µl/ear), phytocomposition (betulin-thymol in PSO) (20 µl/ear), vehicle (mineral oil, 20 µl/ear, positive control) and hydrocortisone 0,5% (0,05 mg/ear). The left ear was considered as contralateral control and received only vehicle or saline solution (negative control, 20 µl/ear). The ear edema was evaluated 1 h after xylene application. Ear edema measurement. Ear thickness was measured before and after induction of the inflammatory response using a digital micrometer. The micrometer was applied near the tip of the ear just distal to the cartilaginous ridges and the thickness was recorded in µm. To evaluate the ear weight, animals were euthanized, and then both ears’ biopsies were removed and individually weighed. Edema was expressed as right ear thickness variation and as the weight difference between the right and left ears. The inhibition edema percentage was calculated as weight reduction in comparison to positive control.

RESULTS AND DISCUSSION Characterization of Pumpkin Seed Oil (Cucurbita Pepo). PSO had the high unsaturated content of 80 /100 g total fatty acids, including linoleic acid (ω-6, 42 %) and oleic acid (ω-9, 38%). [7] These results were close to literature data: linoleic acid (ω-6, 55,83%) and oleic acid (ω- 9, 23,47%).[13] Assay of the carotenoids in PSO was determinated by spectrophotometry using hexane solution of PSO. The Fig. 3a shows the visible spectrum of the solution having two intensive bands with λmax 424 and 434 nm and several weak bands in the region 520, 570 and 620 nm characterizing chlorophill type compounds. Calibration curves were plotted using the dependence of the absorption in the region both λmax 424 and 434 nm on PSO concentration (the insert of Fig. 3).

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а) b)

Аотн y = 0,48х + 0,0016 R2 = 0 ,9998 1 0,3

0,2

0,1

2 0 Absorption Absorption 0 0,25 0,50 0,75 m, г Х Concentration of PSO л о р о ф и Wavelengths λ, nm Wavelengths λ, nm л л Figure 3. Visible spectra of hexane solutions of carotenoids in PSO: a) 1 - PSO extract in hexane, 2 - hexane solution of thymol; b) the solution of 1,2,3 model mixtures: 5% thymol, PSO (25%, 50% and 100%, respectively). Spectra are resulted in the base line. Insert: the dependence of the absorption on the content of PSO in 1,2,3 model mixtures.

Total carotenoid content of pumpkin seed oil was equal to 9.6 mg%, by contrast to data of Parry et al [14], which indicated the total carotinoids content as 4,3 mg% (71 μmol/kg oil). RP-HPLC - analysis of the contents of tocols and of β-sitosterol in pumpkin seed oil. The initially present in PSO carotenoids and chlorophyll were removed by adsorption on the polar sorbent column as described above. It has been estimated by using tocols standard samples, that γ-tocopherol, γ-tocotrienol were the main components – 58-60 mg%, but α-tocopherol concentration was lower significantly – 8-10 mg%. The influence of saponification on the tocol assay was studied for the estimation of tocols content in the dosage form – cream. Fig. 4 shows the change of HPL-chromatograms of pumpkin seed oil after saponification in time by methods of sample preparation for RP-HPLC phytosterol analysis. a) b) mAU mAU 284nm,4nm (1.00) 284nm,4nm (1.00)

850 750

800 700

750

650 9.714/11383614/752908

700 γ-tocopherol 600 650 550

600 10.904/9558077/517107 500 550 α-tocopherol 450 500

450 400

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350 300

γ-tocotrienol7.343/2437840/309568 300 250

250 200

200 150 150

100 100

50 50

0 0 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 мин 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 мин c) d)

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mAU mAU 284nm,4nm (1.00) 284nm,4nm (1.00) 500 750 475 700 γ-tocopherol 450 γ-tocotrienol

425 7.309/3926502/486496 650 9.714/11383614/752908

400 600 375 550 γ-tocotrienol 350 500 325

300 450 275 α-tocopherol 400 250 350 α-tocopherol 225

200 7.343/2437840/309568 300 γ-tocopherol 175 250

150 11.294/1892732/139275

200 125

100 9.671/846250/83939 150 11.465/2010626/148676 75 100 50

50 25

0 0 -25 -50 -50

-100 -75 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 мин 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 мин Peak area Peak height Ratio Name Retention time 30 min 1 hour 30 min 1 hour 30 min 1 hour α- tocopherol 11,47 2010626 1892732 148676 139275 1 1,5 γ- tocopherol 9,71 11383614 846250 752908 83939 6 1 γ- tocotrienol 7,34 2437840 39269502 309568 486496 2 6

Figure 4. HPL-chromatograms(eluent - 44:50:6, v/v/v (acetonitrile/methanol/dichloromethane), flow rate 1.0 ml min- 1 *, UV detector (284 nm) 30ºC): a, b) standard samples of γ-tocopherol and γ-tocotrienol (a), 95% α-tocopherol and 5% α-tocotrienol (b); c, d) after saponification of PSO during 30 minutes (c), and during 1 hour (d)

During saponification for 30 min, ratios of α – to γ- tocopherols and to γ- tocotrienol were equal to 1:6:2, and total tocols concentration was equal to 56 mg%. If saponification time was one hour, the ratio changed to 1.5:1:6 by conservation of total tocol concentration (56 mg%). These results may be explained by the transformation of γ- tocopherol into γ-tocotrienols during saponification. Therefore, probe treatment (saponification) was needed to carry out during 30 min, if cream samples are analyzed. Roasted pumpkin seed oils had higher ratio of γ- tocopherols to γ- tocotrienols which was approximately 1:8, and total tocol content was 62,5 mg%, but it had 2 mg% of Δ- tocopherols according to paper [14]. Analysis of β-sitosterol in PSO was conducted using the samples after saponification and β-sitosterol as the standard. HPL-chromatogram of β-sitosterol and of PSO samples after saponification shown in Fig. 5. The impurities of brassicasterol (peak 1) and campesterol (peak 3) and β-D-β- glucopiranozide form of sitosterol (peak 4) were found according to the literature under comparable conditions analysis. a) b) mAU mAU 210nm4nm (1.00) 210nm4nm (1.00)

110

275 105

100 250

95 19.066/1557044/81256 90

19.187/4537386/224178 sitosterol 225 β- 2 85 2

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150 60

55

125 50

45 100 40

35 75 brassicasterol 30

25 1 50 3 1 20 15 4 25

10

0 5

0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 мин 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 мин

Figure 5. HPL-chromatograms of the samples after saponification: a) standard sample of β-sitosterol (85%) – 2; impurity of brassicasterol (15%) – 1; b) sample of PSO; 1 - impurity of brassicasterol; 2 - β-sitosterol; 3 – campesterol, 4 - β-D-β-glucopiranozide form of sitosterol; eluent - 85:15, v / v (acetonitrile / ethanol 96%), flow rate

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1.0 ml / min, UV detector (210 nm) 40ºC. It has been shown by RP-HPLC-methods, that 0,1-2,0 % betulin addition into PSO did not change tocols, phytosterols and carotinoids concentration. The assay of betulin in the phytocomposition was carried out with the same probe as in the phytosterols analysis, but the eluent differed (90:10, v / v (acetonitrile / water)). So, PSO which had 6 mg% α-tocopherol, about 50 mg% of a mixture of γ-tocopherol and γ- tocotrienol and 105-150 mg% β-sitosterol, may be used as a base of phytocomposition of PSO and betulin with anti-inflammatory, antiallergical and lipid-regulator properties.

Scavenging abilities of phytocomposition. It is well-known that PSO has radical scavenging activity against DPPH•and ABTS•+ and also suppresses human LDL oxidation [12]. However, PSO can oxidize under room temperature storage in the light, therefore it is necessary to put compounds being antioxidants and preservatives into phytocomposition for formulation of dosage forms. Thymol is a component found in Marjoram or Thyme oils, and it has antioxidant properties, so we studied radical scavenging activity using stable 2,2’-diphenyl-1-picrylhydrazyl (DPPH•) radical with thymol in the phytocomposition of betulin in PSO. The phytocomposition of betulin with thymol in PSO and ethanol solution of thymol differed in their DPPH• scavenging abilities (Table 1). The ethanol solution of thymol exhibited the strongest DPPH• scavenging capacity, and quenched 70-75% of radicals in the reaction mixtures 10 minutes after (I = 25-30%), and PSO (25 mg seed oil equivalents per mL) quenched 30-35% radicals in initial reaction mixture (I = 65-70%) (Table 1).

Table 1. DPPH• radical remaining C, % of thymol I, % DPPH• radical remaining Pumpkin seed oil - 65-70 PSO and thymol 6,2 50-53 Ethanol solution of thymol 6,2 25-30 Mixture of PSO, betulin and thymol 6,2 48-53

Radical scavenging activity of thymol in phytocomposition may be explained by mechanism typical for its reaction with •OH and •O·- to produce reducing adduct radicals and oxidizing phenoxyl radicals in lipid medium.[12] It has been estimated using thymol-ascorbate couple by authors [12], that the redox potential value of thymol·/thymol couple (0.98 V vs NHE) obtained by cyclie voltammetry is less than those of physiologically important oxidants, which reveal the antioxidant capacity of thymol by scavenging these oxidants. The repair of the phenoxyl- radicals of thymol with ascorbate together with the redox potential value makes it a potent antioxidant with minimum pro-oxidant effects. The radical scavenging activity of betulin and thymol phytocomposition in PSO is possible due to strong antioxidants - carotinoids, tocols and other phenols which may be oxidized in PSO and then reduced by thymol. Mixture of PSO, betulin and thymol shows the same result as that of PSO and thymol (average 50%). The increase in betulin dissolving. The other problem of dosage form formulation of the betulin and thymol phytocomposition is their low hydro- and oil-solubility. Recent literature data suggest that betulin can be solved through complexation such as formation of «guest-host» complex or inclusion complex. For example, the solubility of betulin can be increased in the presence of hydroxyl-propyl-gamma-cyclodextrin. [15] Two preparation methods (physical mixing and kneading) were used to obtain inclusion complexes - cyclodextrin:betulin - at 1:1 and 1:2 molar ratios. [16] The kneading of betulin with polymer excipience such as polyvinylpirrollidons or polysaccharides ensured inclusion complexes formation and improved betulin water solubility 10-fold. [16] It has been shown, that after kneading of composition of betulin with thymol in molar ratio 1:1 during 1 hour and removing ethanol traces, IR-spectrum of dry reaction mixture was different from initial IR-spectra of betulin and thymol, significantly (Fig. 6). Moreover, new 3550 cm-1 narrow intensive band and 1044 cm-1 band appeared in the spectrum. This fact may mean the formation of thymol and betulin inclusion complex in molar ratio 1:1. At the same time, the formation of inclusion complex makes it possible to increase betulin dissolving in the oil. It has been estimated that solubility of betulin in an inclusion complex with thymol in the PSO increased 1000-fold approximately from 1∙10-6 mg/mL to 1∙10-3 mg/mL (water) and from 2∙10-6 mg/mL to 1,5∙10-3 mg/mL (PSO).

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100 Тимол %T

75

1 8 7 6 ,7 4 2 6 0 1 ,9 7

50

2 7 0 9 ,9 9

1 5 8 5 ,4 9

8 5 4 ,4 7

1 0 5 8 ,9 2 7 3 8 ,7 4

25 1 5 1 6 ,0 5

1 6 2 2 ,1 3

3 0 3 4 ,0 3

3 0 6 6 ,8 2

5 8 8 ,2 9

2 8 6 8 ,1 5

2 9 2 6 ,0 1

9 4 3 ,1 9

1 1 5 7 ,2 9 1 0 9 1 ,7 1

0 1 4 6 0 ,1 1

1 2 8 6 ,5 2

1 4 2 1 ,5 4

3 2 2 6 ,9 1

1 2 4 4 ,0 9

8 0 6 ,2 5 2 9 5 8 ,8 0

4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500 FTIR Measurement 1/cm

Wave number,ν,cm-1 Figure 6. IR-spectra data of betulin (a), thymol (b), mixture of betulin-thymol in molar ratio 1:1 after kneading

This result will accelerate and significantly improve bioavalibality of betulin through skin. Topical anti-inflammatory potential of phytocomposition on acute skin inflammation in mice The phytocomposition formulation (wt%) consisted of betulin 2.1, thymol 6.4, lecithin 2.0 and pumpkin seed oil up to 100. In the acute model of inflammation, caused by xylene on the ear of the mouse, the increase in both ear thickness and tissue weight was a marked result (Fig. 7). Topical application of the vehicles (mineral oil or saline solution) or phytocomposition and its components alone on the ear did not alter the skin thickness significantly (data not shown). However, PSO and phytocomposition inhibited the xylene-induced ear edema in both skin thickness and weight when compared to respective positive controls (P < 0.05) at 1 hour. This inhibition was similar to hydrocortisone and PSO results (Fig. 7, Table 2). a) b)

Figure 7. Topical activity of thymol in mineral oil (T in MO), pumpkin seed oil (PSO), phytocomposition (B-T in PSO), and hydrocortisone 0,5% (HC) on xylene(Xyl)-induced ear edema in mice. Ear edema was measured 1 hour after induction of inflammation in both ear thickness (a) and tissue weight (b). The positive control – when mineral oil applied topically after the challenge with xylene. The bars represent the mean ±SD for nine animals.

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Table 2. Topical anti-inflammatory effect of pumpkin seed oil (PSO) and reference drug in acute model of inflammation at the end of the experimental period* Treatment groups Inflammation inhibition (%) after xylene PSO,100 % 65,17±3,11 Phytocomposition (B-T in PSO) 66,81±4,91 6,4% thymol in mineral oil 21,45±8,24 0,5% hydrocortizone 67,15±7,35 *Results are expressed as mean±SD (n=9) of ears weight reduction in relation to the positive control of model. Recently, the first topical anti-inflammatory activity of both phytocomposition betulin-thymol or activity of PSO has been evaluated in several the cream. The antibacterial effect was achieved if experimental models. [8] In this study authors used as levomicetin was added into the cream. (Table 5) phlogistic agents: xylene, a promoter of neurogenic inflammation; 12-O-tetradecanoylphorbol acetate, a CONCLUSION phorbol ester that activates protein kinase C, leading To sum up, this study represents the results of the to production of lipid-derived mediators, and topical anti-inflammatory action of phytocomposition oxazolone, an inductor of contact delayed-type containing betulin and thymol in Pumpkin Seed Oil hypersensitivity. The results explained the possibility (PSO). The use of PSO was due to richness in natural of an alteration inflammatory response by PSO via antioxidants such as α-tocopherol (6 mg%), mixture modulation of cellular and molecular mediators γ-tocopherol and γ-tocotrienol (about 50 mg%), involved in inflammatory pathways activated by carotenoids (9,6 mg%) and phytosterols, among them theses phlogistic agents. In addition, this oil – PSO – β-sitosterol (105-150 mg%), which were estimated by was able to resolve a persistent inflammatory lesion UV-Vis – spectroscopy and RP- similar to dexamethasone, but any cutaneous HPLChromatography. Bioavailability of betulin was alterations caused by its topical use, akin to improved by the increase in dissolving of betulin in corticosteroids, were not observed. It may be oil by means of thymol that forms a complex with proposed, that positive effects can be attributed not betulin in molar ratio 1:1. The phytocomposition of only to the proper balance of ω-6 and ω-9 unsaturated betulin and thymol in Pumpkin Seed Oil fatty acids present in PSO, as the authors believe [8], demonstrated anti-inflammatory activity by but the inflammatory activity is also caused by high experiments of contact dermatitis model with content of phytosterols, carotinoids and tocols in mice.The cream formulation will allow to use this PSO. Moreover, betulin and thymol in the phytocomposition for topical infection wound phytocomposition can also reduce topical treatment. inflammation in skin disorders. REFERENCES The formulation of cream on the base of the 1. Ajzyatulov R, Yukhimenko V. The value of phytocomposition. risk factors in the occurrence and Cream is the most suitable dosage form on the base progression of psoriatic disease. West. of the phytocomposition for topical rout in the dermatol. 2001, 1, 41-43. treatment of dermatological diseases. We proposed 2. Hyshiktuev B, Tarasenko G, et al. Lipid the formulation of cream: betulin - 0.2, thymol - 0.8, peroxidation in the epidermis of patients PSO - 12.0, the base. Topical anti-inflammatory with psoriasis. Military Medical Journal effect of betulin was estimated by experiments with 2000,7, 40-43. rats and rabbits as compared with Metyluracil and 3. Prohorenkov V, Klemenkov S, Vandysheva Pantotenic acid, too. [17]. Contradictory data about T. Especially fatty acid lipid spectrum of the antibacterial properties of betulin and thymol are affected epidermis in patients with psoriasis. presented in literature [18, 19] , whereas these Sib. Zh. Derm. and Venus. 2001, 1, 36-37. antibacterial effects are important under acute skin 4. Jones P, MacDougall D, Ntanios F, and inflammation treatment. Good antibacterial activity Vanstone C. Dietary phytosterols as of betula birch extracts was shown by several cholesterol-lowering agents in humans. Can. investigators. [18] Nevertheless, other authors don’t J. Physiol. Pharmacol. 1997, 75, 217–227. show this effect. We haven’t shown antibacterial

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Melnikova Nina, et al. / International Journal of Advances in Pharmaceutical Research

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