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Research Paper
Journal of Pharmacy And Pharmacology
Luteolin–phospholipid complex: preparation, characterization and biological evaluation Junaid Khana, Amit Alexandera, Ajazuddinb, Swarnlata Sarafa and Shailendra Sarafa a University Institute of Pharmacy, Pandit Ravishankar Shukla University, Raipur, bRungta College of Pharmaceutical Sciences and Research, Bhilai, Chhattisgarh, India
Keywords inflammation; luteolin; phospholipid complex; solubility Correspondence Shailendra Saraf, University Institute of Pharmacy, Pandit Ravishankar Shukla University, Raipur, Chhattisgarh, 492010, India. E-mail: [email protected] Received January 14, 2014 Accepted May 15, 2014 doi: 10.1111/jphp.12280
Abstract Objectives This study aims to develop novel carrier system incorporating luteolin, a poorly soluble biologically active plant active. Methods We investigated a lipid-based drug delivery system to enhance dissolution and absorption profile of luteolin. Luteolin was complexed with phospholipids, and the preparation was characterized. The formulation was evaluated for physicochemical properties, in-vitro solubility or release studies. In vivo antiinflammatory action of luteolin and its phospholipid complex was evaluated by using carrageenan and 12-O-tetradecanoylphorbol-13-acetate as inducers. Key findings The prepared luteolin–phospholipid complex (LPC) showed drug loading of about 72.64% with average particle size of 152.6 nm. The Fourier transform infrared spectroscopy and thermal studies confirm formation of complex. The solubility of luteolin as LPC was about 2.5 times higher than the solubility of pure luteolin in water. In the diffusion study, LPC showed 95.12% of drug release at the end of 2 h. Animal studies demonstrated significant differences in response of LPC and luteolin. Conclusion LPC was successfully prepared by optimizing the process parameters. The resultant delivery system improved bioavailability and efficacy of luteolin and in the future may become an efficient tool for administration of luteolin.
Introduction With the advancements in herbal technology, plant-derived drugs have gained substantial attention from the medical community as well as patients for treatment of several debilitating ailments.[1] The potentially harmful adverse effects of synthetic drugs have further added to the cause.[2] As a consequence, the active pharmaceutical ingredients of plant origin have drawn the attention of researchers to formulate these molecules in such a form that has qualities like ease of administration, sufficient bioavailability and desirable aesthetic properties. The traditional herbal formulations of Chinese and Indian systems, however, lack these merits. Hence, the development of modern drug delivery systems for plant drugs has now become an essential element of pharmaceutical research. Among the plant-derived medicinally active compounds, polyphenolic structures and specially flavonoids have been extensively studied for their therapeutic potential in humans and animals.[3] These secondary metabolites are widely distributed throughout the plant kingdom and administered as food in the form fruits in our day-to-day
life. However, such administration is insufficient to impart any therapeutically useful effect of flavonoids.[4] Their consumption has remained limited in the form of food product as there are only some rarely available pharmaceutical formulations containing these therapeutically active compounds. Larger molecular size or weight, low aqueous or lipid solubility, and poor membrane permeability are some of the major factors that limit the clinical utility of these natural drugs.[5,6] Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a polyphenolic flavonoidal compound present in variety of medicinal plants and has been isolated from a number of floras, which include Reseda luteola, Delonix elata, Elsholtzia rugulosa and others.[7–9] Luteolin has strong scavenging properties for superoxide radicals. It is an established antioxidant and has shown antioxidant potential better than vitamin E and butylated hydroxytoluene.[10] It is also known to inhibit cholesterol synthesis, affect blood coagulation and has the ability to increase coronary blood flow.[11] It is considered to be a cancer preventive agent and has shown to exhibit strong antiproliferative activity against different
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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human cancer cell lines.[12] Luteolin is a component of herbs with anti-inflammatory action and has itself demonstrated effectiveness against inflammatory processes. It has been reported as an active inhibitor of inflammatory mediators, 12-lipoxygenase and 5-lipoxygenase.[13] Luteolin is also known for its cardioprotective potential and affects the pathogenesis of atherosclerosis by inhibiting cholesterol biosynthesis at different levels, increasing elimination of cholesterol from liver and inhibiting LDL oxidation. Moreover, various herbal drugs containing luteolin are used for prevention or treatment of diabetes, obesity and other metabolic disorders.[14–16] Pure luteolin is a yellow microcrystalline powder, is sparingly soluble in water and is soluble in organic solvents. Inside the body, it is excreted rapidly from kidneys.[17,18] The poor aqueous solubility of luteolin hinders its systemic absorption, and extensive presystemic metabolism has further restricted the use of this highly potent and efficacious molecule to be utilized as a medicine by humans in fighting the menace of several debilitating disorders. To successfully utilize the different physiological effects of luteolin as a therapeutically active agent and to overcome its poor oral absorption, it is necessary to develop new formulation of luteolin, which can increase its oral absorption and add to its therapeutic potential. In recent years, complexation of poorly soluble polyphenolic plant actives with dietary phospholipids has emerged as an efficient tool to improve their oral/topical bioavailability and clinical efficacy.[19] Several potent plant constituents like curcumin,[20] silybin,[21] quercitin[22] and others have been successfully complexed with phospholipids and have shown better and improved solubility, bioavailability and efficacy.[23–25] The dietary or essential phospholipids used for complexation uniquely resemble the mammalian cellular phospholipids and hence are sufficiently biocompatible.[26] These phospholipids are known to be non-carcinogenic and non-mutagenic.[27] Phospholipid complex of plant actives are known to improve the solubility, stability and hence, the therapeutic efficacy of hydrophobic drugs.[28] Several polyphenolic herbal drugs have been combined with dietary phospholipids to form soluble micellar complexes resulting in new products of improved oral bioavailability and efficacy.[29] Moreover complexation of polyphenolic plant actives with essential phospholipids have shown biological value addition because of the inherent hepatoprotective potential of phospholipids.[30] However, comprehensive facts about the formulation of these complexes are insufficient, and this technique is still in its emerging phase. Various studies on complexation of polyphenolic compounds with dietary/soya phospholipids have stressed on characterization of prepared complexes with lesser concern on improvement of biological response of the drug as 2
complex.[31] The stability evaluation of phospholipid complexes has also remained almost neglected in most of the studies.[32] Hence, this study was undertaken with the objective to prepare and characterize phospholipid complex of luteolin and assess its moisture-absorbing capacity when exposed to humidity. Rather than the conventional approach of taking equal w/w ratio of lipids and phytoconstituents, we used factorial design in our study to optimize the key process parameters for preparing phyto-phospholipid complexes. Moreover, we also established the correlation between the improvement in solubility and in-vitro diffusion profile of luteolin by luteolin–phospholipid complex (LPC) with its enhanced biological response in laboratory animals. For this, we evaluated the in-vivo anti-inflammatory potential of LPC in comparison with pure luteolin.
Materials and Methods Materials Luteolin and carrageenan were purchased from Sigma Aldrich (Mumbai, India). Hydrogenated soya phosphatidylcholine (PHOSPHOLIPON 90H) was obtained as a gift sample from LIPOID GmbH (Ludwigshafen, Germany). All other chemicals of analytical grade were procured from local chemical suppliers.
Experimental animals Wistar rats (150–250 g) and Swiss albino mice (25–35 g) of either sex were used for in-vivo studies. The animals were obtained from animal house of Rungta College of Pharmaceutical Sciences and Research, Bhilai, Chhattisgarh, India. The animals were kept in groups of eight per cage under standard conditions (12 : 12 h light : dark cycle at 25 ± 2°C) and were provided with standard pellet diet and water given ad libitum. The studies were approved by the Institutional Animal Ethics Committee (1189/PO/a/08/CPCSEA) and were performed as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, India.
Preparation of complex A fixed amount of drug and lipids were taken in a 500-ml flask containing 250 ml of ethanol. The mixture was kept at different temperatures ranging from 40 to 50°C for 3 h with magnetic stirring. The resultant solution was evaporated under vacuum (RV 10, IKA, Staufen, Germany) to remove the solvent. The resultant LPC was kept in an ambercoloured glass bottle flushed with nitrogen. All the procedures were performed under aseptic conditions. The formulation parameters of LPC were optimized for better yield and drug loading capacity of product using response surface quadratic model (Design Expert version 9,
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Stat-Ease, Minneapolis, MN, USA). The LPC thus formulated appeared as almost completely dispersed particles in water and was found to be transparent. However, the dispersion of pure luteolin appeared to be highly turbid. The physicoshemical and biological characteristics of the prepared LPC were studied in relation to the pure luteolin and the physical mixture of luteolin and phospholipids prepared by simple mechanical mixing.
Characterization of LPC Spectrophotometric characterization was carried out by ultraviolet (UV)-visible and Fourier transform infrared spectroscopy (FTIR) studies. The samples of luteolin, phospholipid, physical mixture and LPC were dissolved in methanol to prepare dilutions of known concentration and then scanned between wavelength range of 200–400 nm using Shimadzu UV 1800 spectrophotometer (Shimadzu, Kyoto, Japan). FTIR of luteolin, phospholipon 90H and prepared LPC were obtained using a FTIR-8400 spectrophotometer (Shimadzu). A 1% w/w of sample with Potassium bromide (KBr) was triturated to fine powder and compressed into disc under a hydraulic press at 10 000 psi for 30 s. Each KBr disc was scanned over a wave number region of 4000–500/cm. The particle size and ζ-potential of LPC were determined at 25°C using photon correlation spectroscopy (Malvern Zeta-Size Nano ZS90, Worcestershire, UK). A proper dilution of the suspension was prepared with double-distilled water before each analysis. Differential scanning calorimetry (DSC) analysis was used to characterize the thermal behaviour of the luteolin, phospholipon 90H, physical mixture of luteolin with lipid and prepared LPC. DSC thermograms were obtained using an automatic thermal analyser system (Pyris 6 DSC, Perkin Elmer, Waltham, MA, USA). Samples were crimped in standard aluminium pans and heated at rate of 10°C/min within a temperature range of 50–400°C under constant purging of dry nitrogen (30 ml/min).
Hygroscopicity study Moisture-retaining capacities of luteolin and LPC were studied as per previously described method.[33] Solid samples were stored under controlled temperature and humidity conditions to investigate the moisture-absorbing capacity of luteolin and LPC on storage. The relative humidity (RH) at 25°C was generated using saturated inert solutions of known RH values. Definite amount of samples were weighed and placed in open glass bottles that were exposed to the desired RH inside desiccators.
Solubility and release studies Apparent solubility was determined by adding excess of luteolin and LPC to 5 ml of water or n-octanol in sealed
Luteolin–phospholipid complex
glass containers at room temperature. The liquid was agitated for 24 h on rotatory shaker then centrifuged for 15 min at 5000g to remove excessive luteolin. The supernatant was filtered through membrane filter. One millilitre of the filtrate was then mixed with 9 ml of distilled water to prepare dilutions, and these samples were measured for absorbance at wavelength of 254 nm by using UVspectrophotometer, and the concentration of luteolin in each sample was determined. For in-vitro release study of luteolin and LPC, dialysis method was applied using cellophane membrane. Activation of cellophane membrane was done to remove sulfur and other impurities. Two millilitres of luteolin and LPC were transferred to sample holders in triplicate placed in receiving compartments containing 18 ml of phosphate buffer (pH 7.4). The whole operation was performed at constant speed of 150 g at 25°C. Samples were measured at λmax 254 nm.
In-vivo studies The prepared LPC was evaluated for acute toxicity in rats. The study was carried out as per the Organisation for Economic Co-operation and Development guidelines 420. Female Wistar rats weighing 150–180 g were used for the study. The animals were continuously observed for the autonomic and behavioural changes and for mortality up to 14 days after drug administration. After 14 days, the animals were sacrificed. The organs were excised, weighed and examined macroscopically. For evaluation of the antiinflammatory potential of LPC in relation to the response of pure luteolin, two different animal models were used, namely 12-O-tetradecanoylphorbol-13-acetate (TPA)induced ear oedema in mice and carrageenan-induced paw oedema in rats. For TPA-induced mouse ear oedema, Swiss albino mice of either sex were used. Oedema was induced in both ears of each mouse by the topical application of 2 μg TPA dissolved in 20 μl of acetone to both the inner and outer ear surfaces. Thirty minutes after the application of TPA, the inner and outer surface of each ear was treated with 10 μl of 5% of Tween 80 and 0.5 mg/ear of indomethacin, luteolin and LPC, respectively, to different groups of animals (n = 8). After completion of 4 h, each animal was sacrificed under anaesthesia, and treated ears were removed and weighed.[34] The ears were then fixed in 10% buffered formalin solution, implanted in paraffin wax and cut into 5-mm sections. The sections were then stained with H&E and were observed under light microscopy. To assess the effect of LPC on carrageenan-induced acute inflammatory response, wistar rats of either sex (175–200 g) were used. Animals were administered different treatments, namely solvent, luteolin 10 mg/kg, LPC equivalent to 10 mg/kg of luteolin and indomethacin 10 mg/kg. Initial paw volume was measured using a digital plethysmometer, PLM
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Statistical analysis Data of animal studies were analysed with repeated measure analysis of variance and post-hoc test. All data were termed statistically significant with P-values less than 0.05.
Results and Discussion Luteolin–phospholipid complex To successfully develop any novel drug delivery system, yield and drug content (loading) are the key factors that represent the efficiency and acceptability of method followed.[38] Both of these aspects of formulation were taken into consideration in our study. We tried to prepare the complex of luteolin with phospholipids utilizing different solvents like ethanol, methanol, acetone, tetrahydrofuran and diethylether. The amount of luteolin loaded in the complex was at higher end with ethanol and methanol as solvents. Taking in to consideration the processing time and toxicity, ethanol was finally selected as the solvent medium. 4
(a) 72.6414
Loading (%)
80 70 60 50 40 30 50.00 47.50
45.00 B: temperature 42.50 (C)
2.50
(b)
90 85 80 75 70 65
3.00
2.00
40.00 1.00
Yield (%)
01 PLUS (Orchid Scientific, Nasik, India). At 1 h after oral administration of drug, 0.1 ml 1% carrageenan in normal saline was administered in the subplantar region of the rat hind paw. Paw volume was measured up to 6 h after carrageenan administration.[35] For estimation of nitric oxide (NO) and malondialdehyde (MDA), another set of rats were orally administered with different treatments as stated above after carrageenan injection into right hind paws. After 2 h, the hind paws of the animals were surgically removed. The collected paw tissue was rinsed in ice-cold normal saline and immediately placed in cold normal saline and homogenized at 4°C. The homogenate was centrifuged at 13 000 g for 5 min. The supernatant was obtained and stored at −20°C for further estimation of NO and MDA. The production of NO was evaluated as per Griess reaction.[36] Briefly, 100 μl of supernatant from each treatment groups was mixed with equal volumes of sulphanilamide (1% in 5% phosphoric acid) and 0.1% naphthylenediamine dihydrochloride. The resultant samples were then incubated at room temperature for 10 min. The absorbance was recorded at 550 nm, and nitrite production was determined against a NaNO2 standard curve. For the estimation of MDA content in paw fluid, aqueous acid extraction thiobarbituric acid method was used with tetramethoxy propane as working standard.[37] Briefly, the supernatant of treated paw fluid was mixed with 10% of trichloroacetic acid and centrifuged for 5 min at 5000 g. To 1 ml of this supernatant, 1 ml of 0.67% TBA in 0.05 mol/L of NaOH was added. The above aliquots were heated in boiling water bath for 25 min and then cooled at room temperature. Absorbance was measured at 532 nm, and concentration of MDA was determined and expressed as nanomoles per millilitre of fluid.
1.50 A: PLP–Drug ratio (mg)
85.7912
3.00 2.50 A: PLP–Drug 2.00 ratio (mg) 1.50 1.00 50.00
42.50 45.00 47.50 B: temperature (C)
40.00
Figure 1 Response surface plot showing the influence of (a) phospholipid–drug ratio (X1), (b) reaction temperature (X2) on percentage yield (Y1) and drug loading (Y2).
To optimize the formulation parameters, the phospholipid to drug ratio and the reaction temperature were kept as the independent variables against which the dependent variables, that is, yield (%) and drug loading (%) were determined. The response surface statistical model demonstrated the effect of lipid to drug ratio and reaction temperature on the quality of end product (Figure 1). According to the data generated from the study, a response surface quadratic model suited fit for optimization of drug loading while a linear model was used for obtaining maximum yield. The independent variables were varied against the two dependent variables using two different equations:
Y1 = 73.02 + 3.99 X1 + 8.52 X 2 − 0.13 X1 X 2 − 8.44 X12 − 15.28 X 22 Y2 = 81.7 + 0.89 X1 + 5.99 X 2
(1) (2)
Where, Y1 and Y2 are the drug loading % and yield % of complex, respectively, and X1 and X2 are the coded levels of the independent variables, namely phospholipid–drug ratio and reaction temperature (°C) respectively. The % yield of
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Luteolin–phospholipid complex
LPC was calculated by formula (LU – LF)/LU × 100 where, LU was the fixed amount of luteolin used for preparing LPC and LF was the content of luteolin in free form. The drug loading % of LPC was estimated by the formula LC/L × 100, where LC was the amount of luteolin that formed a complex and L was the weight of the LPC. The experimental results concerning the tested variables on the combination of luteolin in the complex are shown in Table 1.
The experimental results of nine batches showed a wide variation in yield of LPC from 67.19% to 89.78%, and in the per cent of drug loading in the complex ranging from 38.75% to 75.32%. According to the results of the quadratic response surface methodology, the LPC was prepared using the optimal process variable settings, where X1 and X2 were equal to 2.33°C and 50°C respectively. The per cent yield of prepared LPC was 85.79%, and the content of luteolin in the complex was 72.64%.
Ultraviolet and infrared analysis Table 1 Response values of different variables for the optimization of luteolin–phospholipid complex (LPC) preparation Formulation
Lipid: drug (w/w)
Temperature
Yield (%)
Drug loading (%)
1 2 3 4 5 6 7 8 9
1.00 2.00 3.00 1.00 2.00 3.00 1.00 2.00 3.00
40.00 40.00 40.00 45.00 45.00 45.00 50.00 50.00 50.00
78.25 67.19 79.95 81.39 83.24 83.91 85.23 89.78 86.33
38.75 45.29 46.75 59.21 75.32 67.65 53.25 67.89 60.74
(a) 1.0
(b) 1.0
0.8 0.6
We evaluated any possible interaction between the drug and the phospholipids to confirm the formation of complex. The results of UV spectra showed only minor variations in the peaks of luteolin and its phospholipid complex, suggesting some interaction between the two moieties rather than formation new chemical entity. The UV spectra are shown in Figure 2. The phospholipids showed only end absorptions close to 200 nm. The absorption curves of luteolin, the physical mixture and LPC were nearly the same. Two characteristic absorption bands of luteolin were observed at 254 and 349 nm. To explore the type of interaction between luteolin and phospholipid, FTIR studies were performed.
0.8 254 nM268 nM
349 nM 0.6
0.4
0.4
0.2
0.2
0.0 190
272
354
436 nM
(c) 1.0
254 nM268 nM
436 nM
350 nM 0.6 0.4
0.2
0.2
Figure 2
354
0.8 349 nM
0.4
0.0 190
272
(d) 1.0
0.8 0.6
0.0 190
200 nM
272
354
436 nM
0.0 190
254 nM 268 nM
272
354
436 nM
Ultraviolet (UV) spectra of (a) luteolin, (b) phospholipids, (c) physical mixture and (d) luteolin–phospholipid complex (LPC).
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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The infrared (IR) spectrum of LPC showed significant changes as compared with the spectra of luteolin and phospholipid. The results of IR study indicated formation of complex between luteolin and phospholipid as there are changes in peaks in complexes and positions from that of luteolin (Figure 3). The IR spectrum of luteolin showed –OH vibration band, at about 3144/cm, which is most probably the result of –OH vibrations of the phenolic groups. An intense band at 1616/cm was also noted, which seems to be the result of C = O vibrations from the central heterocyclic ring. The lipid demonstrated characteristic bands at 3619 and 2994/cm. The IR of LPC showed shift in phenolic –OH to 3425/cm. There are carbonyl groups belonging to phospholipids that allow the formation of hydrogen bonds with the phenolic hydroxyl groups of luteolin. This was evident from the FTIR graph where the phenolic –OH of luteolin shifted from its position and so
the P = O absorption band of the lipid, which suggests possible interaction of the phospholipid molecule with these phenolic –OH groups to form complexes.
Particle size determination The average particle size of LPC as estimated by the ζ sizer was found to be 152.6 nm, with a polydispersity index (PDI) of 0.675. The ζ-potential of LPC prepared in our experiment was found to be –27.6 ± 2.1 mV, which can be attributed to the type and composition of the phospholipids. The lower value of ζ-potential suggests possibility of incipient instability of LPC on storage. The PDI, however, is acceptable and closer to unity, indicating a sufficiently low critical micellar concentration.[39] The average particle size of the prepared complex is closer to 150 nm, which is suitable for retention at inflammatory sites with a reduced overall clearance.[40]
(a)
Thermal studies
100
3298 /cm
50
3144 /cm
Figure 4 shows the DSC thermogram of (a) pure luteolin, (b) pure phospholipid and (c) LPC. The thermogram of luteolin showed a single sharp peak starting from 321.45°C with maximum intensity on 329.34°C, which can be attributed to its melting point. DSC thermogram of phospholipids showed two major peaks at 74.15°C and 106.24°C. The first peak of phospholipids at 74.25°C is of mild intensity, which is probably due to the heat-induced movement of polar head groups. The second peak at 106.24°C is very sharp, and it appears to be because of phase transition from gel-like consistency to liquid crystalline state.[41] The thermogram of the complex (LPC) exhibits a single peak with onset of 47.7°C and maximum intensity at 63.5°C. The thermogram of physical mixture shows two peaks similar to that of luteolin and LPC. The DSC peaks of luteolin and LPC differed markedly. This shows that luteolin may interact with phospholipids, and the interaction is hydrophobic in nature. There is also some contribution of hydrogen bonding apart from hydrophobic interaction in the luteolin–phospholipids interaction. The –OH groups of the phenol rings of luteolin are involved in hydrogen bonding whereas the aromatic rings could be involved in hydrophobic interaction.
1174 /cm 1616 /cm
0 4000
3000
2000
1000
2000
1000
(b) 100
50
3519 2789
0
2994
4000
3000
(c) 100 2345
50
Hygroscopicity study 3425 1692
0 4000
3000
2000
1000
Figure 3 Fourier transform infrared spectroscopy (FTIR) spectra of (a) luteolin, (b) phospholipids and (c) luteolin–phospholipid complex (LPC).
6
The results demonstrated moisture-absorbing capacity of LPC with increase in RH at room temperature in open to air environment. The increase in weight per cent of luteolin stored at 25°C are shown in Figure 5. The LPC gained considerable weight on exposure to atmosphere ranging from 1.85% to 16.07% as compared with the bulk drug, which
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Luteolin–phospholipid complex
(a)
(b)
321.45°C
74.15°C 329.34°C 108.24°C 0
50
100
150
200
250
300
350
0
400
50
100
150
200
250
100
150
200
250
(d)
(c)
47.7
321.45°C
47.7°C
63.9
330.01°C 63.5°C 0
50
Figure 4
100
150
200
250
300
350
400
0
DSC thermograms of (a) luteolin, (b) phospholipids, (c) physical mixture and (d) luteolin–phospholipid complex (LPC).
18 16 14 12 10 8
LPC
6
Luteolin
4 2 0 20
40
60
80
100
% Drug release
Weight gain (%)
50
110 100 90 80 70 60 50 40 30 20 10 0
LPC Mixture Luteolin
15
Relative humidity (%)
30
45
60 75 90 Time (min)
105 120
Figure 5 Moisture adsorption curves of luteolin and luteolin– phospholipid complex (LPC).
Figure 6 In-vitro release curves of luteolin, physical mixture and luteolin–phospholipid complex (LPC).
gained marginal weight to about 2.05% at the end of the study. The critical RH of the complex was found to be about 78.9% at room temperature. The higher moistureabsorbing behaviour of LPC can be taken as a major
limitation that needs careful counter-action by varying the content, purity and nature of lipid being used for formulation and also demands for storage of the complex in dryer conditions.
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Solubility and release profile The extent and rate of dissolution has always remained an issue of concern for designing of pharmaceutical formulations. The poor aqueous solubility of polyphenolic plant drugs exhibit dissolution rate-limited absorption. The results of solubility study demonstrated marked improvements in solubility of luteolin as LPC in water and n-octanol. The solubility of luteolin in water increased from 0.93 ± 0.028 mg/ml to 2.36 ± 0.089 mg/ml and in n-octanol from 4.95 ± 0.097 mg/ml to 5.97 ± 0.074 mg/ml as LPC. However, the physical mixture of luteolin and phospholipids failed to show any significant increase in solubility of luteolin. The highly hydrophilic nature of LPC tends to decrease the surface tension between the waterinsoluble drug and water or aqueous gastrointestinal fluids. Because phospholipids are having adequate solubility in aqueous and oily phase, LPC showed improved solubility in both the phases. The results of in-vitro diffusion study showed improved dissolution–diffusion profile than the pure drug (Figure 6). The free drug exhibited first-order pharmacokinetic profile with gradual increase in drug release percentage against time. However, the increase in amount of drug absorbed was only marginal to a total amount of 41.12% till the end of 2 h. The physical mixture of drug and lipids exhibited a release profile similar to that of pure drug. LPC showed 95.12% of drug release at the end of 120 min. The lower per cent drug release from physical mixture and comparatively higher drug release by the LPC corroborates with the claim of soluble complex formation of luteolin with phospholipids in LPC. Moreover, the release of luteolin from complex was biphasic in nature with an initial rush followed by sustained release, suggesting entrapment of drug with lipid molecules. The changes in solubility and release profile of luteolin in our product justify the formation of the LPC.
Acute toxicity study Both the pure drug and LPC did not show any signs of toxicity up to the dose of 2000 mg/kg, which was taken as the end point. Physical observations indicated no changes in skin, eyes, body posture and behavioural pattern of animals under study. No significant changes were observed in relative weights of vital organs in the study (Table 2). The gross necropsy observations showed no major changes in luteolin-/LPC-treated animals as compared with control group. Only minor sporadic findings were observed in all the treatment groups (Table 3).
Table 2 Relative organ weights of rats receiving single dose of luteolin and luteolin–phospholipid complex (LPC) for acute toxicity study Relative weight Organ
Control
Luteolin (2000 mg/kg)
LPC (2000 mg/kg)
Lung Heart Spleen Liver Uterus Kidneys
0.76 ± 0.05 0.54 ± 0.04 0.35 ± 0.02 3.27 ± 0.15 0.21 ± 0.02 0.98 ± 0.07
0.69 ± 0.04 0.61 ± 0.01 0.47 ± 0.02 3.89 ± 0.19 0.21 ± 0.01 1.01 ± 009
0.79 ± 0.03 0.47 ± 0.02 0.38 ± 0.03 3.10 ± 0.10 0.30 ± 0.05 1.05 ± 0.09
n = 6.
Table 3 Necropsy findings after oral administration of luteolin and luteolin–phospholipid complex (LPC) (2000 mg/kg) for 14 days
Organ Lung Heart Spleen Liver Uterus
Treatment
Necropsy observations
Control
Luteolin
LPC
Normal Congested Normal Hypertrophied Normal Hypertrophied Normal Hypertrophied Normal Oedema
5/6 1/6 4/6 2/6 5/6 1/6 6/6 0/6 5/6 1/6
6/6 0/6 4/6 2/6 3/6 3/6 4/6 2/6 6/6 0/6
5/6 1/6 5/6 1/6 6/6 0/6 5/6 1/6 4/6 2/6
n = 6.
tion. The results of our study showed that pretreatment with luteolin, LPC and indomethacin significantly suppressed mouse ear oedema induced by TPA (Figure 7). The weight of ear was found to be reduced from 9.69 ± 0.79 mg in TPA-treated group to 7.05 ± 0.25 (P < 0.05), and 4.37 ± 0.41 mg (P < 0.001) in luteolin- and LPC-treated groups, respectively. The response of LPC was also found to be significant as compared with the response of pure luteolin. Histopathological evaluation of ear after TPA treatment showed substantial increase in infiltration of mononuclear and polymorphonuclear inflammatory cells and epidermal hyperplasia compared with untreated control group (Figure 8). The increase in ear weight of TPAtreated animals can be attributed to the intense ear oedema induced by TPA.[42] Luteolin and LPC treatments markedly reduced the ear thickness and associated pathological indicators. However, the changes in LPC-treated group were more prominent and were comparable with the response of indomethacin.
Ear oedema
Paw oedema
TPA-induced ear oedema is a widely accepted method for screening of anti-inflammatory activity on topical applica-
Carrageenan induced paw oedema is a well-established model to test effect of drugs against acute inflammatory
8
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Luteolin–phospholipid complex
12
Weight of ear (mg)
10 ∗
8 6
# ∗∗
# ∗∗
4 2
ci n
C In d
om et ha
LP
lin Lu
te o
TP A
nt ro l Co
N
or m al
0
Figure 7 Effect of luteolin and luteolin–phospholipid complex (LPC) on TPA-induced ear oedema in mice. Values are expressed as mean ± standard error of the mean (SEM) (n = 8). *P < 0.05 and **P < 0.01 compared with the control group, #P < 0.05 compared with luteolin (one-way analysis of variance (ANOVA) with least significant difference (LSD) post-hoc test).
response. The inflammatory response generated in this model is considered to be biphasic in nature with a preliminary oedema generation caused primarily by the effect of hypersensitivity mediators like histamine, serotonin and bradykinin on vascular endothelium, which is followed by a delayed response by the release of prostaglandins and NO.[43] Both luteolin and LPC significantly inhibited the increase in paw volume. However, the effect of LPC was found to be more potent than luteolin and was comparable with the effect of indomethacin (Figure 9). The paw volume reduced from 1.66 ± 0.09 (control) to 1.35 ± 0.08 (luteolin) 0.97 ± 0.05 (LPC) after 6 h of treatment. The effect of LPC on paw oedema was also found significant in relation to the effect of luteolin (P < 0.05). To assess the antioxidant potential of LPC relative to luteolin, the content of cell-damaging moieties like NO and MDA were analysed in the inflamed tissue fluid. Inflammatory response stimulates the leukocytes to release reactive oxygen species. NO plays a major role in recruitment of immune cells at the site of inflammation whereas MDA is the major degradation product of lipid hydroperoxides that accumulates at the inflammatory site. The results demonstrated
(a)
(b)
(c)
(d)
(e)
Figure 8 Representative histological stained sections of (a) normal mouse ear, (b) 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation, (c) 0.5 mg/ear indomethacin, (d) 0.5 mg/ear luteolin and (e) 0.5 mg/ear luteolin–phospholipid complex (LPC) treated (n = 8). © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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significant decrease in the tissue levels of NO and MDA by indomethacin, luteolin and LPC against the control group. However, the effect of LPC was more profound (P < 0.01 for NO and P < 0.001 for MDA) than luteolin (P < 0.05) and was closely comparable with indomethacin (Figure 10). This strong suppression of inflammatory mediators by LPC
2
Paw volume (ml)
1.8 1.6
Control
1.4
Luteolin
1.2
advocates an improvement in antioxidant and associated anti-inflammatory action of luteolin by the LPC.
Conclusions Phospholipid complex of luteolin was successfully formulated with optimized process parameters. The resultant preparation was found to be having desired physicochemical properties for oral drug delivery and was more effective than the pure drug against inflammation. The LPC can thus be a successful candidate for clinical trials. However, the stability of LPC on storage remains an issue of concern for future research.
Declaration
LPC
Conflict of interest
1
Indomethacin
The Author(s) declare(s) that they have no conflicts of interest to disclose.
0.8 0.6 0.4
Acknowledgement
0.2 2
1
4 6 Time (h)
Figure 9 Effect of luteolin and luteolin–phospholipid complex (LPC) on carrageenan induced oedema in rats. Values are expressed as mean ± standard error of the mean (SEM) (n = 8). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control group, #P < 0.05 and ##P < 0.01 compared with luteolin (one-way analysis of variance (ANOVA) with least significant difference (LSD) post-hoc test).
(b)
6 4 2
ac i et h
m al or
n
0
in
In do
et ha c
LP C In
do m
in Lu te
ol
l nt ro Co
N
or
m al
0
∗∗
m
0.2
∗∗
LP C
∗∗∗
in
∗∗∗
0.4
8
N
0.6
∗
10
te ol
∗
12
Lu
1 0.8
14
ol
1.2
Tissue No concentration (μM)
Tissue MDA concentration (nM/mg)
1.4
tr
(a)
The authors acknowledge the Department of Science and Technology (No. SR/FST/LSI-434/2010), New Delhi (Science and Engineering Research Council Division), India for providing financial assistance under the Fund for Improvement of S&T Infrastructure in Higher Educational Institutions (DST-FIST) scheme, as well as the Maulana Azad National Fellowship (MANF), University Grants Commission (UGC), New Delhi, India for providing financial assistance.
Co n
0
Figure 10 Effect of luteolin and least significant difference (LPC) on concentration of (a) nitric oxide (NO) and (b) malondialdehyde (MDA) in paw oedema fluids. Values are expressed as mean ± standard error of the mean (SEM) (n = 8). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control group.
10
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© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Research Paper
Journal of Pharmacy And Pharmacology
Luteolin–phospholipid complex: preparation, characterization and biological evaluation Junaid Khana, Amit Alexandera, Ajazuddinb, Swarnlata Sarafa and Shailendra Sarafa a University Institute of Pharmacy, Pandit Ravishankar Shukla University, Raipur, bRungta College of Pharmaceutical Sciences and Research, Bhilai, Chhattisgarh, India
Keywords inflammation; luteolin; phospholipid complex; solubility Correspondence Shailendra Saraf, University Institute of Pharmacy, Pandit Ravishankar Shukla University, Raipur, Chhattisgarh, 492010, India. E-mail: [email protected] Received January 14, 2014 Accepted May 15, 2014 doi: 10.1111/jphp.12280
Abstract Objectives This study aims to develop novel carrier system incorporating luteolin, a poorly soluble biologically active plant active. Methods We investigated a lipid-based drug delivery system to enhance dissolution and absorption profile of luteolin. Luteolin was complexed with phospholipids, and the preparation was characterized. The formulation was evaluated for physicochemical properties, in-vitro solubility or release studies. In vivo antiinflammatory action of luteolin and its phospholipid complex was evaluated by using carrageenan and 12-O-tetradecanoylphorbol-13-acetate as inducers. Key findings The prepared luteolin–phospholipid complex (LPC) showed drug loading of about 72.64% with average particle size of 152.6 nm. The Fourier transform infrared spectroscopy and thermal studies confirm formation of complex. The solubility of luteolin as LPC was about 2.5 times higher than the solubility of pure luteolin in water. In the diffusion study, LPC showed 95.12% of drug release at the end of 2 h. Animal studies demonstrated significant differences in response of LPC and luteolin. Conclusion LPC was successfully prepared by optimizing the process parameters. The resultant delivery system improved bioavailability and efficacy of luteolin and in the future may become an efficient tool for administration of luteolin.
Introduction With the advancements in herbal technology, plant-derived drugs have gained substantial attention from the medical community as well as patients for treatment of several debilitating ailments.[1] The potentially harmful adverse effects of synthetic drugs have further added to the cause.[2] As a consequence, the active pharmaceutical ingredients of plant origin have drawn the attention of researchers to formulate these molecules in such a form that has qualities like ease of administration, sufficient bioavailability and desirable aesthetic properties. The traditional herbal formulations of Chinese and Indian systems, however, lack these merits. Hence, the development of modern drug delivery systems for plant drugs has now become an essential element of pharmaceutical research. Among the plant-derived medicinally active compounds, polyphenolic structures and specially flavonoids have been extensively studied for their therapeutic potential in humans and animals.[3] These secondary metabolites are widely distributed throughout the plant kingdom and administered as food in the form fruits in our day-to-day
life. However, such administration is insufficient to impart any therapeutically useful effect of flavonoids.[4] Their consumption has remained limited in the form of food product as there are only some rarely available pharmaceutical formulations containing these therapeutically active compounds. Larger molecular size or weight, low aqueous or lipid solubility, and poor membrane permeability are some of the major factors that limit the clinical utility of these natural drugs.[5,6] Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a polyphenolic flavonoidal compound present in variety of medicinal plants and has been isolated from a number of floras, which include Reseda luteola, Delonix elata, Elsholtzia rugulosa and others.[7–9] Luteolin has strong scavenging properties for superoxide radicals. It is an established antioxidant and has shown antioxidant potential better than vitamin E and butylated hydroxytoluene.[10] It is also known to inhibit cholesterol synthesis, affect blood coagulation and has the ability to increase coronary blood flow.[11] It is considered to be a cancer preventive agent and has shown to exhibit strong antiproliferative activity against different
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human cancer cell lines.[12] Luteolin is a component of herbs with anti-inflammatory action and has itself demonstrated effectiveness against inflammatory processes. It has been reported as an active inhibitor of inflammatory mediators, 12-lipoxygenase and 5-lipoxygenase.[13] Luteolin is also known for its cardioprotective potential and affects the pathogenesis of atherosclerosis by inhibiting cholesterol biosynthesis at different levels, increasing elimination of cholesterol from liver and inhibiting LDL oxidation. Moreover, various herbal drugs containing luteolin are used for prevention or treatment of diabetes, obesity and other metabolic disorders.[14–16] Pure luteolin is a yellow microcrystalline powder, is sparingly soluble in water and is soluble in organic solvents. Inside the body, it is excreted rapidly from kidneys.[17,18] The poor aqueous solubility of luteolin hinders its systemic absorption, and extensive presystemic metabolism has further restricted the use of this highly potent and efficacious molecule to be utilized as a medicine by humans in fighting the menace of several debilitating disorders. To successfully utilize the different physiological effects of luteolin as a therapeutically active agent and to overcome its poor oral absorption, it is necessary to develop new formulation of luteolin, which can increase its oral absorption and add to its therapeutic potential. In recent years, complexation of poorly soluble polyphenolic plant actives with dietary phospholipids has emerged as an efficient tool to improve their oral/topical bioavailability and clinical efficacy.[19] Several potent plant constituents like curcumin,[20] silybin,[21] quercitin[22] and others have been successfully complexed with phospholipids and have shown better and improved solubility, bioavailability and efficacy.[23–25] The dietary or essential phospholipids used for complexation uniquely resemble the mammalian cellular phospholipids and hence are sufficiently biocompatible.[26] These phospholipids are known to be non-carcinogenic and non-mutagenic.[27] Phospholipid complex of plant actives are known to improve the solubility, stability and hence, the therapeutic efficacy of hydrophobic drugs.[28] Several polyphenolic herbal drugs have been combined with dietary phospholipids to form soluble micellar complexes resulting in new products of improved oral bioavailability and efficacy.[29] Moreover complexation of polyphenolic plant actives with essential phospholipids have shown biological value addition because of the inherent hepatoprotective potential of phospholipids.[30] However, comprehensive facts about the formulation of these complexes are insufficient, and this technique is still in its emerging phase. Various studies on complexation of polyphenolic compounds with dietary/soya phospholipids have stressed on characterization of prepared complexes with lesser concern on improvement of biological response of the drug as 2
complex.[31] The stability evaluation of phospholipid complexes has also remained almost neglected in most of the studies.[32] Hence, this study was undertaken with the objective to prepare and characterize phospholipid complex of luteolin and assess its moisture-absorbing capacity when exposed to humidity. Rather than the conventional approach of taking equal w/w ratio of lipids and phytoconstituents, we used factorial design in our study to optimize the key process parameters for preparing phyto-phospholipid complexes. Moreover, we also established the correlation between the improvement in solubility and in-vitro diffusion profile of luteolin by luteolin–phospholipid complex (LPC) with its enhanced biological response in laboratory animals. For this, we evaluated the in-vivo anti-inflammatory potential of LPC in comparison with pure luteolin.
Materials and Methods Materials Luteolin and carrageenan were purchased from Sigma Aldrich (Mumbai, India). Hydrogenated soya phosphatidylcholine (PHOSPHOLIPON 90H) was obtained as a gift sample from LIPOID GmbH (Ludwigshafen, Germany). All other chemicals of analytical grade were procured from local chemical suppliers.
Experimental animals Wistar rats (150–250 g) and Swiss albino mice (25–35 g) of either sex were used for in-vivo studies. The animals were obtained from animal house of Rungta College of Pharmaceutical Sciences and Research, Bhilai, Chhattisgarh, India. The animals were kept in groups of eight per cage under standard conditions (12 : 12 h light : dark cycle at 25 ± 2°C) and were provided with standard pellet diet and water given ad libitum. The studies were approved by the Institutional Animal Ethics Committee (1189/PO/a/08/CPCSEA) and were performed as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, India.
Preparation of complex A fixed amount of drug and lipids were taken in a 500-ml flask containing 250 ml of ethanol. The mixture was kept at different temperatures ranging from 40 to 50°C for 3 h with magnetic stirring. The resultant solution was evaporated under vacuum (RV 10, IKA, Staufen, Germany) to remove the solvent. The resultant LPC was kept in an ambercoloured glass bottle flushed with nitrogen. All the procedures were performed under aseptic conditions. The formulation parameters of LPC were optimized for better yield and drug loading capacity of product using response surface quadratic model (Design Expert version 9,
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Stat-Ease, Minneapolis, MN, USA). The LPC thus formulated appeared as almost completely dispersed particles in water and was found to be transparent. However, the dispersion of pure luteolin appeared to be highly turbid. The physicoshemical and biological characteristics of the prepared LPC were studied in relation to the pure luteolin and the physical mixture of luteolin and phospholipids prepared by simple mechanical mixing.
Characterization of LPC Spectrophotometric characterization was carried out by ultraviolet (UV)-visible and Fourier transform infrared spectroscopy (FTIR) studies. The samples of luteolin, phospholipid, physical mixture and LPC were dissolved in methanol to prepare dilutions of known concentration and then scanned between wavelength range of 200–400 nm using Shimadzu UV 1800 spectrophotometer (Shimadzu, Kyoto, Japan). FTIR of luteolin, phospholipon 90H and prepared LPC were obtained using a FTIR-8400 spectrophotometer (Shimadzu). A 1% w/w of sample with Potassium bromide (KBr) was triturated to fine powder and compressed into disc under a hydraulic press at 10 000 psi for 30 s. Each KBr disc was scanned over a wave number region of 4000–500/cm. The particle size and ζ-potential of LPC were determined at 25°C using photon correlation spectroscopy (Malvern Zeta-Size Nano ZS90, Worcestershire, UK). A proper dilution of the suspension was prepared with double-distilled water before each analysis. Differential scanning calorimetry (DSC) analysis was used to characterize the thermal behaviour of the luteolin, phospholipon 90H, physical mixture of luteolin with lipid and prepared LPC. DSC thermograms were obtained using an automatic thermal analyser system (Pyris 6 DSC, Perkin Elmer, Waltham, MA, USA). Samples were crimped in standard aluminium pans and heated at rate of 10°C/min within a temperature range of 50–400°C under constant purging of dry nitrogen (30 ml/min).
Hygroscopicity study Moisture-retaining capacities of luteolin and LPC were studied as per previously described method.[33] Solid samples were stored under controlled temperature and humidity conditions to investigate the moisture-absorbing capacity of luteolin and LPC on storage. The relative humidity (RH) at 25°C was generated using saturated inert solutions of known RH values. Definite amount of samples were weighed and placed in open glass bottles that were exposed to the desired RH inside desiccators.
Solubility and release studies Apparent solubility was determined by adding excess of luteolin and LPC to 5 ml of water or n-octanol in sealed
Luteolin–phospholipid complex
glass containers at room temperature. The liquid was agitated for 24 h on rotatory shaker then centrifuged for 15 min at 5000g to remove excessive luteolin. The supernatant was filtered through membrane filter. One millilitre of the filtrate was then mixed with 9 ml of distilled water to prepare dilutions, and these samples were measured for absorbance at wavelength of 254 nm by using UVspectrophotometer, and the concentration of luteolin in each sample was determined. For in-vitro release study of luteolin and LPC, dialysis method was applied using cellophane membrane. Activation of cellophane membrane was done to remove sulfur and other impurities. Two millilitres of luteolin and LPC were transferred to sample holders in triplicate placed in receiving compartments containing 18 ml of phosphate buffer (pH 7.4). The whole operation was performed at constant speed of 150 g at 25°C. Samples were measured at λmax 254 nm.
In-vivo studies The prepared LPC was evaluated for acute toxicity in rats. The study was carried out as per the Organisation for Economic Co-operation and Development guidelines 420. Female Wistar rats weighing 150–180 g were used for the study. The animals were continuously observed for the autonomic and behavioural changes and for mortality up to 14 days after drug administration. After 14 days, the animals were sacrificed. The organs were excised, weighed and examined macroscopically. For evaluation of the antiinflammatory potential of LPC in relation to the response of pure luteolin, two different animal models were used, namely 12-O-tetradecanoylphorbol-13-acetate (TPA)induced ear oedema in mice and carrageenan-induced paw oedema in rats. For TPA-induced mouse ear oedema, Swiss albino mice of either sex were used. Oedema was induced in both ears of each mouse by the topical application of 2 μg TPA dissolved in 20 μl of acetone to both the inner and outer ear surfaces. Thirty minutes after the application of TPA, the inner and outer surface of each ear was treated with 10 μl of 5% of Tween 80 and 0.5 mg/ear of indomethacin, luteolin and LPC, respectively, to different groups of animals (n = 8). After completion of 4 h, each animal was sacrificed under anaesthesia, and treated ears were removed and weighed.[34] The ears were then fixed in 10% buffered formalin solution, implanted in paraffin wax and cut into 5-mm sections. The sections were then stained with H&E and were observed under light microscopy. To assess the effect of LPC on carrageenan-induced acute inflammatory response, wistar rats of either sex (175–200 g) were used. Animals were administered different treatments, namely solvent, luteolin 10 mg/kg, LPC equivalent to 10 mg/kg of luteolin and indomethacin 10 mg/kg. Initial paw volume was measured using a digital plethysmometer, PLM
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Statistical analysis Data of animal studies were analysed with repeated measure analysis of variance and post-hoc test. All data were termed statistically significant with P-values less than 0.05.
Results and Discussion Luteolin–phospholipid complex To successfully develop any novel drug delivery system, yield and drug content (loading) are the key factors that represent the efficiency and acceptability of method followed.[38] Both of these aspects of formulation were taken into consideration in our study. We tried to prepare the complex of luteolin with phospholipids utilizing different solvents like ethanol, methanol, acetone, tetrahydrofuran and diethylether. The amount of luteolin loaded in the complex was at higher end with ethanol and methanol as solvents. Taking in to consideration the processing time and toxicity, ethanol was finally selected as the solvent medium. 4
(a) 72.6414
Loading (%)
80 70 60 50 40 30 50.00 47.50
45.00 B: temperature 42.50 (C)
2.50
(b)
90 85 80 75 70 65
3.00
2.00
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Yield (%)
01 PLUS (Orchid Scientific, Nasik, India). At 1 h after oral administration of drug, 0.1 ml 1% carrageenan in normal saline was administered in the subplantar region of the rat hind paw. Paw volume was measured up to 6 h after carrageenan administration.[35] For estimation of nitric oxide (NO) and malondialdehyde (MDA), another set of rats were orally administered with different treatments as stated above after carrageenan injection into right hind paws. After 2 h, the hind paws of the animals were surgically removed. The collected paw tissue was rinsed in ice-cold normal saline and immediately placed in cold normal saline and homogenized at 4°C. The homogenate was centrifuged at 13 000 g for 5 min. The supernatant was obtained and stored at −20°C for further estimation of NO and MDA. The production of NO was evaluated as per Griess reaction.[36] Briefly, 100 μl of supernatant from each treatment groups was mixed with equal volumes of sulphanilamide (1% in 5% phosphoric acid) and 0.1% naphthylenediamine dihydrochloride. The resultant samples were then incubated at room temperature for 10 min. The absorbance was recorded at 550 nm, and nitrite production was determined against a NaNO2 standard curve. For the estimation of MDA content in paw fluid, aqueous acid extraction thiobarbituric acid method was used with tetramethoxy propane as working standard.[37] Briefly, the supernatant of treated paw fluid was mixed with 10% of trichloroacetic acid and centrifuged for 5 min at 5000 g. To 1 ml of this supernatant, 1 ml of 0.67% TBA in 0.05 mol/L of NaOH was added. The above aliquots were heated in boiling water bath for 25 min and then cooled at room temperature. Absorbance was measured at 532 nm, and concentration of MDA was determined and expressed as nanomoles per millilitre of fluid.
1.50 A: PLP–Drug ratio (mg)
85.7912
3.00 2.50 A: PLP–Drug 2.00 ratio (mg) 1.50 1.00 50.00
42.50 45.00 47.50 B: temperature (C)
40.00
Figure 1 Response surface plot showing the influence of (a) phospholipid–drug ratio (X1), (b) reaction temperature (X2) on percentage yield (Y1) and drug loading (Y2).
To optimize the formulation parameters, the phospholipid to drug ratio and the reaction temperature were kept as the independent variables against which the dependent variables, that is, yield (%) and drug loading (%) were determined. The response surface statistical model demonstrated the effect of lipid to drug ratio and reaction temperature on the quality of end product (Figure 1). According to the data generated from the study, a response surface quadratic model suited fit for optimization of drug loading while a linear model was used for obtaining maximum yield. The independent variables were varied against the two dependent variables using two different equations:
Y1 = 73.02 + 3.99 X1 + 8.52 X 2 − 0.13 X1 X 2 − 8.44 X12 − 15.28 X 22 Y2 = 81.7 + 0.89 X1 + 5.99 X 2
(1) (2)
Where, Y1 and Y2 are the drug loading % and yield % of complex, respectively, and X1 and X2 are the coded levels of the independent variables, namely phospholipid–drug ratio and reaction temperature (°C) respectively. The % yield of
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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LPC was calculated by formula (LU – LF)/LU × 100 where, LU was the fixed amount of luteolin used for preparing LPC and LF was the content of luteolin in free form. The drug loading % of LPC was estimated by the formula LC/L × 100, where LC was the amount of luteolin that formed a complex and L was the weight of the LPC. The experimental results concerning the tested variables on the combination of luteolin in the complex are shown in Table 1.
The experimental results of nine batches showed a wide variation in yield of LPC from 67.19% to 89.78%, and in the per cent of drug loading in the complex ranging from 38.75% to 75.32%. According to the results of the quadratic response surface methodology, the LPC was prepared using the optimal process variable settings, where X1 and X2 were equal to 2.33°C and 50°C respectively. The per cent yield of prepared LPC was 85.79%, and the content of luteolin in the complex was 72.64%.
Ultraviolet and infrared analysis Table 1 Response values of different variables for the optimization of luteolin–phospholipid complex (LPC) preparation Formulation
Lipid: drug (w/w)
Temperature
Yield (%)
Drug loading (%)
1 2 3 4 5 6 7 8 9
1.00 2.00 3.00 1.00 2.00 3.00 1.00 2.00 3.00
40.00 40.00 40.00 45.00 45.00 45.00 50.00 50.00 50.00
78.25 67.19 79.95 81.39 83.24 83.91 85.23 89.78 86.33
38.75 45.29 46.75 59.21 75.32 67.65 53.25 67.89 60.74
(a) 1.0
(b) 1.0
0.8 0.6
We evaluated any possible interaction between the drug and the phospholipids to confirm the formation of complex. The results of UV spectra showed only minor variations in the peaks of luteolin and its phospholipid complex, suggesting some interaction between the two moieties rather than formation new chemical entity. The UV spectra are shown in Figure 2. The phospholipids showed only end absorptions close to 200 nm. The absorption curves of luteolin, the physical mixture and LPC were nearly the same. Two characteristic absorption bands of luteolin were observed at 254 and 349 nm. To explore the type of interaction between luteolin and phospholipid, FTIR studies were performed.
0.8 254 nM268 nM
349 nM 0.6
0.4
0.4
0.2
0.2
0.0 190
272
354
436 nM
(c) 1.0
254 nM268 nM
436 nM
350 nM 0.6 0.4
0.2
0.2
Figure 2
354
0.8 349 nM
0.4
0.0 190
272
(d) 1.0
0.8 0.6
0.0 190
200 nM
272
354
436 nM
0.0 190
254 nM 268 nM
272
354
436 nM
Ultraviolet (UV) spectra of (a) luteolin, (b) phospholipids, (c) physical mixture and (d) luteolin–phospholipid complex (LPC).
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Luteolin–phospholipid complex
Junaid Khan et al.
The infrared (IR) spectrum of LPC showed significant changes as compared with the spectra of luteolin and phospholipid. The results of IR study indicated formation of complex between luteolin and phospholipid as there are changes in peaks in complexes and positions from that of luteolin (Figure 3). The IR spectrum of luteolin showed –OH vibration band, at about 3144/cm, which is most probably the result of –OH vibrations of the phenolic groups. An intense band at 1616/cm was also noted, which seems to be the result of C = O vibrations from the central heterocyclic ring. The lipid demonstrated characteristic bands at 3619 and 2994/cm. The IR of LPC showed shift in phenolic –OH to 3425/cm. There are carbonyl groups belonging to phospholipids that allow the formation of hydrogen bonds with the phenolic hydroxyl groups of luteolin. This was evident from the FTIR graph where the phenolic –OH of luteolin shifted from its position and so
the P = O absorption band of the lipid, which suggests possible interaction of the phospholipid molecule with these phenolic –OH groups to form complexes.
Particle size determination The average particle size of LPC as estimated by the ζ sizer was found to be 152.6 nm, with a polydispersity index (PDI) of 0.675. The ζ-potential of LPC prepared in our experiment was found to be –27.6 ± 2.1 mV, which can be attributed to the type and composition of the phospholipids. The lower value of ζ-potential suggests possibility of incipient instability of LPC on storage. The PDI, however, is acceptable and closer to unity, indicating a sufficiently low critical micellar concentration.[39] The average particle size of the prepared complex is closer to 150 nm, which is suitable for retention at inflammatory sites with a reduced overall clearance.[40]
(a)
Thermal studies
100
3298 /cm
50
3144 /cm
Figure 4 shows the DSC thermogram of (a) pure luteolin, (b) pure phospholipid and (c) LPC. The thermogram of luteolin showed a single sharp peak starting from 321.45°C with maximum intensity on 329.34°C, which can be attributed to its melting point. DSC thermogram of phospholipids showed two major peaks at 74.15°C and 106.24°C. The first peak of phospholipids at 74.25°C is of mild intensity, which is probably due to the heat-induced movement of polar head groups. The second peak at 106.24°C is very sharp, and it appears to be because of phase transition from gel-like consistency to liquid crystalline state.[41] The thermogram of the complex (LPC) exhibits a single peak with onset of 47.7°C and maximum intensity at 63.5°C. The thermogram of physical mixture shows two peaks similar to that of luteolin and LPC. The DSC peaks of luteolin and LPC differed markedly. This shows that luteolin may interact with phospholipids, and the interaction is hydrophobic in nature. There is also some contribution of hydrogen bonding apart from hydrophobic interaction in the luteolin–phospholipids interaction. The –OH groups of the phenol rings of luteolin are involved in hydrogen bonding whereas the aromatic rings could be involved in hydrophobic interaction.
1174 /cm 1616 /cm
0 4000
3000
2000
1000
2000
1000
(b) 100
50
3519 2789
0
2994
4000
3000
(c) 100 2345
50
Hygroscopicity study 3425 1692
0 4000
3000
2000
1000
Figure 3 Fourier transform infrared spectroscopy (FTIR) spectra of (a) luteolin, (b) phospholipids and (c) luteolin–phospholipid complex (LPC).
6
The results demonstrated moisture-absorbing capacity of LPC with increase in RH at room temperature in open to air environment. The increase in weight per cent of luteolin stored at 25°C are shown in Figure 5. The LPC gained considerable weight on exposure to atmosphere ranging from 1.85% to 16.07% as compared with the bulk drug, which
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Junaid Khan et al.
Luteolin–phospholipid complex
(a)
(b)
321.45°C
74.15°C 329.34°C 108.24°C 0
50
100
150
200
250
300
350
0
400
50
100
150
200
250
100
150
200
250
(d)
(c)
47.7
321.45°C
47.7°C
63.9
330.01°C 63.5°C 0
50
Figure 4
100
150
200
250
300
350
400
0
DSC thermograms of (a) luteolin, (b) phospholipids, (c) physical mixture and (d) luteolin–phospholipid complex (LPC).
18 16 14 12 10 8
LPC
6
Luteolin
4 2 0 20
40
60
80
100
% Drug release
Weight gain (%)
50
110 100 90 80 70 60 50 40 30 20 10 0
LPC Mixture Luteolin
15
Relative humidity (%)
30
45
60 75 90 Time (min)
105 120
Figure 5 Moisture adsorption curves of luteolin and luteolin– phospholipid complex (LPC).
Figure 6 In-vitro release curves of luteolin, physical mixture and luteolin–phospholipid complex (LPC).
gained marginal weight to about 2.05% at the end of the study. The critical RH of the complex was found to be about 78.9% at room temperature. The higher moistureabsorbing behaviour of LPC can be taken as a major
limitation that needs careful counter-action by varying the content, purity and nature of lipid being used for formulation and also demands for storage of the complex in dryer conditions.
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Luteolin–phospholipid complex
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Solubility and release profile The extent and rate of dissolution has always remained an issue of concern for designing of pharmaceutical formulations. The poor aqueous solubility of polyphenolic plant drugs exhibit dissolution rate-limited absorption. The results of solubility study demonstrated marked improvements in solubility of luteolin as LPC in water and n-octanol. The solubility of luteolin in water increased from 0.93 ± 0.028 mg/ml to 2.36 ± 0.089 mg/ml and in n-octanol from 4.95 ± 0.097 mg/ml to 5.97 ± 0.074 mg/ml as LPC. However, the physical mixture of luteolin and phospholipids failed to show any significant increase in solubility of luteolin. The highly hydrophilic nature of LPC tends to decrease the surface tension between the waterinsoluble drug and water or aqueous gastrointestinal fluids. Because phospholipids are having adequate solubility in aqueous and oily phase, LPC showed improved solubility in both the phases. The results of in-vitro diffusion study showed improved dissolution–diffusion profile than the pure drug (Figure 6). The free drug exhibited first-order pharmacokinetic profile with gradual increase in drug release percentage against time. However, the increase in amount of drug absorbed was only marginal to a total amount of 41.12% till the end of 2 h. The physical mixture of drug and lipids exhibited a release profile similar to that of pure drug. LPC showed 95.12% of drug release at the end of 120 min. The lower per cent drug release from physical mixture and comparatively higher drug release by the LPC corroborates with the claim of soluble complex formation of luteolin with phospholipids in LPC. Moreover, the release of luteolin from complex was biphasic in nature with an initial rush followed by sustained release, suggesting entrapment of drug with lipid molecules. The changes in solubility and release profile of luteolin in our product justify the formation of the LPC.
Acute toxicity study Both the pure drug and LPC did not show any signs of toxicity up to the dose of 2000 mg/kg, which was taken as the end point. Physical observations indicated no changes in skin, eyes, body posture and behavioural pattern of animals under study. No significant changes were observed in relative weights of vital organs in the study (Table 2). The gross necropsy observations showed no major changes in luteolin-/LPC-treated animals as compared with control group. Only minor sporadic findings were observed in all the treatment groups (Table 3).
Table 2 Relative organ weights of rats receiving single dose of luteolin and luteolin–phospholipid complex (LPC) for acute toxicity study Relative weight Organ
Control
Luteolin (2000 mg/kg)
LPC (2000 mg/kg)
Lung Heart Spleen Liver Uterus Kidneys
0.76 ± 0.05 0.54 ± 0.04 0.35 ± 0.02 3.27 ± 0.15 0.21 ± 0.02 0.98 ± 0.07
0.69 ± 0.04 0.61 ± 0.01 0.47 ± 0.02 3.89 ± 0.19 0.21 ± 0.01 1.01 ± 009
0.79 ± 0.03 0.47 ± 0.02 0.38 ± 0.03 3.10 ± 0.10 0.30 ± 0.05 1.05 ± 0.09
n = 6.
Table 3 Necropsy findings after oral administration of luteolin and luteolin–phospholipid complex (LPC) (2000 mg/kg) for 14 days
Organ Lung Heart Spleen Liver Uterus
Treatment
Necropsy observations
Control
Luteolin
LPC
Normal Congested Normal Hypertrophied Normal Hypertrophied Normal Hypertrophied Normal Oedema
5/6 1/6 4/6 2/6 5/6 1/6 6/6 0/6 5/6 1/6
6/6 0/6 4/6 2/6 3/6 3/6 4/6 2/6 6/6 0/6
5/6 1/6 5/6 1/6 6/6 0/6 5/6 1/6 4/6 2/6
n = 6.
tion. The results of our study showed that pretreatment with luteolin, LPC and indomethacin significantly suppressed mouse ear oedema induced by TPA (Figure 7). The weight of ear was found to be reduced from 9.69 ± 0.79 mg in TPA-treated group to 7.05 ± 0.25 (P < 0.05), and 4.37 ± 0.41 mg (P < 0.001) in luteolin- and LPC-treated groups, respectively. The response of LPC was also found to be significant as compared with the response of pure luteolin. Histopathological evaluation of ear after TPA treatment showed substantial increase in infiltration of mononuclear and polymorphonuclear inflammatory cells and epidermal hyperplasia compared with untreated control group (Figure 8). The increase in ear weight of TPAtreated animals can be attributed to the intense ear oedema induced by TPA.[42] Luteolin and LPC treatments markedly reduced the ear thickness and associated pathological indicators. However, the changes in LPC-treated group were more prominent and were comparable with the response of indomethacin.
Ear oedema
Paw oedema
TPA-induced ear oedema is a widely accepted method for screening of anti-inflammatory activity on topical applica-
Carrageenan induced paw oedema is a well-established model to test effect of drugs against acute inflammatory
8
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Junaid Khan et al.
Luteolin–phospholipid complex
12
Weight of ear (mg)
10 ∗
8 6
# ∗∗
# ∗∗
4 2
ci n
C In d
om et ha
LP
lin Lu
te o
TP A
nt ro l Co
N
or m al
0
Figure 7 Effect of luteolin and luteolin–phospholipid complex (LPC) on TPA-induced ear oedema in mice. Values are expressed as mean ± standard error of the mean (SEM) (n = 8). *P < 0.05 and **P < 0.01 compared with the control group, #P < 0.05 compared with luteolin (one-way analysis of variance (ANOVA) with least significant difference (LSD) post-hoc test).
response. The inflammatory response generated in this model is considered to be biphasic in nature with a preliminary oedema generation caused primarily by the effect of hypersensitivity mediators like histamine, serotonin and bradykinin on vascular endothelium, which is followed by a delayed response by the release of prostaglandins and NO.[43] Both luteolin and LPC significantly inhibited the increase in paw volume. However, the effect of LPC was found to be more potent than luteolin and was comparable with the effect of indomethacin (Figure 9). The paw volume reduced from 1.66 ± 0.09 (control) to 1.35 ± 0.08 (luteolin) 0.97 ± 0.05 (LPC) after 6 h of treatment. The effect of LPC on paw oedema was also found significant in relation to the effect of luteolin (P < 0.05). To assess the antioxidant potential of LPC relative to luteolin, the content of cell-damaging moieties like NO and MDA were analysed in the inflamed tissue fluid. Inflammatory response stimulates the leukocytes to release reactive oxygen species. NO plays a major role in recruitment of immune cells at the site of inflammation whereas MDA is the major degradation product of lipid hydroperoxides that accumulates at the inflammatory site. The results demonstrated
(a)
(b)
(c)
(d)
(e)
Figure 8 Representative histological stained sections of (a) normal mouse ear, (b) 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation, (c) 0.5 mg/ear indomethacin, (d) 0.5 mg/ear luteolin and (e) 0.5 mg/ear luteolin–phospholipid complex (LPC) treated (n = 8). © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
9
Luteolin–phospholipid complex
Junaid Khan et al.
significant decrease in the tissue levels of NO and MDA by indomethacin, luteolin and LPC against the control group. However, the effect of LPC was more profound (P < 0.01 for NO and P < 0.001 for MDA) than luteolin (P < 0.05) and was closely comparable with indomethacin (Figure 10). This strong suppression of inflammatory mediators by LPC
2
Paw volume (ml)
1.8 1.6
Control
1.4
Luteolin
1.2
advocates an improvement in antioxidant and associated anti-inflammatory action of luteolin by the LPC.
Conclusions Phospholipid complex of luteolin was successfully formulated with optimized process parameters. The resultant preparation was found to be having desired physicochemical properties for oral drug delivery and was more effective than the pure drug against inflammation. The LPC can thus be a successful candidate for clinical trials. However, the stability of LPC on storage remains an issue of concern for future research.
Declaration
LPC
Conflict of interest
1
Indomethacin
The Author(s) declare(s) that they have no conflicts of interest to disclose.
0.8 0.6 0.4
Acknowledgement
0.2 2
1
4 6 Time (h)
Figure 9 Effect of luteolin and luteolin–phospholipid complex (LPC) on carrageenan induced oedema in rats. Values are expressed as mean ± standard error of the mean (SEM) (n = 8). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control group, #P < 0.05 and ##P < 0.01 compared with luteolin (one-way analysis of variance (ANOVA) with least significant difference (LSD) post-hoc test).
(b)
6 4 2
ac i et h
m al or
n
0
in
In do
et ha c
LP C In
do m
in Lu te
ol
l nt ro Co
N
or
m al
0
∗∗
m
0.2
∗∗
LP C
∗∗∗
in
∗∗∗
0.4
8
N
0.6
∗
10
te ol
∗
12
Lu
1 0.8
14
ol
1.2
Tissue No concentration (μM)
Tissue MDA concentration (nM/mg)
1.4
tr
(a)
The authors acknowledge the Department of Science and Technology (No. SR/FST/LSI-434/2010), New Delhi (Science and Engineering Research Council Division), India for providing financial assistance under the Fund for Improvement of S&T Infrastructure in Higher Educational Institutions (DST-FIST) scheme, as well as the Maulana Azad National Fellowship (MANF), University Grants Commission (UGC), New Delhi, India for providing financial assistance.
Co n
0
Figure 10 Effect of luteolin and least significant difference (LPC) on concentration of (a) nitric oxide (NO) and (b) malondialdehyde (MDA) in paw oedema fluids. Values are expressed as mean ± standard error of the mean (SEM) (n = 8). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control group.
10
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Luteolin–phospholipid complex
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