Quercetin Phospholipid Complex

Current Drug Discovery Technologies, 2012, 9, 17-24 17

1875-6220/12 $58.00+.00 © 2012 Bentham Science Publishers

Quercetin-Phospholipid Complex: An Amorphous Pharmaceutical System in Herbal Drug Delivery

Devendra Singh1, Mohan S.M. Rawat1,*, Ajay Semalty2 and Mona Semalty2

1Department of Chemistry, H. N. B. Garhwal University, Srinagar (Garhwal) 246 174, Uttarakhand, India; 2Department of Pharmaceutical Sciences, H. N. B. Garhwal University, Srinagar (Garhwal) 246 174, Uttarakhand, India

Abstract: Development of amphiphilic drug-lipid complexes is a potential approach for improving therapeutic efficacy of the drugs by increasing solubility, release profile and oral bioavailability. Quercetin (3, 3', 4', 5, 7-pentahydroxyflavone), a polyphenolic flavonoid, shows several biological effects like anti-inflammatory, anti-cancer, antiproliferative, anti-mutagenic and apoptosis induction but its use is limited due to its low aqueous solubility. To overcome this limitation, a value added phospholipid complex of quercetin was developed to improve its aqueous solubility for better absorption through the gastrointestinal tract and this might result in improved bioavailability. The quercetin-phospholipid complex thus prepared was evaluated for various physico-chemical parameters like infra red spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray powder diffractometry (XRPD), scanning electron microscopy (SEM) and solubility study. The In vitro antioxidant activity was also studied. The phospholipid complex of quercetin was found to be fluffy and porous with rough surface in SEM. FTIR, DSC and XRPD data confirmed the formation of phospholipid complex. The water solubility of quercetin was improved by 12 folds (from 3.44 �g/ ml to 36.81 �g/ ml) in the prepared complex. There was no statistical difference between the quercetin complex and quercetin in the In vitro anti-oxidant activity, indi-cating that the process of complexation did not adversely affect the bioactivity of the active ingredient.

Keywords: Amorphous product, antioxidant activity, phosphatidylcholine, phospholipid complex, quercetin, solubility behavior.

1. INTRODUCTION

Upon oral ingestion, a drug is dissolved into the gastric fluid (hydrophilic environment) initially followed by the permeation across the biological membranes (lipophilic envi-ronment) and finally reaches the blood stream. Most of the biologically active polyphenolic phytoconstituents are asso-ciated with the problem of either poor absorption or the poor permeation through the biological membrane, thereby limit-ing their absorption and overall availability to the body sys-tem [1, 2]. Poor absorption may be due to their poor water solubility. While the poor permeation may be due to the na-ture of structure of the drug (multiple-ring molecules like many herbal drugs may be too large to be absorbed by simple diffusion) or due to the poor miscibility with oils and other lipids thereby, severely limiting their ability to pass across the lipid-rich outer membranes of the enterocytes of the small intestine [3, 4]. For good bioavailability, a herbal drug must have an adequate hydrophilicity (for dissolution in to gastrointestinal fluid) as well as an adequate lipophilicity (to permeate across the lipidic biomembrane).

Flavonoids are a widely distributed group of polypheno-lic compounds characterized by a common benzo-pyrone structure. Flavonoids have a broad pharmacological profile

*Address Correspondence to this author at the Department of Chemistry, H. N. B. Garhwal University, Srinagar (Garhwal) 246 174, Uttarakhand, India; Tel: +91 1346 252229; Fax: +91 1346 252174; E mail: [email protected]

such as anti-lipoperoxidant, anti-inflammatory, anticancer and chemo preventive activities. Among these, quercetin (3,3',4',5,7-pentahydroxyflavone, Fig. 1a) is a naturally oc-curring flavone mostly found in onion leaves, black and green tea (Camellia sinensis), papaya shoots, green vegeta-bles [5] and shows several biological activities like, anti-inflammatory [6], anti-cancer [7, 8], renoprotective, neuro-protective and hypertensive effects [9-11]. In spite of this wide spectrum of pharmacological properties, its absorption and hence the bioavailability is limited due to its low aque-ous solubility. The rate of release of a drug is a function of its intrinsic solubility and is influenced by particle size, crys-tallinity, drug derivatization and formation of more-soluble complexes [12-18]. Recently, natural polymers like phos-pholipids, polysaccharides and proteins have received much attention in the pharmaceutical field owing to their good biocompatibility and biodegradability [19-22].

Phospholipids (like phosphatidylcholine, Fig. 1b) play a major role in drug delivery due to their amphiphilic nature that can modify the solubility behavior and rate of drug re-lease for the enhancement of drug absorption across the bio-logical barriers. Development of amphiphilic drug-lipid complexes may prove to be a potential approach for improv-ing therapeutic efficacy of the drugs by modifying the solu-bility and release (sustained or controlled release) for im-provement of oral bioavailability. These amphiphilic drug-lipid complexes are stable and more bioavailable with low interfacial tension between the system and the gastrointesti-nal (GI) fluid, thereby facilitating membrane, tissue, or cell

18 Current Drug Discovery Technologies, 2012, Vol. 9, No. 1 Singh et al.

wall transfer, in the organism [4, 23, 24]. Among the phos-pholipids, phosphatidylcholine (PC) is an important natural carrier which can play a major role to improve solubility and dissolution profile of the drug. It is compatible with pharma-ceuticals, highly bioavailable and also exerts its own thera-peutic benefits (like hepatoprotection). Moreover, it is also an excellent emulsifier that enhances the bioavailability of constituents with which it is co-administered [23, 25].

The present study deals with the development of quer-cetin-phospholipid complex with the aim of improving the aqueous solubility of quercetin for better absorption through the GI tract, which might result in improved bioavailability. The prepared complex was characterized for various phys-ico-chemical parameters FTIR, DSC, XRD, Solubility, SEM and the In vitro antioxidant activity.

Fig. (1). (a) Quercetin (b) Phosphatidylcholine.

2. MATERIALS AND EXPERIMENTAL METHODS

2.1. Materials

Quercetin (95%) was purchased from Sigma Aldrich Mumbai (India). Soya phosphatidylcholine (LIPOID S-80) was obtained as a gift sample from LIPOID, Germany. Buty-lated hydroxy anisole (BHA) and 2, 2 diphenyl 1-picryl hy-drazyl (DPPH) were purchased from E. Merck, Mumbai. All chemical reagents were of analytical grade.

2.2. Method of Preparation

The quercetin-phospholipid complex was prepared by refluxing the quercetin and phosphatidylcholine in (1:1) mo-lar ratio. Both the reactants were placed in 100 ml round bottom flask containing 20 ml of dichloromethane and the reaction proceeded by refluxing the reaction mixture at 45-50°C until all the quercetin had dissolved. Thereafter the volume of resulting solution concentrated to 2-3 ml and then the sufficient amount of n-hexane was added to get the com-plex as amorphous product. The complex was filtered, washed, dried under vacuum and stored in an air tight con-tainer until further use.

2.3. Infrared Spectroscopy

The IR spectra were recorded on a Perkin Elmer FTIR RX-1 spectrophotometer in KBr tablets.

2.4. Differential Scanning Calorimetry

Thermograms of quercetin, phosphatidylcholine (80%) and the quercetin-PC complex were recorded using a differential scanning calorimeter (2910 Modulated DSC V4.4E, TA Instruments, US). The thermal behavior was studied by heating 2.0 ± 0.2 mg of each individual sample in a covered sample pan under nitrogen gas flow. The investi-gations were carried out over the temperature range 0-300 ºC with a heating rate of 10ºC min-1.

2.5. X-Ray Powder Diffractometry

The crystalline state of drug (quercetin) in the different samples was evaluated with X-ray powder diffraction. Dif-fraction patterns were obtained on a Bruker Axs-D8 Dis-cover Powder X-ray diffractometer (Germany). The X-ray generator was operated at 40 KV tube voltages and 40 mA of tube current, using the K � lines of copper (wavelength 0.1541 nm) as the radiation source. The scanning angle ranged from 5 to 50o of 2� in step scan mode (step width 1o/min).

2.6. Scanning Electron Microscopy

SEM imaging of the complex was performed using a Scanning Electron Microscope (JEOL JSM 5600).

2.7. Apparent Solubility Study

Apparent solubility was determined by adding excess of quercetin and quercetin-phospholipid complex to 5ml of water or n-octanol in sealed glass containers at room tem-perature (25-30ºC). The liquid was agitated for 24 hours then centrifuged for 20 min at 1,000 rpm to remove excess of quercetin. The supernatant was filtered through a membrane filter (0.45 �m) then 1 ml filtrate was mixed with 9 ml of distilled water or n-octanol to prepare dilutions and these samples were measured at 322 nm UV spectrophotometri-cally.

2.8. Anti-Oxidant Activity

The free radical scavenging activity of quercetin and quercetin complex was measured and compared with the activity of BHA for free radical-scavenging ability using the stable free radical DPPH [26, 27]. The free radical scaveng-ing activities of quercetin, quercetin complex and BHA were measured by decrease in the absorbance of methanolic solu-tion of DPPH at 517 nm.

0.1 mM solution of DPPH in methanol was prepared and 1.5 ml of this solution was added to 3.5 ml methanolic solu-tion of quercetin, quercetin-phospholipid complex and BHA of different concentration (20-250 �g/ml). Thirty minutes later, the absorbance was measured at 517 nm. Lower absor-bance of the reaction mixture indicates higher free radical scavenging activity. The capability to scavenge the DPPH free radical was calculated using the following equation: DPPH Scavenged (%) = [(Acont - Atest) / Acont] x 100 Where, Acont = Absorbance of the control and Atest = Absorbance in the presence of the sample of the drug/complex.

O

O

OH

OHHO

OHOH

Quercetin

R1

C

OCH2

CHO

C

R2O

CH2 O O

CH2

CH2

N

CH3

CH3

CH3

O

P

Phosphatidylcholine

O

O

Quercetin-Phospholipid Complex Current Drug Discovery Technologies, 2012, Vol. 9, No. 1 19

3. RESULTS AND DISCUSSION

Due to the amorphous characteristics and improved solu-bility, the quercetin phospholipid complex may provide an increased clinical value and absorb better than the free crys-talline state of the drug in the gastrointestinal tract, which could improve the overall availability of the drug within the body.

3.1. Infrared Absorption

The possible interaction between quercetin and PC in the phospholipid complex was studied by IR spectroscopy and presented in Fig. (2). In IR spectra the characteristic C-H stretching band of long fatty acid chain at 2918cm-1 and 2850cm-1, carbonyl stretching band at 1738cm-1, P=Ostretching band at 1236cm-1, P-O-C stretching band at 1091 cm-1 and N+ (CH3)3 stretching at 970cm-1 in phosphatidylcho-line molecule.

In case of quercetin, the main characteristic bands for the hydroxyl (O-H) stretching at 3325cm-1, C=O stretching at 1614cm- 1and benzene ring vibrations near about 1522cm- 1

were observed. The FTIR of the complex showed the signifi-cant changes in the spectrum and the absorption peaks of hydroxyl (O-H) and keto (C=O) group of the quercetin have been shifted to higher wave number, whereas the P=O ab-sorption band of the phosphatidylcholine remarkably broad-ened.

The spectrum of the physical mixture was quite different from the spectrum of the complex and showed same vibra-tional frequencies as that of the individual components and seemed to be only a summation of both the constituents. Therefore, the spectroscopic changes showed that, the shift-ing of hydroxyl and keto group frequencies of quercetin from their original place accounts for the interaction of quer-cetin to polar end of the phospholipid.

Fig. (2). IR spectra: (a) Phospholipid (b) Quercetin (c) Quercetin.

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.00.0

5

10

15

20

25

30

35

40

45

50.0

cm-1

%T

Phospholipiod-1

3435.63

2918.11

2850.22

1738.81

1468.74

1236.301091.50

(a)

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.045.0

50

55

60

65

70

75

80

85

89.0

cm-1

%T

Que rcetin

3325.06

2360.41

1614.99

1522.24

1385.28

1319.87 1264.77

1199.93

1169.08

1015.31 824.16

(b)

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.025.0

30

35

40

45

50

55

60

65

70

75

8082.1

cm-1

%T

Quercetin Complex (DCM)

3391.88

2918.272850.49

2360.34

1733.84

1653.661471.62

1199.951087.44

822.69

(c)

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.00.0

5

10

15

20

25

30

35

40

45

50

53.5

cm-1

%T

Quercetin-Pc Phy. Mix.

3413.46

2917.822850.06

1739.56

1468.02

1237.071091.28

970.76

721.52

(d)


3.2. Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) is a fast and reliable method to detect drug-excipient compatibility to provide maximum information regarding the possible inter-actions. In DSC, an interaction is concluded by elimination of endothermic peaks, appearance of new peaks and changes in various parameters of thermogram (like peak shape, its onset, peak temperature/ melting point and relative peak area or enthalpy). The thermal curves of pure components (quer-cetin), phospholipid and of the drug-phospholipid system are shown in Fig. (3).

Phospholipid showed two major endothermic peaks at 83.21ºC and 107.90ºC and a small peak at 64.45ºC. The first one peak of phospholipids is mild peak (at 64.45ºC), which is probably due to the heat-induced movement of phosphol-ipids polar head group. The second (83.21ºC) peak is very sharp and it appears due to phase transition from gel to liquid crystalline state. The non-polar hydrocarbon tail of phos-

pholipid may be melted during this phase, yielding a sharp peak. This melting might have occurred in two phases which subsequently gave another peak (107.90ºC) which is rela-tively less sharp. Quercetin in the DSC curve showed a sharp endothermic peak at 122.38ºC. On the other hand, the com-plex showed a complete disappearance of the melting endo-thermic peaks of the individual components (quercetin, phospholipid) with reduced enthalpy and melting points. It showed a noticeable reduction in the enthalpy of 71.48 J/g along with its lowest melting point (60.02ºC) in comparison with free quercetin, indicating the formation of the complex and supports to the previous study [28, 29].

Reduction in melting point and enthalpy account for in-creased solubility and reduced crystallinity of the drugs [30, 31]. This phenomenon can be assumed the interactions be-tween component and the phospholipid in the complex sys-tem and can be considered as indicative of drug amorphiza-tion and/ or complex formation as supported by IR spectros-copy results also.

(a)

(b)

(c)

Fig. (3). DSC Thermograms: (a) Phospholipid (b) Quercetin (c) Quercetin- Phospholipid Complex


3.3. X-ray Powder Diffractometry

Fig. (4) shows the X-ray diffraction patterns of the quer-cetin, phospholipid and the complex. In the X-ray diffracto-gram quercetin showed intense diffraction peaks of crystal-linity at a diffraction angle of 2� and suggested that the drug is present as a crystalline material. The phospholipid showed a single diffraction peak. A total drug amorphization was induced by complex formation where X-ray diffraction pat-terns of quercetin-phospholipid complex were characterized only by large diffraction peaks in which it is no longer pos-sible to distinguish the characteristic peaks of the drug. The results, confirmed that quercetin is no longer present as a crystalline material and its phospholipid complex is in the

amorphous state. Thus, XRD data supports the DSC studies which indicated the reduced crystallinity of drug in the pre-pared complex by exhibiting lower values of enthalpy and melting points and well supported by previous literature [32-35]. As the amorphous form of the drug absorb better than their crystalline counterpart [36, 37], the quercetin-phospholipid complex may serve as a potential approach for effective oral delivery of their parental analogue.

3.4. Scanning Electron Microscopy

The scanning electron micrographs of quercetin and the complex are given in Fig. (5). The quercetin was character-ized as needle-like crystals of smaller size and regular shape

(a)

(b)

(c)

Fig. (4). X-ray diffraction patterns: (a) Phospholipid (b) Quercetin (c) Quercetin-Phospholipid Complex.


with an apparently smooth surface. In contrast, a drastic change in the morphology and shape of particles was ob-served in the phospholipid complex and showed fluffy, po-rous and rough surface, revealing an apparent interaction in the solid-state which might have resulted in improved solu-bility and enhanced dissolution rate as compared to pure drug.

3.5. Solubility Study

Quercetin is extremely hydrophobic (log P, 1.82 ± 0.32) [38] in nature and slightly soluble in aqueous media. Its poor solubility leads to its poor absorption/ permeation across the intestinal epithelial cells of the gastrointestinal (GI) tract leading to low bioavailability of the drug. Aqueous solubility of quercetin (3.44 �g/ml) improved by 12 folds (36.81 �g/ ml) in the prepared complex (Table 1). As an amphiphilic surfactant, phospholipids could increase the solubility of the drug by the action of wetting, dispersion and micellisation; the phospholipid complexation increased the solubility be-havior of herbal active due to the amphiphilic and amor-phous nature of the product. Micellisation is an important approach capable of solubilizing a hydrophobic drug in a hydrophilic environment comprised of biodegradable drug carrier micelle and a hydrophobic drug wherein the drug is compatible and not strongly bonded to the polymeric drug carrier micelle. The complex showed enhanced solubility due to its amphiphilicity, which in turn may show the im-proved absorption across the biological barriers for improv-ing oral bioavailability of the active component.

3.6. Anti-Oxidant Activity

Quercetin and its complex showed 90.76% and 89.82% inhibition of DPPH at a concentration of 20 �g/ml and 90.82%, 90.70% at a concentration of 200�g/ml respec-tively. There was no statistical (n = 6, p � 0.01) difference in the percent inhibition of DPPH between quercetin and its complex, on all the dose levels (Fig. 6). Therefore, the anti-oxidant activity of the quercetin remains unchanged even after the complex formation and it can be concluded that the process of complexation did not adversely affect the antioxi-dant activity of quercetin.

4. CONCLUSIONS

Based on the results of the study, it can be concluded that the phospholipid complex may be considered as promising drug delivery system for improving the bioavailability of the quercetin molecule. The physicochemical properties of quer-cetin changed significantly after it was complexed with the phospholipid and these characteristics especially, the im-proved solubility might contribute to improve oral absorp-

Fig. (5). SEM micrographs: (a) Quercetin (b) Quercetin-Phospholipid Complex.

Table 1. Solubility Study (H2O/ n-Octanol) at 25°C

Sample Aqueous Solubility (�g/ml)

n-Octanol Solubility (�g/ml)

Quercetin 3.44 52.17

Quercetin complex 36.81 51.92

(a) (b)


tion of the drug. Further, it was found that, there was no sta-tistical difference in the percent inhibition of DPPH between quercetin and its complex on all the dose levels. This indi-cates that, the bioactivity was maintained when quercetin was complexed with phospholipid and the production proc-ess of the complex did not change or destroy the molecular structure of active ingredient (quercetin) in the complex. As these amphiphilic drug-lipid complexes have been reported to be stable and more bioavailable, the phospholipid com-plex of quercetin may serve as a value added herbal drug delivery system.

CONFLICT OF INTEREST

Declared none.

ACKNOWLEDGEMENTS

The authors are thankful to the Department of Science and Technology, Government of India for the research grant (SRSO/ HS/ 72/ 2006). The authors acknowledge LIPOID GmbH Germany for providing the gift sample of phosphati-dylcholine for the research work. Facilities provided by UGC-DAE Consortium for Scientific Research, Indore (In-dia), are gratefully acknowledged.

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Received: April 01, 2011 Revised: April 21, 2011 Accepted: April 26, 2011

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Quercetin Phospholipid Complex