Freeze dried Multicomponent Inclusion Complexes of Piperine with Cyclodextrin and Hydrophilic Polymers: Physicochemical Characterization and In vivo Anti-inflammatory Activity


Anita S. Kulkarni1, Remeth J. Dias2*, Vishwajeet S. Ghorpade3, Kailas K Mali4

1Department of Pharmaceutical Chemistry, Government College of Pharmacy, Karad, Maharashtra, India.

2Department of Pharmacy, Government Polytechnic, Jalgaon, Maharashtra, India.

3Department of Pharmaceutics, School of Pharmaceutical Sciences, Sanjay Ghodawat University,

Kolhapur, Maharashtra, India.

4Department of Pharmaceutics, Adarsh College of Pharmacy, Bhavaninagar, Kundal Road,

Vita. Taluka - Khanapur, District - Sangli. (Maharashtra)-415311.

*Corresponding Author E-mail:



In order to improve the physicochemical properties and anti-inflammatory activity of piperine (PIP), its multicomponent inclusion complexes were prepared with β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPβCD) in presence of a ternary component such as polyvinylpyrrolidone K30 (PVP) and poloxamer 188 (POLO) by lyophilization. The initial phase solubility studies were carried out for determination of the stability and complexation efficiency of the prepared complexes. The complexes were evaluated for the saturation solubility, drug content and in-vitro dissolution, and characterized using ATR-FTIR, DSC, XRPD and SEM. Phase solubility studies revealed that PIP-βCD and PIP-HPβCD complexes exhibited 1:1 stoichiometry. The ternary systems showed good solubilizing and dissolution efficiency than the binary systems. The ATR-FTIR and DSC analysis showed interaction of polymers with PIP and cyclodextrins whereas XRPD analysis revealed amorphization of the ternary complexes. The PIP-HPβCD-POLO ternary complex showed high solubility and improved dissolution than the binary complexes. The in vivo anti-inflammatory activity of the PIP-HPβCD-POLO ternary complex was found to be maximum than pure PIP. Overall results indicated that freeze dried PIP-HPβCD-POLO ternary system can enhance the anti-inflammatory activity of PIP than the binary complexes.


KEYWORDS: Piperine; β-cyclodextrin; Hydroxypropyl-β-cyclodextrin; Lyophilization; Multicomponent inclusion complex.




Piperine, 1-[5-(1, 3-benzodioxol- 5-yl)-1-oxo-2, 4-pentadienyl] piperidine (PIP) is an alkaloid amide mainly present in the dried mature fruits of black pepper Piper nigrum, family Piperaceae1. The use of PIP as spice in the dietaries is common. Besides, it has great potential as a drug having medicinal value.


PIP shows different pharmacological actions such as anti-inflammatory2,3, antidiarrhoeal4, anticonvulsant, antiepileptic5, antioxidant6,7 as well as antimycobacterial8. It also exhibits anticancer/antitumor immunomodulatory activity9. It acts as bioavailability enhancer for several drugs10. However, piperine has low aqueous solubility which might be a limiting factor for its absorption process. The literature available till date reveals few formulation approaches explored for enhancing water solubility and dissolution rate of PIP. Some of these include the development of the self-emulsifying drug delivery system of PIP11 and hot melt extrusion technique12.


Cyclodextrins (CDs) are cyclic oligosaccharides consisting of (α-1, 4)-linked α-D-glucopyranose units with a lipophilic central cavity. CD molecules are shaped like cones because of chair conformation of the glucopyranose units with secondary hydroxy groups extending from the wider edge and the primary groups from the narrow edge. This gives CD molecules a hydrophilic outer surface and a lipophilic central cavity. It is a well-known fact that CDs enhance the stability and solubility of drugs by the formation of inclusion complexes with them13–15. Formation of inclusion complexes usually results in modifications in the physicochemical properties of the drug with improvement in its solubility, stability, dissolution rate, and bioavailability16,17. Binary inclusion complexes of PIP and βCD were prepared by co-grinding method and evaluated for physicochemical properties and dissolution behavior18. As βCD has low solubility, its large amount is usually needed for solublizing small amount of a hydrophobic drug19. The low solubility of βCD is attributable to the formation of internal hydrogen bonds in between the secondary hydroxyl groups and high crystal lattice energy. In order to improve the solubility of βCD, various derivatives have been synthesized by modifying the hydroxyl groups of βCD. However, their use in the formulations is limited due to their toxicity and high cost. It has been found that incorporation of small quantities of certain hydrophilic auxiliary substances to the aqueous solutions of CDs can improve their solubilizing efficiency. These auxiliary substances may be polymers20, hydroxy acids21 and/or amino acids22–24. The aqueous solutions of the multicomponent inclusion complexes (MICs) comprised of poorly soluble drug, CD and the auxiliary substance can remain stable for longer duration. The hydrophilic polymers are known for stabilizing the CD aggregates in the aqueous solutions. Also, they can reduce the mobility of the CD molecules and increase the solubility of drug-CD complexes by altering the hydration properties of CD molecules. Till date, no one has investigated the effect of incorporation of hydrophilic polymers on the PIP solubilizing efficiency of CDs and the anti-inflammatory effect of the resultant complex.


In the present study, ternary inclusion complexes of PIP with CDs and hydrophilic polymers such as polyvinylpyrrolidone K30 (PVP) and poloxamer 188(POLO) were prepared by lyophilization technique. PVP has shown promising results as a ternary component when used in the preparation of MICs25. Besides, in a recent study related to the solid dispersions, it was found superior in enhancing the solubility of PIP26. POLO, on the other hand, can form nano-aggregates with CD resulting in the formation of micelles, which in turn can contribute in improvement of drug solubility27. The binary complexes were prepared for comparison. Initial phase solubility studies were conducted to determine the stability, Gibbs free energy and stoichiometry of the complexes formed. The prepared complexes were evaluated for the saturation solubility and drug content, and characterized by differential scanning calorimetry (DSC), X-ray powder diffractometry (XRPD), Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and scanning electron microscopy (SEM). The optimized complex was subjected to in vivo anti-inflammatory activity studies.




Piperine (PIP) was purchased from Sigma Aldrich, India. Hydroxypropyl-β-cyclodextrin (HPβCD) was obtained as a gift sample from Gangwal Chemicals, Mumbai, India. β-cyclodextrin (βCD) and polyvinylpyrrolidone K30 (PVP-K30) were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India. Poloxamer-188 (POLO) was obtained from Signet Chemicals, Mumbai, India. The analytical grade reagents and glass distilled water were used for the experimental procedures.


Phase solubility studies:

The method described by Higuchi and Connors28 was used for the phase solubility studies in distilled water at room temperature (25 ± 2oC).PIP, in excess quantity (50 mg), was added to a series of 20 mL of aqueous solutions containing various concentrations of βCD or HPβCD (0, 0.002, 0.004, 0.006, 0.008 and 0.01 M) in the presence or absence of auxiliary substance PVP (0.5% w/w) or POLO (0.5% w/v). The suspensions were subjected to shaking on rotary shaker (Lab HOSP – Shaker) for 72 h at 150 rpm to attain equilibrium. The samples were filtered through Whatman filter paper No. 41 and appropriately diluted and analyzed spectrophotometrically (Shimadzu 1800, Japan) at 341.6 nm to determine the concentration of PIP. The results were obtained in triplicate. The phase solubility curves were obtained by plotting the concentration of dissolved PIP (moles/ liter) against the respective concentration of CDs (moles/ liter). The stability constants (KS) of the binary and ternary complexes were calculated using the equation15.



Ks=  –––––––––––                                                             (1)

          S0 (1-Slope)


Where, S0 is the solubility of PIP in distilled water in absence of CDs


The complexation efficiency (C.E.)/ solubilization efficiency (S.E.) of CDs was determined by the following equation15.



C.E. or S.E. Ks=  –––––––––––                                       (2)

                           S0 (1-Slope)


Gibbs free energy change () in Joules/mole was also calculated for the phase solubility analysis to assess the thermodynamics of the solution and complexation process, using the equation given below:


∆Gtr= -2.303 RT log Sc/S0                                                 (3)


Where, Sc is molar solubility of PIP in aqueous solution of HPβCD or βCD in the presence or absence of auxiliary substance (0.5%, w/v POLO and 0.5 w/v PVP), S0 is molar solubility of PIP in distilled water in absence of HPβCD and βCD, R is gas constant and T is temperature in Kelvin.


Preparation of freeze-dried solid inclusion complexes:

To prepare binary complex systems, the weighed quantities of PIP and βCD or HPβCD, in the molar ratio 1:1. were dissolved separately in 20 ml of methanol and 60 ml of distilled water, respectively. The solutions were mixed together after sonicating for 15 min. The resultant solutions were stirred using a magnetic stirrer (2MLH, Remi laboratory Instruments, Mumbai), for 72 h at room temperature (25±2oC) and 150 rpm to attain an equilibrium and were frozen in deep freezer (ELCOLD) at -80oC for 24 hours. Then, the frozen solutions were lyophilized (DELVAC-mini Lyodel) and stored in desiccators till further use.


In case of ternary complex systems of PIP, above procedure was followed with the addition of the auxiliary substance, either 0.5 %w/w PVP or 0.5%w/w POLO, to the complexation media (aqueous solutions of CDs).


Characterization of freeze-dried inclusion complexes:

Differential scanning calorimetry (DSC):

DSC analysis of PIP, βCD, HPβCD, polymers and all complexes were performed using DSC analyzer (TA instruments Q600 SDT USA). A sample (5mg) was sealed in an aluminum pan and subjected to heating at a rate of 10oC /min from 30 to 400ºC under nitrogen atmosphere


Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR):

Attenuated Total Reflectance (ATR), in association with Fourier transform infrared spectroscopy (FTIR) assists in direct analysis of the solid or liquid samples and does not require any complex sample preparation procedure. Infrared spectra of all samples were obtained by using ATR- FTIR (BRUKER–ECO-ATR-ALPHA, Germany). The samples were directly placed on ATR crystal and analyzed from 600 to 4000 cm-1 spectral range with 24 scans.

X-ray powder diffractometry (XRPD):

The XRPD patterns of all formulations were obtained by using X-ray diffractometer (PW 1723, Philips, Netherland) with tube anode Cu over the interval 05-60◦ (2θ). Generator tension (voltage) 40 kV, Generator current 30mA, and scanning speed 2◦/min. were maintained during the operation.


Determination of drug content:

For calculation of content of PIP, the prepared complexes equivalent to 5 mg of PIP were dissolved in 5 ml methanol in 50 ml volumetric flasks, stirred well and the volume was made with distilled water. The solutions were filtered through Whatman filter paper No. 41, suitably diluted and analyzed spectrophotometrically at 341.6nm.


Saturation solubility studies:

For saturation solubility studies, the method described by Higuchi and Connors25 was followed. Excess quantity of pure PIP and all the prepared inclusion complexes were added to conical flasks each containing 10ml of distilled water and sealed. The mixtures were subjected to shaking in rotary flask shaker (Lab HOSP – Shaker) for 24 h at room temperature to attain equilibrium. Appropriate fractions were withdrawn and filtered through Whatman filter paper No. 41 and analyzed spectrophotometrically at 341.6nm.


In-vitro dissolution study:

The dissolution study of pure PIP and the prepared complexes was performed in 900 ml of distilled water maintained at 37±0.5oC, using USP Type II dissolution apparatus (Electrolab TDT-061, Mumbai, Maharashtra, India). The stirring speed of the paddle was set at 50 rpm. The powder sample equivalent to 10 mg of PIP was clamped in between the infusion filter paper and introduced into the dissolution medium. The aliquots were withdrawn at 5, 10, 20, 30, 40, 50 and 60 min, respectively, followed by filtration. The filtrates were subjected to the spectrophotometric analysis (lmax = 341.6nm) for determination of the amount of PIP in the dissolution medium.18,29,30


Anti-inflammatory activity:

The anti-inflammatory activity of PIP and its CD complexes was evaluated in vivo as per the approval from Institutional Animal Ethics Committee (IAEC) of College of Pharmacy, Bhor, Maharashtra, India (Approval No. RDCOP/IAEC/Approval/2016-17/03 dated 08/08/2016). Carrageenan-induced rat paw edema method was used as reported previously2,3. The Wistar albino rats, 150-180g. of either sex were randomly divided into four groups of six rats in each. To produce acute inflammation, 0.1ml of 1% freshly prepared Carrageenan solution in normal saline was injected in right hind paw of rats by sub-plantar route. Control group rats were treated with normal saline, reference standard group with indomethacin (10 mg/Kg body weight per oral), third group with pure PIP 10 mg/ Kg body weight per oral and the test group with PIP/HPBCD/POLO (FHTPO) complex; quantity equivalent to 10 mg of PIP/Kg body weight per oral respectively one hour prior to carrageenan injection. The paw volume was measured using plethysmometer at an interval of 1, 2, 3, 4 and 5 hrs after Carrageenan injection and increase in mean paw volume in ml (Mean ± Standard Error) and % inhibition of paw inflammation was calculated.


The percentage inhibition of paw inflammation was calculated using the formula:


                                                   (Control mean – Treated mean)

% Inhibition of inflammation= ––––––––––––––––––––––––––×100

                                                               Control mean


Statistical Analysis:

All values were shown as Mean ± Standard Error Mean (S.E.M.). Statistical analysis was performed using one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.



Figure 1. Phase solubility diagram of PIP-βCD (a) and PIP-HPβCD (b) system in absence and presence of 0.5% PVP and POLO.



Phase solubility studies and interaction of PIP with CDs

The phase solubility curves of PIP in aqueous solutions of βCD and HPβCD in the presence or absence of an auxiliary substance 0.5% w/v POLO or 0.5% w/v PVP are shown in Fig. 1 (a and b). A linear relationship (AL-type) was observed between the concentration of dissolved PIP and concentration of aqueous solutions of CDs. The slopes of the phase solubility diagrams were less than unit indicating establishment of 1:1 stoichiometry in between PIP and CDs28.


The results obtained regarding Gibbs free energy change, stability constants, regression coefficients, slope of the equation and complexation efficiency are presented in Table 1 and Table 2.


Amongst the binary systems, PIP-HPβCD showed high value of Kc and complexation efficiency. This indicates that PIP showed greater affinity towards HPβCD possibly due to high solubility of HPβCD than βCD and better complexing property31. The incorporation of POLO and PVP to the complexation media resulted in increase in the slope and complexation efficiency of both the CDs towards PIP. The value of Kc increased with addition of PVP to the PIP-βCD and PIP-HPβCD systems indicating increase in the stability of the complex formed. On addition of POLO, Kc increased in case of PIP-βCD system but decreased for PIP-HPβCD system. This may be due to displacement of PIP by POLO, from the cavity of HPβCD. POLO is known to form pseudo-polyrotaxanes with CDs which can reduce the stability of PIP-HPβCD complex.32,33 Despite of decrease in Kc, the solubility of PIP was found to be enhanced to a greater extent in presence of HPβCD and POLO.


Table 1. Phase solubility data of binary and ternary inclusion complexes of PIP with βCD and HPβCD.

PIP/CD systems

Phase solubility parameters


Kc,1:1 (M-1)






PIP/βCD/PVP (0.5%w/w)




PIP/βCD/POLO (0.5%w/w)








PIP/HPβCD/PVP (0.5%w/w)




PIP/HPβCD/POLO (0.5%w/w)




Kc,1:1: Stability constant; PIP: piperine; βCD: β-cyclodextrin; HPβCD: hydroxypropyl-β-cyclodextrin; PVP: polyvinylpyrrolidone K30; POLO: poloxamer


As the concentration of CDs increased the value of Gtr was found to be decreased. The change in the values of Gtr with respect to increase in the CD concentration indicated the spontaneous nature of PIP solubilization.


Table 2. Gibbs free energy change) in Joules/ mole.

Moles of CDs



PVP (0.5%w/w)











































CDs: cyclodextrins; PIP: piperine; βCD: β-cyclodextrin; HPβCD: hydroxypropyl-β-cyclodextrin; PVP: polyvinylpyrrolidone K30; POLO: poloxamer


This clearly suggests that the reaction became more favorable with the increase in the concentration of CDs. The Gtr values indicated the exothermic nature of the complexation process which became more favorable with the addition of POLO and PVP. The highest increase in the solubilizing efficiency of PIP- HPβCD-POLO system can be attributed to the formation of nano-aggregates comprised of HPβCD and POLO27.


Characterization of the inclusion complexes:

DSC analysis:

DSC analysis is widely used to ascertain the interaction between CDs and drug molecules by comparing their individual thermograms with that of inclusion complexes. The interaction usually results in either shifting of endothermic peaks to a different temperature or their disappearance34. DSC patterns of PIP and all systems are shown in Fig. 2A. The thermogram of PIP showed a characteristic endothermic peak at 133.52oC close to its melting point 131.0oC indicating presence of crystalline phase and decomposition was observed at 339.51oC. The DSC thermogram of βCD displayed a broad endotherm at 99.95oC, due to loss of water of hydration. A melting endotherm is observed at 335.32oC. In case of HPβCD, a broad endothermic peak at 75.48oC, corresponding to a loss of water due to dehydration process, was observed35. It shows melting endotherm at 366.62oC corresponding to its melting point.


The DSC thermogram of PVP showed a broad endotherm between 70oC to 100oC because of the presence of residual moisture; while a sharp endothermic peak at 60.28oC suggests crystalline nature of POLO.


Figure 2. DSC thermograms (A) and ATR-FTIR (B) of PIP, βCD, HPβCD, PVP, POLO, binary and ternary complexes.


In the βCD binary complex system of PIP the melting endotherm of PIP is found to be diffused at 130.09oC., while the melting endotherm of βCD is shifted to 352.48oC. The thermogram of HPβCD binary complex of PIP showed diffused endotherm at 130.09oC and the melting endotherm of HPβCD is shifted to 368.12oC. This clearly indicates the formation of inclusion complex in between PIP and CDs.


In the ternary systems FBTP and FBTPO, the characteristic endothermic peak of PIP was completely diffused at 128.46oC and 129.32oC, respectively. Similarly, the ternary systems FHTP and FHTPO showed diffused endothermic peaks at 128.18oC and 131.61oC. An increase in the diffusivity of the endothermic peaks of the ternary systems revealed the involvement of PVP and POLO in stabilizing the binary inclusion complexes via hydrogen bonding25,36.


ATR-FTIR analysis:

ATR-FTIR was used to ascertain the probable interaction between PIP and CDs in the presence or absence of auxiliary substances. The IR spectra of all samples are shown in Fig. 2B. The spectrum of PIP showed principle absorption peaks at 3731.92 cm−1, 3647.53 cm−1 (aromatic C-H stretching), 1628.13 cm−1, 1578.05 cm−1,and 1486.27 cm−1 (symmetric and asymmetric stretching of conjugated diene C=C), 1437.39 cm−1(CH2 bending) 1247.58 cm−1 (asymmetric stretching of =C-O-C), 1126.20 cm−1 (in plane bending of phenyl C-H), 1023.98 cm−1 (symmetrical stretching of =C-O-C), 994.52 cm−1 (C-H bending of trans –CH=CH), and 921.95 cm−1 ( C-O stretching ) 8,37.


The peaks of βCD were recorded at 3276.49 cm−1 (O-H stretch), 1152.61 cm−1, 1078.03 cm−1, 1023.99 cm−1 (C- O-C stretch), 997.86 cm−1, 937.10 cm−1. While absorption peaks of HPβCD were observed at 3737.23 cm−1, 3348.45 cm−1 (O-H, stretch), 2929.2 cm−1 (C- H, Stretch), 1026.41 cm−1 (C- O –C stretch).


IR spectrum of POLO shows distinct peaks at 2878.80 cm-1(C-H stretching aliphatic), 1446.23 cm-1, 1341.71 cm-1 (in plane O-H bending) 1279.23, 1241.06, 1100.17 cm-1 (C-O stretch). In case of PVP, the principle peaks at 1644.61 cm-1 (C=O stretching), 1422.28 cm-1, 1281.05 cm-1 and a broad peak between 3000 to 3700 cm-1 (O-H stretching vibrations of absorbed water) were obtained.


Alterations in the characteristic IR bands of PIP and CDs were noted in case of all binary and ternary systems. In case of PIP-βCD binary system, the peaks of PIP at 1247.58 cm-1, 1189.84 cm-1 and 1126.20 cm-1, were shifted to 1250.91 cm-1, 1156.24 cm-1 and 1134.05 cm-1 respectively while all other peaks were disappeared. Broad peak of βCD at 3276.49 cm-1was disappeared, while peaks at 1078.03 cm-1 and 1023.99 cm-1 and 937.10 cm-1 were shifted to 1080.26 cm-1 and 1028.15 cm-1 and 935.97 cm-1 resp. For PIP-HPβCD complex, the principal peaks of PIP were either disappeared or smoothened, while peaks at 3731.92 cm-1, 3647.53 cm-1 and 1247.58 cm-1, were shifted to 3733.53 cm-1, 3648.72 cm-1 and 1250.47 cm-1 respectively. The peaks of HPβCD at 1149.44 cm-1 and 1026.41 cm-1 were shifted to 1152.12 cm-1 and 1030.72 cm-1 respectively, while peaks at 1364.89 cm-1 and 1329.57 cm-1 disappeared completely.


For ternary system with βCD and PVP (FBTP), the principle peaks of PIP disappeared completely or smoothened; while peaks of βCD at 1152.61 cm-1, 1078.03 cm-1 and 1023.99 cm-1, were shifted to 1154.51 cm-1, 1080.15 cm-1 and 1027.38 cm-1 respectively. In case of ternary system with βCD and POLO (FBTPO), the peaks of PIP disappeared completely while peaks of βCD at 1152.61 cm-1, 1078.03 cm-1, and 1023.99 cm-1 were shifted to 1155.12 cm-1, 1080.08 cm-1 and 1026.76 cm-1 respectively. In case of ternary system with HPβCD and PVP (FHTP), the principle peaks of PIP have either disappeared completely or smoothened or appeared with decreased intensity; while peaks of HPβCD at 1149.44 cm-1, 1026.41 cm−1 and 940.65 cm−1 were shifted to 1152.16 cm−1 and 1029.35 cm−1 and 945.22 cm−1 respectively. For ternary system with HPβCD and POLO (FHTPO), the peaks of PIP at 3731.92 cm-1, 1513.94 cm-1 were shifted to 3735.04 cm-1 and 1513.73 cm-1 respectively. All other peaks had disappeared. The broad peak at 3348.45 cm-1 of HPβCD had disappeared completely while peaks at 1026.41 cm-1 and 1149.44 cm-1 were shifted to 1030.32 cm-1 and 1149.66 cm-1, respectively.


The peaks in the spectra of complexes were observed to be either shifted to different wavelengths or smoothened or appeared with decreased intensity with no any new peak was observed indicating non covalent interaction in inclusion complexes, more specifically, the ternary complexes38.


XRPD analysis:

Fig. 3 presents XRPD patterns of pure PIP, βCD, HPβCD, PVP, and POLO as well as of all the prepared inclusion complexes. The diffraction pattern of PIP exhibited sharp peaks at 14.2, 14.7, 14.8, 14.9, 22.3 and 25.8 (2θ0) with peak intensities, 982, ,1954, 2315, ,1504, 680, and 911 respectively. The peak intensities for βCD at 2θ0 equivalent to 12.6, 12.7, 17.2, 18.9, 20.9, 23.0 and 27.2 recorded were 1689, 1751, 1371, 2025, 1577, 1329 and 1459 respectively. This reveals the crystalline nature of PIP and βCD. The diffractogram of HPβCD exhibited halo pattern indicating its amorphous nature. It showed peaks at 12.1, 17.2, 16.2, 18.3, 21, and 21.8 with peak intensities 798, 1462, 1090, 1786, 1087, 1087 and 1120 respectively. The intensity of the characteristic peaks of PIP in the diffractogram of FBB and FBH was found to be reduced. A marked reduction in the PIP peak intensities was observed in case of FBTP, FBTPO, FHTP and FHTPO. This suggests that HPβCD based binary and ternary complexes exhibit maximum amorphization.


Figure 3. XRPD pattern of PIP, βCD, HPβCD, PVP, POLO, binary and ternary complexes.


Percentage drug content and saturation solubility studies:

The percentage drug content and saturation solubility for PIP and all the complexes is presented in Table 3.


Table 3. Percentage drug content, saturation solubility and DE of the complexes.

PIP/CD complexes

Drug content (%)*

Solubility in water at 25oC mg/mL*


Pure PIP


0.0112± 0.0004



98.15 ±2.58
















77.56 ± 0.94







*indicates mean of 3 readings ± standard deviation; ! indicates significant value (p<0.05) as compared to pure PIP; w indicates significant value (p<0.05) as compared to FBB; x indicates significant value (p<0.05) as compared to FBTP; y indicates significant value (p<0.05) as compared to FBTPO; z indicates significant value (p<0.05) as compared to FHB. DE- dissolution efficiency.


The uniformity of drug content was observed in all the complexes. All the binary and ternary complexes exhibited an enhancement in solubility with respect to pure drug. The ternary inclusion complex of PIP with HPβCD and POLO exhibited the highest solubility. This can be attributed to the amorphization of PIP due to the formation of PIP-HPβCD inclusion complexes along with formation of HPβCD-POLO nano-aggregates which can encapsulate the non-complexed PIP27.


In-vitro dissolution of PIP and freeze-dried complexes:

The effect of formation of multicomponent inclusion complexes of PIP on its dissolution behavior was studied by performing in-vitro dissolution study of PIP in distilled water. The dissolution profile of PIP and the prepared complexes is shown in Fig. 4A. At the end of 60 min, only 8.37±0.39% of PIP was found to be dissolved. Although an increase was observed in the dissolution of PIP in case of FBB and FBTP, this increase was insignificant (p>0.05).


Figure 4. Dissolution profile of pure PIP and the freeze-dried complexes in distilled water (A) and Anti-inflammatory activity of PIP by Carrageenan-induced rat paw edema method.


#: significant value (p<0.05) as compared to carrageenan; †: significant value (p<0.01) as compared to carrageenan; ‡: significant value (p<0.05) as compared to PIP.

The binary and ternary complexes comprised of HPβCD (FHB, FHTP and FHTPO) showed marked improvement (p<0.05) in the dissolution of PIP when compared with the dissolution profile of pure PIP. The amount of PIP dissolved at the end of 60 min was found to be high in case of FHTPO. Also, the percent dissolution efficiency (%DE) of FHTPO was maximum as compared to the other complexes (see Table 3). FHTPO exhibited significant improvement (p<0.05) in dissolution of PIP as compared to the complexes based on βCD. This can be attributed to the maximum solubility of FHTPO, as shown in Table 3.


Anti-inflammatory activity:

The results for anti- inflammatory activity with respect to change in paw volume and % inhibition of paw inflammation (Mean±Std. Error) by using Carrageenan-induced rat paw edema method are given in Fig. 4B. The positive control i.e. indomethacin decreased the paw volume by 50.74% after five hours. The percent inhibition of paw volume in case of FHTPO is 49.67%which is more than that of pure PIP (24.67 %).


This confirms the enhancement in the bioavailability of PIP due to improvement in its solubility and formation of stable inclusion complex between PIP and HPβCD in presence of POLO.



In this study, the lyophilization technique was successfully employed for the preparation of binary and ternary complexes of PIP with both βCD and HPβCD. The synergistic effect of POLO and PVP in the formation ternary inclusion compounds of PIP with both the CDs effected modifications in physicochemical properties of pure PIP. An enhancement in solubility and improvement in dissolution of PIP was in regard to the overall effect of complexation, use of auxiliary substance and development of amorphous phase achieved through lyophilization technology. The physicochemical and characterization studies support the results. The increased in vivo anti-inflammatory activity of prepared complexes confirms the formation of stable complexes showing increased bioavailability as compared to pure PIP. Thus stable multicomponent inclusion complexes of PIP with both CDs in the presence or absence of PVP and POLO can be obtained through lyophilization technique, resulting in improved physicochemical properties with increased anti-inflammatory activity as compared to pure drug. HPβCD ternary system with 0.5% w/w POLO exhibited better performance among all the systems.



The authors are thankful to Shivaji University, Kolhapur and Savitribai Phule Pune University, Pune for providing analytical facilities to perform characterization studies. The authors are thankful to the Principal, Government College of Pharmacy, Karad for providing necessary laboratory facilities and to the Principal, Rajgad Dnyanpeeth’s College of Pharmacy, Bhor, Dist. Pune for providing necessary facilities for animal studies. The authors gratefully acknowledge Gangwal Chemicals, Mumbai for providing gift sample of HPβCD.



The authors declared no conflict of interest.



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Received on 26.06.2019           Modified on 19.11.2019

Accepted on 24.04.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(10):4916-4924.

DOI: 10.5958/0974-360X.2020.00864.1