Fabrication of Apigenin loaded gellan gum–chitosan hydrogels (GGCH-HGs) for effective diabetic wound healing

International Journal of Biological Macromolecules 91 (2016) 1110–1119

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Fabrication of Apigenin loaded gellan gum–chitosan hydrogels (GGCH-HGs) for effective diabetic wound healing Rajesh Shukla a , Sushil K. Kashaw b,∗ , Alok Pal Jain c , Santram Lodhi d a

Department of Pharmaceutical Science, Suresh Gyan Vihar University, Jaipur, RJ, India Department of Pharmaceutical Sciences, Dr. H. S. Gour Central University, Sagar, MP, 470003, India Department of Pharmacy, RKDF College of Pharmacy, SRK University, Bhopal, MP, India d Department of Pharmacy, Guru Ramdas Khalsa Institute of Science & Technology, Jabalpur, MP, India b c

a r t i c l e

i n f o

Article history: Received 27 April 2016 Received in revised form 13 June 2016 Accepted 23 June 2016 Available online 23 June 2016 Keywords: Hydrogels Apigenin Morus alba Wound healing Antioxidant

a b s t r a c t The Apigenin (APN) was isolated from ethanolic extract of M. alba leaves and screened by in-vivo wound models (Diabetic and Dead space) in rats. Apigenin loaded hydrogel (HGs) was prepared using gellan gumchitosan (GGCH) with PEG as a cross linker and characterized for various parameter like AFM, swelling property, entrapment efficiency and drug release. Further performance of hydrogel was evaluated by wound healing activity tested against wound contraction, collagen content, dried granuloma weights and antioxidant activity. The percent entrapment efficiency of optimized hydrogel found to be 87.15 ± 1.20. APN loaded GGCH-HGs were able to release 96.11% APN in 24 h. The level of superoxide dismutase (SOD) and catalase were found increased significantly in granuloma tissue of APN treated group. APN GGCHHGs found higher wound healing effect in diabetic as well as normal wound tissues with significant antioxidant activity. Results proven the utility of prepared hydrogel (APN loaded GGCH-HGs) seems to be highly suitable for wound healing due to its unique properties of biocompatibility, biodegradability, moist nature and antioxidant effectiveness. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The replacement of injured epidermal tissues by structural and functional integrity is the process of wound healing. It involves healing process of wounded tissue by inflammation, proliferation and migration of connective tissue cells, production of extracellular matrix including collagen synthesis, epithelial cells migration and proliferation most important to neovascularization [1]. Imperfect collagen metabolism in diabetics is considered as a factor in delayed wound healing. Injured endothelial with potential occlusion of capillary vessels as well as hyperglycemia-induced leukocyte dysfunction, phagocytosis and decreased chemotaxis resulting in impaired wound healing. Wound healing in diabetes is impaired by many factors that results thickening of the basement membrane of the capillaries and arterioles. It is frequently occurs in individuals with diabetes, resulting in an impaired wound healing and pushy ulcer formation [2]. It has been reported that hyperglycaemia produce a deleterious effect on wound healing through the formation of

∗ Corresponding author. E-mail address: [email protected] (S.K. Kashaw). http://dx.doi.org/10.1016/j.ijbiomac.2016.06.075 0141-8130/© 2016 Elsevier B.V. All rights reserved.

advanced glycation end-products which induce the production of inflammatory molecules (TNF-, IL-1) and interfere with collagen synthesis [3]. In addition, the presence of high glucose level also changes to cellular morphology, granulation tissue was deficient in collagen, decreased proliferation, and abnormal differentiation of keratinocytes [2,4]. Morus alba Linn. (Moraceae) is commonly known as white Mulberry. In Ayurvedic system of medicine, it has been used by the tribals for ailments such as asthma, cough, bronchitis, edema, sleeplessness, diabetes, influenza, eye infections and nose bleeds [5]. Leaves of M. alba have been reported by various researchers for antihyperglycemic [6,7], antioxidant [8], anticancer [9], free radical scavenger, antibacterial [10], anthelmintic and antiulcer [11] activities. Its leaves contain fixed oil, carbohydrate, protein, tannin [5], alkaloids [12], flavonoids i.e. apigenin, quercetin and rutin [13], glycosides [14] and saponin. The various types of formulation have been used for potential wound healing like ointment, creams, nanoparticles, patches, wound dressings and recently very popular hydrogels (HGs). Apigenin (APN), is a bioactive flavones under the category of flavonoid. It is found in a wide variety of plants and vegetables. It has various biomedical applications especially for antiproliferative, antiviral, antioxidant, antibacterial, wound healing and anti-inflammatory activity [15,16]. A study reported that

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administration of apigenin to alloxan-induced diabetic mice ameliorated hyperglycemic and improved antioxidants such as CAT, SOD and GSH content [17]. In addition, Suh et al. [18] investigated that apigenin attenuates dRib induced cell damage in pancreatic ␤-cells via oxidative stress-related signaling. Apigenin also preserves the cellular structure of vital tissues towards normal in STZ-induced diabetic rats. Furthermore, apigenin could enhanced GLUT4 translocation and have glucose lowering as well as ␤-cell preserving efficacy [19,20]. However, the limitation of APN is its poor water solubility and low bioavailability [21]. Therefore, it is necessary to develop new drug delivery system or formulations to improve APN bioavailability. The solubility and dissolution rate of poorly water-soluble drugs can be improved by reducing particle size, enhancing wettability and porosity. There are many formulations fro APN are reported by researchers like liposomal, PLGA nanoparticles, polymeric micelles, and lipid nanocapsules [21]. Hydrogel (HGs) is the networks of three-dimensional polymer which swollen by huge amounts of a solvent. The HGs have flexible, rubbery and soft texture, resembles on human tissues with influential contention as wound application [22]. In addition, they kept the wound environment moist because of their excellent water absorption properties [23]. Hydrogels offer convenient drug delivery matrices composed of natural polymers or crosslinked synthetic macromolecules (e.g. polyethylene glycol, polyvinyl alcohol), which offer biocompatibility and desirable physical properties [24]. However, to overcome undesired burst release of drugs, polymer was shrinkage post-crosslinking. In addition, these systems also facilitate wound healing, allowing cell infiltration and retention of viability. Such materials can also be used to hydrophobic drugs for controlled release and entrap bioactive molecules. Gellan gum is exploited in the field of controlled release of bioactive molecules. Interpenetrating polymer networks or cocrosslinked polymer networks based on gellan gum and other polysaccharide systems have also been developed as drug delivery matrices [25]. Chemical hydrogels of Gellan gum are prepared via chemical crosslinking of preformed physical networks, to enhance their mechanical properties, and to obtain slower drug release. Chitosan acts as a penetration enhancer by opening epithelial tight-junctions. Due to its positive charges at physiological pH, chitosan work as bioadhesive, this increases retention at the site of application. Chitosan also promotes wound-healing and has bacteriostatic effects [26]. Due to very abundant and low cost production, selected chitosan is ecologically interesting. In present study, chemical hydrogels are formed by irreversible covalent crosslinked chitosan hydrogels. Hydrogel has appropriate physical and mechanical properties to prevent secondary infection and outstanding physiological environment to facilitate cell adhesion, proliferation and differentiation. Thus hydrogels are particularly advantageous because they can prevent water loss and secondary infection, absorb wound fluids and provide adequate gaseous exchange. Due to the amorphous structure of the Gellan gum-Chitosan (GGCH) Hydrogel (HGs) can take water up to 99% its own weight [27]. The apigenin was isolated from ethanol extracts of M. alba leaves by column chromatography and spectroscopic methods. Accordingly, the present study was aimed to prepare, characterize and investigate, gellan gum-chitosan polymer based apigenin loaded HGs for chronic wound healing (especially in diabetic) and antioxidant properties.

2. Materials and methods 2.1. Chemicals and reagents Gellan gum and chitosan were purchased from Otto Chemie pvt. Ltd., Mumbai, India. Standard Streptozotocin was purchased from

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Sigma-Aldrich (USA). Ethyl acetate, petroleum ether, silica, benzene, chloroform, DMSO (Dimethyl sulfoxide), PEG (Poly ethylene glycol), EDAC (1-ethyl-3-(3-dimethylaminopropyl)) carbodiimide hydrochloride and NHS (N-Hydroxysuccinimide) was purchased from HiMedia Lab, Mumbai, India and Methanol purchased from Merck Ltd, New Delhi, India.

2.2. Extraction and separation of Apigenin The dreid powdered leaves (1.0 kg) of M. alba taken for extraction and defatted with petroleum ether and extracted with ethyl alcohol for 72 h. Solvent recovered by distillation and remaining solvent was removed under vacuum. The semisolid ethanol extract was suspended in distilled water and extracted with ethyl acetate in a separating funnel repeatedly. Ethyl acetate M. alba fraction (MAF) was dried under vacuum produced (2.6% w/w) a brown powdered product that tested for presence of flavonoids. The MAF was subjected to separate flavonoid by column chromatography. By suspending silica (60–120 mesh; Merck) for 24 h in benzene, the stationary phase Silica bed was prepared. The slurry was loaded in flash glass chromatographic column (35 cm × 1.5 cm) and bed (final geometry 28 cm × 1.5 cm) was allowed to settle. A sample of 2.0 g MAF fraction was prepared and transfer in a column. The elution was performed by using the 100% benzene and 20 eluates of 5 ml each were collected. The TLC of each fraction was performed by using the benzene:chloroform:methanol (14:16:2) solvent system. The fraction12 to 20 were referred to as compound 1 because of a single spot observed in the TLC profile. Re-crystallized the compounds (0.63 mg) in methanol and characterized by spectroscopic techniques [28]. The isolated pure compound was collected and the residue was dissolved in methanol for identified by UV, infra red (IR), proton nuclear magnetic resonance (1H NMR), and mass spectroscopy (MS). UV analysis was performed on a double-beam Agilent Technology UV-1620 CARY (USA). IR spectra were recorded on a spectrum 100 FT-IR spectrometer supplied (Cary- 630 FTIR, Agilent Technologies USA) with Attenuated Total Reflectance (ATR). The 1 HNMR analysis done by a FT-NMR spectrometer 400 MHz (Bruker) using deuterated chloroform (CDCl3 ) as solvent and TMS (tetramethylsilane) as internal standard, ESI–MS spectrum was taken at 70 eV on a Water MSD Ion Trap XCT instrument (Japan).

2.3. Preparation of gellan gum-PEG-chitosan hydrogels (GGCH-HGs) Gellan gum (50 mg) was dissolved in 10 ml of distilled water and then fivefold excess 250 mg of bifunctional PEG (COOH-PEGNH2 ) was added with continuous stirring, followed by addition of 250 mg of EDAC and 50 mg NHS. The reaction was performed at 110 ◦ C for 2 h. Chitosan (50 mg) was dissolved in 10 ml of water, and then chitosan solution was added to the activated polymeric solution of gellan gum-PEG-COOH with continuous stirring. Apigenin (30 mg) was added into Gellan gum-PEG-Chitosan dispersion and magnetically stirred (Remi, Mumbai, India) for 12 h. The reaction mixture was extensively dialyzed (MWCO 12–14 kDa) to separate GG-PEG-Chitosan from unreacted GG and chitosan [29]. The obtained copolymer named GGCH was dried under vacuum in lyophilizer (Tanco Laboratory Equipment, PG-302). Synthesis of GGCH was confirmed by IR and NMR spectroscopy. IR Spectra of GGCH were recorded with a FT-IR spectrophotometer (Cary- 630 FTIR, Agilent Technologies USA). Nuclear Magnetic Resonance (NMR, Bruker DRX, USA) spectroscopy of GGCH was performed at 300 MHz, after dissolving in DMSO. The flowchart (Fig. 1) represents the formulation of GGCH-HGs.

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HO

HO

HO

OH

O

O

O

O

O

OH

OH

OH

OH

CH2OH

COOH

CH2OH

CH3

Gellan Gum O NHS

EDAC

+

O

H 2N

O

OH

n

Amine-PEG-COOH °C Temp. 30-120 pH 4.5- 6.0

HO O

O

O

CH2OH

CO

CH2OH

CH3

OH

O

HO

HO

O

OH

OH

OH

OH

HN

O

O O

OH

n

Gellan Gum-PEG-COOH Complex

Mix with chitosan solution OH

OH O

O O

HO

N H

O

CO CH3

HO

NH2

Chitosan

HO

HO O

O OH O

HO O

O

OH

O O

O

CO O

NH

O HO

O

CH2OH

CH2 OH

CH3

OH

OH

OH

OH

OH

N H

CO CH3

HO

N H

O

Gellan gum-PEG-Chitosan Fig. 1. Flowchart for development of GGCH-HGs.

2.4. In vitro characterization 2.4.1. Atomic force microscopic (AFM) The shape and surface topography of GGCH-HGs were determined by using AIST-NT Smart SPM 1000, CA atomic force microscope (AFM). AFM of the HGs was carried out at glass substrate in AC mode to characterize the surface roughness which is regarded as one of the most vital surface properties that plays a significant role in membrane permeability and abhorrent behavior.

2.4.2. Swelling measurements The swelling property was carried out by immersing an accurate weighed quantity of dry sample in PBS solution (pH 7.4) at a given time interval. The excess of water was removed using filter paper and highly swollen samples weight was taken. Same experiment was repeated for three times, and the average value was noted as swelling ratio. The degree of swelling was calculated from the following formula [30]. DS = [

WW − Wd ] × 100 Wd

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In the above formula Ww and Wd are the weights of wet and dry Hydrogel respectively. 2.4.3. Entrapment efficiency The drug (APN) entrapped per gram of the sample was determined by pre-weighed piece of completely dry HGs. The dry sample was placed in 25 ml of aqueous drug solution containing different drug concentration and equilibrated for a period of 24 h to ensure equilibrium loading. Further, the quantity of entrapped drug was analyzed with HPLC (Agilent Technologies, 1220 infinity LS, UK) method. The HPLC analysis was done by variable wavelength detector, a zorbax 5 ␮ C18 column (250 × 4.60 mm) was used for the sample analysis at 334 nm. The mobile phase was water: methanol (95:5 v/v) pumped at a flow rate of 1 ml/min at 25◦ C. The amount of drug entrapped per gram of HGs s was determined using the expression: Drug entrapment =

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formation on implanted tube was dissected out on the ninth postwounding day. Dry granuloma tissue weight from each tube was determined and a part was stored in 10% formalin for the biochemical assay [32]. 2.6. Wound healing evaluation 2.6.1. Wound contraction measurement Wound contraction is the reduction rate of unhealed area throughout the healing process. Thus, the fast rate of wound closer indicates the better efficacy of drug. An excision wound margin was traced after wound creation by using transparent paper. The percent wound contraction was measured in each 2 days interval, until complete healing and expressed in percentage of healed

Initial drug content in solution − Final drug content in solution ␮g/g Weight of dry sample

2.4.4. Drug release Accurately weighed quantity of drug loaded HGs was placed in 25 ml of physiological fluid at 37 ◦ C. The samples (0.5 ml) were withdrawn at 1–8, 10, 12 and 24 h. The amount of drug released at different time intervals was analyzed by and chromatographic conditions were same as mentioned in entrapment study. Calibration curve was prepared for the drug solutions of known concentrations in the appropriate range, to determine the amount of drug released. 2.5. In-vivo wound healing study 2.5.1. Animals grouping Inbred house wistar rats (200–250 g) of either sex were acclimatized to laboratory hygienic circumstances for 10 days early of the experiment. The animals were fed commercial pellet diet (Hindustan Lever Pvt, Bangalore, India), and water ad libitum. In vivo study was performed at Guru Ramdas Khalsa Institute of Science and Technology (Pharmacy) Barela, Jabalpur (MP) with prior approval from Institutional Animal Ethical Committee (Registration No. 1471/PO/a/11/CPCSEA, India). Animals were divided into three groups for each model consisting of six animals each group. The control group received vehicle (Simple GGCH-HGs), test group received APN loaded GGCH-HGs and reference group received marketed formulation Betadin® (Win-Medicare Pvt. Ltd., New Delhi, India). All treatments were given topically. 2.5.2. Diabetic wound creation Streptozotocin was prepared in cold citrate buffer (0.1 M, pH 4.5) for intraperitoneal administration after overnight fasting. The streptozotocin (60 mg/kg) was injected in experiment animals for induction of diabetic condition six weeks prior. For blood glucose measurement blood was drawn from the tail vein and estimated by using the glucometer (CONTEC BC 300 Auto analyzer) after three days later. A circular excision wound was made on dorsal portion of rats showing elevated blood glucose (more than 135 mg/dL) [31]. Blood glucose level was estimate at the time of wounds creation and after treatment. Wound contraction, antioxidant level and histopathology study were performed in diabetic wound model. 2.5.3. Dead space wound creation Study of granuloma tissue formation in dead space wound model was done for the determination of dry granulation weight and estimation of biochemical parameters. Animals were anaesthetized by light ether before wound creation. A wound was made by implantation of a polypropylene tube (2.0 × 0.5), in the lumber region, at the dorsal portion of each animal. Granuloma tissue

wound area. The epithelialization time was measured from preliminary day [33]. The percentage wound contraction was calculated as given below formula: Percent wound contraction =

healed area × 100 total area

2.6.2. Collagen content measurement Wound tissues were analyzed on 18th day for hydroxyproline content that is a basic constituent of collagen. Tissues were dried at 60–70 ◦ C up to constant weight and samples were hydrolyzed with 6N HCl for 4 h at 130 ◦ C. The hydrolysate was neutralized (pH 7) then subjected to Chloramine-T oxidation for 20 min. The colored adduct formed with Ehrlich reagent at 60 ◦ C [34] was read at 557 nm. Standard hydroxyproline was also run and values reported as ␮g/mg dry weight of tissue. 2.6.3. Protein estimation and granuloma weight On the post wounding days 18th the protein content of skin tissues were determined by method of Lowry et al. [35]. The tissue lysate was treated with a mixture of sodium tartrate, copper sulphate and sodium carbonate. The mixture was left to stand for 10 min and then treated with Folin-Ciocalteau reagent which gives a bluish color in 20–30 min. The absorbance was taken at 650 nm using Spectrophotometer. 2.6.4. Estimation of antioxidants level in skin tissues The granuloma tissues were collected from full thickness wound and analysed for antioxidants assay. Catalase was estimated following the breakdown of hydrogen peroxide. Superoxide dismutase (SOD) assay was based on the inhibition of epinephrine autoxidation by the enzyme. Reduced glutathione (GSH) level was determined by method of Moron et al. [36] Tissue homogenates were immediately precipitated with 0.1 ml of 25% TCA and separated by centrifugation. The assay of free-SH groups in 3 ml of sample done by the addition of 2 ml of 0.6 mM DTNB and 0.9 ml 0.2 mM sodium phosphate buffer (pH 8.0) to 0.1 ml of the supernatant and the absorbance was read at 412 nm using a UV spectrophotometer. 2.6.5. Histopathological studies Animals were anaesthetized before taking skin sample using diethyl ether. On 18th day wound tissue sample from each group were collected. Samples were fixed in 10% buffered formalin, processed, blocked with paraffin and then cut into 6 ␮m thickness

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Fig. 2. FTIR spectra of the isolated compound from ethanolic extract M. alba.

sections and stained with hematoxylin & eosin (HE) stains [37]. The tissues were examined by light microscope.

2.7. Statistical analysis Data are presented as the mean ± standard deviation. Treated groups were compared with the standard group. The results were analyzed statistically using Student’s t-test for the comparison. The data were considered significant at p < 0.01.

3. Results and discussion 3.1. Separation of Apigenin Phytochemical work revealed that ethanolic extract of M. alba leaves contains flavonoids including apigenin which have been studied for antibacterial activity [38], hypoglycemic activity [10] and potent antioxidant. Separated compound was yellowish-green needle-shaped crystals, soluble in acetone and methanol, with 108–110 ◦ C melting point. The FTIR spectroscopic results confirmed the absorption band of CH2 group found at 2958 cm−1 , and bending vibration approximately at 1460 cm−1 . In the spectrum of OH vibration, at approximately 3574 cm−1 and others at 3096 cm−1 that are most probably of vibration of phenol OH group. The intensive band at 1648 cm−1 is most probably the result of C O vibration of group from central heterocyclic ring, while the C O vibration occurs at 1124 cm−1 (Fig. 2). The 1H NMR spectrum exhibited a broad characteristic signal of aromatic hydroxyl groups at 8.94-10.58 (2H, 4 and 7-H), 12.83 (1H, singlet, 5-H). The other protons signal at ı 7.83 (2H, double doublet, J = 8.89 Hz), ı 7.23 (2H triplet, J = 7.96), ı 6.87 (1H singlet, J = 8.85), ı 6.87 (1H double, J = 3.21), ı 6.13 (1H double, J = 2.43) were found (Fig. 3). The molecular ion peak (m/z) 270.81 calculated against 270 m/z for MH+ of apigenin (C15 H10 O5 ). By comparison of the spectral data with those of 5,7-dihydroxy-2(4-methoxyphenyl) chromen-4-one, compound was identified as apigenin [33].

Table 1 Entrapment efficiency of optimized GGCH, GG and CH hydrogels. S. No.

Formulation

% Entrapment Efficiency

1 2 3

GGCH-HGs CH-HGs GG-HGs

87.15 ± 1.20 79.08 ± 1.15 77.19 ± 0.5

3.2. In-vitro characterization of gellan gum-PEG-chitosan hydrogels (GGCH-HGs) The morphology and shape of GGCH HGs was visualized under AFM and HGs were found in micrometric size range having gelly structure shown in Fig. 4(A and B). Photomicrograph showed the tapping mode atomic force microscopic images of freshly prepared GGCH-HGs. Using the AFM, GGCH-HGs were visualized and found to be relatively constant height, which revealed the size of HGs. Unlike other microscopic techniques, the AFM offers visualization in three dimensions. The results of atomic force microscopy (AFM) confirm the good qualities of HGs are prepared. The swelling property was studied up to 5 h and the result showed that the GGCH-HGs demonstrated significantly higher water absorption property than the GG and CH-HGs. In each case, the swelling property of the HGs increases with time. The GGCHHGs reaches to equilibrium condition within 3 h, whereas in the case of each GG and CH-HGs the degree of swelling value reaches to equilibrium condition after around 4 h shown in Fig. 5. The swelling behavior of the polymeric HGs is based on the theory that there is a network of chains of polyelectrolyte containing ionizable groups, and the mobile counter ions in the gels develop a large swelling pressure due to some intermolecular non-covalent interaction, such as, hydrogen bonding and polar forces [39]. Thus, the polymeric interactions increase and cause a very high sorption rate. The drug entrapment efficiency of optimized HGs are shown in Table 1. The percent entrapment efficiency of optimized GGCHHGs was found to be 87.15 ± 1.20%. Whereas the entrapment efficiency of, GG-HGs and CH-HGs was found to be 77.19 ± 0.5 and 79.08 ± 1.15% (Table 1) respectively. The drug entrapment efficiencies increased progressively with increasing the drug concentration resulting in the formation of dense and higher entrapping the

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Fig. 3.

1

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H NMR spectra of the isolated compound from ethanolic extract M. alba.

greater amount of the drug. This may be the greater degree of cross-linking by PEG as the amount of APN increased. Consequently, the crosslinking of the polymer and compactness of the formed insoluble dense matrices also increased, resulting in more drug entrapment in the GGCH-HGs. Controlled and sustained drug release offers many advantages over the current delivery methods for skin. Controlled local release systems gives the desired constant drug concentrations at the delivery site, lower systemic drug level and a reduced potential for deleterious side effect [40]. Especially, delivery systems which act as biodegradable polymers do not require removal from the skin at the end of treatment period. As said above, HGs is a promising wound healing material due to its chemical purity, physical and mechanical properties. Fig. 6 shows that the HGs appeared to show rapid initial release of APN followed by constant release over a longer period of time. The release of the APN from the GGCH showed a pronounced time prolongation from GGCH system. GGCH-HGs was able to release 96.11% APN in 24 h whereas plain GG-HGs and CH-HGs able to release 82.67%% and 85.9%% APN in 8 h shown in Fig. 6. This is due to the breaking the strong hydrogen bonding between gallen gum-PEG-chitosan chains and GG-water molecules to release carboxyl groups for the crosslinking reaction with amine group of bifunctional PEG (COOH-PEG-NH2 ). After the consumption of carboxyl groups by PEG, the crosslinked GG polymer chains

become less soluble and formation of HGs. Higher swelling of terminal GG in PBS (pH 7.4) may be attributed for prolonged release and highlighted that the rate of drug release depends on the water content of the swollen HGs [35]. The highly porous structure easily permits the loading of drugs into the gel matrix and subsequent drug release at a rate depending on the diffusion coefficient of the small molecule or macromolecule through the gel network [41]. 3.3. In-vivo study of gellan gum-PEG-chitosan hydrogels (GGCH-HGs) Wound repair is a complex, integrated series of biochemical, cellular and physiological process [33]. Angiogenesis plays an important role in wound healing and newly formed blood vessels comprise 60% of the repair tissue. Angiogenesis helps hypoxic wounds to attain the normoxic conditions. Improved angiogenesis, therefore, would be contributing significantly to wound healing activity of the test drug [34]. This study, introduced novel topical formulation for an efficient wound healing. The utility of natural products has been a major booming strategy for the discovery of new medicines. Number of reports has indicating that flavonoids possess multiple biological effects as well as inhibition for cell migration and contraction [42,43]. Generally, HGs with is helpful as medical biomaterials in tissue engineering

80.46 ± 1.78 100.75 ± 1.78* 100.23 ± 1.29* 71.13 ± 2.03 94.46 ± 1.92* 95.05 ± 1.78* 62.23 ± 2.21 82.48 ± 2.09* 87.52 ± 1.57* 54.21 ± 1.32 69.82 ± 2.72 74.67 ± 2.37 n = 6 albino rats per group, value represents Mean ± S.D * p < 0.01, when compared each treated group with control group

vehicle control 14.47 ± 1.52 Apigenin loaded GGCH-HGs 13.47 ± 1.38 Reference Ointment 15.82 ± 1.43

2

4

29.73 ± 2.07 25.28 ± 1.87 31.48 ± 1.78

6

37.46 ± 2.08 41.50 ± 1.56 46.72 ± 1.59

8

40.89 ± 2.05 48.92 ± 2.04 51.38 ± 1.85

47.52 ± 1.78 58.61 ± 2.12 67.20 ± 2.61

20 18 16 10

12

14

3.3.1. Wound contraction measurement Wound area was measured in each 2 days interval by tracing on a transparent paper. The healed area was calculated by subtracting from the original wound area. On day 6, the wound contraction of APN loaded GGCH-HGs treated groups was found to be significantly (p < 0.01) increased which leading to faster wound healing as confirmed by decreased epithelialization period in comparison to control group. On day 18, APN loaded GGCH-HGs treated group was

Post wounding days (Percent wound contraction)

and wound healing. The results of in-vitro study was supported in agreement with high interconnection network and small pore size HGs which directed for in-vivo wound healing investigations [44].

Groups

Fig. 5. Swelling behaviour of GG, CH and GGCH Hydrogels.

Table 2 Effect of prepared Apigenin loaded GGCH HGs and reference ointment on percent wound contraction area of diabetic wound in rats.

Fig. 4. AFM microphotographs of (A) 3D Hydrogel-network structure (B) pore size of GGCH Hydrogels.

86.25 ± 2.04 23 – 18 – 18

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Epithelia-lization period

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Fig. 6. In-vitro apigenin release from CH, GG and GGCH hydrogels.

almost at complete healing stage, whereas control group showed 86.25% healing on day 20 (shown in Table 2 and Fig. 7). It was also observed that epithelialization period of APN loaded GGCHHGs and standard groups were less in comparison to control group (Table 2). 3.3.2. Collagen content, protein level and granuloma weight measurement In case of dead space wound model, the hydroxylproline level of APN loaded GGCH-HGs group found significantly increased when compared to control group (Table 3). The protein content for APN loaded GGCH-HGs was 71.41 ± 1.43 and reference ointment group was 72.84 ± 1.56 group of animals found significantly greater than control group (45.61 ± 2.32). The granuloma weight for APN loaded GGCH-HGs (36.71 ± 2.05 mg) and reference ointment treated (34.80 ± 2.27 mg) group of animals found significant (p < 0.01) higher when compare to control group. The protein content of granulation tissues indicates the levels of protein synthesis as well as cellular proliferation. Increase in protein contents of the treated wounds compared to control group suggest that APN, stimulates cellular proliferation through an unknown mechanism. Protein synthesis is essential for granuloma tissue formation. Inflammation phase involves infiltration of macrophages, neu-

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trophils and fibroblasts proliferation, which are the basic sources for granuloma formation [45]. Thus increase in granuloma weight indicates presence of higher protein content as well as the progression of the proliferative phase. Collagen is a major component that contributes to the wound strength and important constituent of extracellular matrix. Hydroxyproline and its peptides are produced after breakdown of collagen [46]. The estimation of this hydroxyproline is used as an index for collagen turnover. The new synthesized collagen molecules are laid down at the wound site and occurring cross linking to form fibers. The wound strength is acquired by remodeling of collagen and the formation of intra and inter-molecular cross links. A study also confirmed that administration of apigenin (5 ␮mol/L) to the mice, exhibited clearly increased dermal thickness and collagen density compared with DMSO-treated mice [47]. Thus the increased hydroxyproline level of the APN loaded GGCHHGs treated tissues was indicated increased collagen turnover and this lead to rapid healing of the treated wounds. 3.3.3. Estimation of antioxidants level in skin tissue The APN loaded GGCH-HGs posses potent antioxidant activity by increase in the SOD (18.43 ± 2.05 ␮g/50 mg tissue), GSH (22.76 ± 2.32 ␮mol/50 mg tissue) and catalase level (28.52 ± 2.43 ␮mol/50 mg tissue) in the granuloma tissues during wound healing process (Table 4). The significant improvement in SOD, GSH and CAT level in APN loaded GGCH-HGs and reference ointment groups were found on 9th day in diabetic wounds. Reduced glutathione is a strong free radical scavenger. The depletion of GSH results in enhanced lipid peroxidation. This can cause increased GSH utilization and can be correlated to the increase in the level of oxidized glutathione [46]. Treatment with of APN loaded GGCH-HGs resulted the increase in GSH levels, which protect the cell membrane against oxidative damage by control the redox status of protein in the membrane. SOD and CAT are enzymes play a significant role in providing antioxidant defenses to an organism that destroys the peroxides. The functions of all enzymes are interconnected. The lowering of enzymes activities results, accumulation of lipid peroxides and increased oxidative stress in wounded site. Treatment with APN formulation were increased the activity of these enzymes and thus may help to overcome free radicals production during chronic wounds.

Fig. 7. Wound areas of different groups i.e. Vehicle control (without Apigenin GGCH-HGs), Apigenin loaded GGCH-HGs and reference group (marketed formulation Betadin® ointment) in diabetic wound model.

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Table 3 Effect of Apigenin loaded GGCH-HGs and reference ointment on different biochemical parameters of Dead space wound in rats. Groups Vehicle control Apigenin loaded GGCH-HGs Reference Ointment

Hydroxyproline (mg/ g tissue) 18.08 ± 2.09 28.52 ± 1.03* 29.34 ± 1.13*

Protein content (mg/g tissue) 45.61 ± 2.32 71.41 ± 1.43* 72.84 ± 1.56*

Granuloma weight (mg) 17.03 ± 3.18 36.71 ± 2.05* 34.80 ± 2.27*

n = 6 albino rats per group, value represents Mean ± S.D. * p < 0.01, when compared each treated group with control group. Table 4 Effect of Apigenin loaded GGCH-HGs and reference ointment on different biochemical parameters of diabetic wound in rats. Groups Vehicle control Apigenin loaded GGCH-HGs Reference Ointment

SOD (␮g/50 mg tissue) 11.72 ± 2.07 18.43 ± 2.05* 18.05 ± 2.32*

CAT (␮mol/50 mg tissue) 16.22 ± 2.34 28.52 ± 2.43* 28.40 ± 2.97*

GSH (␮mol/50 mg tissue) 10.13 ± 3.12 22.76 ± 2.32* 21.57 ± 2.85*

n = 6 albino rats per group, value represents Mean ± S.D. * p < 0.01, when compared each treated group with control group.

3.3.4. Histopathological study Histopathological examination of stained sections from different treatment groups were exhibited different healing conditions of the wounded tissues. The results provide a good evidence of suitability of the hydrogel formulation for promoting healing in diabetic as well as normal wounds (Fig. 8). The observations showed that the original tissue regeneration found efficiently in the wound treated with APN loaded GGCH-HGs and reference group without any edema and congestion. Both group showed proliferation of epithelial tissue covering the wound area. In vehicle control group, dermal modeling process was very slow which was confirmed by the lower epithelialization time. The granulation tissue section of control animals showed lower epithelialization, fibrosis and aggregation of macrophages with less collagen fibers, indicating incomplete healing of wounds. Histopathological studies, confirms an increase in blood vessels, increased collagen fibers and fibroblast cells growth. It has reported that, active oxygens are cytotoxic and to damage cells by inactivating cellular components. Natural antioxidants are biological molecules that can act direct or indirectly as antioxidants by quenching a free radical scavenging [48]. In present study, we isolated apigenin (APN) from ethanol extract of M. alba leaves and investigated for wound healing application of prepared APN-GGCH-HGs. The results of the study showed enhanced rate of wound contraction and reduced healing time in animals treated with APN HGs. These findings are consistent with other reports of wound healing activity of Morus alba [49–51]. The naturally occurring flavonoids, in the M. alba leaves extract, have shown antioxidant activity in different model systems [52].

Flavonoids are used for numerous therapeutic properties such as antioxidant, antifungal, anti-inflammatory and wound healing [53]. Flavonoids are also known to promote the rapid wound healing due to their antimicrobial property [54]. Therefore, wound healing potential of M. alba may be attributed to the chemical constituents identified in the leaves, due to their synergistic effect that accelerates the proliferation phase of wound healing. 4. Conclusion The results showed that prepared hydrogels with gallen gum, chitosan, by using PEG as cross linker showed excellent results over the reported chitosan based hydrogels towards wound healing applications. Apart from improved drug entrapment, good swelling capability and sustained release property which is most important factors for reducing the risk of wound dehydration. The improved wound healing results also supported with showed good antioxidant activity. It is also an evidence that biodegradable polymer-based hydrogels acts in the remodeling phase of the wound healing process, by promoting extracellular matrix remodeling and accelerating the wound closure of injured tissue. In conclusion, the apigenin loaded gellan gum–chitosan hydrogel (GGCH-HGs) was effectively stimulates wound contraction and significantly increased collagen content in diabetic as well as normal wound tissues. There was further confirmed by increase hydroxyproline content and protein levels, significantly. The collagen maturation was improved may be due to increased cross-linking and an increase in dry granuloma weight that indicates higher protein content. These finding could justify the inclusion of apigenin

Fig. 8. Photomicrograph of rat skin tissues after post-wounding days for different treatment groups in diabetic wound model. Hematoxylin and eosin, ×100; (A) Vehicle control (without Apigenin GGCH-HGs); (B) Apigenin loaded GGCH-HGs (C) Reference group (marketed formulation Betadin® ointment); F: Fibroblast cells; C: Collagen fibers, Bv: Blood vessels.

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