The experimental study was started after getting approval from Institutional Animal Ethics Committee from Bharati Vidyapeeth Deemed University Medical College, Pune, Maharashtra, India, (BVDUMC/30-78/2015/001/005) and conducted according to CPCSEA guidelines. The study duration was 40 days (March and April 2015). Albino rats used for the study were housed in separate cages with free access of rodent food and aquaguard water.

Chemicals: CFA (Sigma-Aldrich), Pterocarpus santalinus bark wood (made into powder after authentication), injection pentobarbitone (LOBA-CHEMIE INDOAUSTRANAL CO., BOMBAY), diclofenac gel (Cipla Pharmaceuticals Ltd.,), carbopol 934P as gelling agent (Thomas Baker Pvt. Ltd.,), triethanolamine (Thomas Baker Pvt. Ltd.,).

Authentication and preparation of study material: Pterocarpussantalinus bark wood was obtained from ayurvedic medical stores (Vaidya Khadiwale) and sent for authentication to Agharkar Research Institute, Maharashtra, India. The sample was critically studied with macroscopic, organoleptic, microscopic and TLC profile and authenticated by AS Upadhye, Scientist, Plant Drug Authentication Service, Botany group, Plant Sciences Division as mentioned in the certificate number Auth.15-072. It was confirmed to be the heartwood of Pterocarpus santalinus Lf (Family: Fabaceae). The bark wood was then dried in shade and powdered finely to be used for preparation of the study formulations.

Preparation of P. santalinus paste formulation: A 10 gm of powder of P. santalinus was added to 10 ml of distilled water and mixed to form a uniform paste. Equal amount of paste (1.5 gm) was applied topically on the arthritic joint of the animal.

Preparation of P. santalinus gel formulation: Weighed (0.1 gm) quantity of carbopol 934P was taken and added to distilled water (5 ml). About 10 gm powder of P. santalinus was mixed with 20 ml distilled water and transferred to appropriate quantity of aqueous dispersion of carbopol 934P. The mixture was then stirred gradually by means of a stirrer until a cloudy, lump free dispersion was obtained. Then it was allowed to stand for air bubbles to separate. Nearly, 0.1 ml triethanolamine then added to neutralize the carbopol 934P solution and to form the gel [9]. This preparation was divided into six equal parts on the tile and one part was applied topically to each rat every day.

Induction of inflammation: Induction of inflammation was done with CFA as described below and evaluation of the inflammatory activity was done by measurement of oedema size resulting from sub-plantar CFA injection unilaterally in the left hind paw region of the body. Animals were anesthetized by injecting pentobarbitone intraperitoneally in the dose of 6 mg/100 gm body weight. Then 0.1 ml of CFA was injected in the sub-plantar tissue of the left hind paw. According to Bendele A, acute inflammatory reaction develops rapidly but chronic inflammation appears in the later stages (day 12+) as severe paw swelling [10].

Groups: Albino rats of either sex weighing 150-200 gm were divided into five groups as follows with six rats in each group-Group I – Negative control (distilled water)

Group II – Vehicle control (Gel base)

Group III –P. santalinus paste

Group IV –P. santalinus gel

Group V– Diclofenac gel (standard treatment)

Topical treatment was started, as per the respective groups from day 12 till day 28. After applying the drug on the diseased part, that area was covered by sterile gauze to prevent the inadvertent consumption of the medication by the animals.

Keeping the ethical issues of 3R’s (Replacement, Reduction and Refinement) in review, the animals were handled by competent, empathetic capture using the cupping method and not lifted by their tail. While injecting the inducing agent, anaesthesia was provided to reduce the suffering in the animals. We ensured that they could easily reach water and food provided to them in the cage. We also implemented husbandry refinements in the form of long nozzles on drinking bottles and soft sawdust bedding material. They were provided appropriate environmental enrichment and housed them in groups of three animals per cage [13].

Data was analysed using Graph Pad Prism version 5. Paired and Unpaired students t-test was used for comparison within and among groups respectively. The p<0.05 was considered statistically significant.

Body weight of the study animals was observed on day 12 when the chronic inflammation model developed and also on day 28 to see the response of the drug treatment on this parameter. There was significant reduction in body weight in rats treated with P.santalinus gel (p=0.02) when compared to other groups on day 28 of the experiment [Table/Fig-1].

[Table/Fig-1]:

Effect of topical Pterocarpus santalinus formulations and diclofenac on body weight in rat chronic inflammation model (Day 12 - Day 28).

*p = 0.02 using unpaired t-test when compared to negative control group (I)

Paw volume calculated using plethysmometer also showed reduction in paw volume in all the groups on day 28 as compared to day 12; but this parameter was statistically significant (p=0.03) only in rats treated with P.santalinus gel as compared to control group and standard treatment group [Table/Fig-2,3].

[Table/Fig-2]:

Reduction in paw volume with topical Pterocarpus santalinus formulations and diclofenac in rat chronic inflammation model (Day 12 - Day 28).

** p = 0.03 using unpaired t-test when compared to negative control group (I). $$ p= 0.03 using unpaired t-test when compared to diclofenac gel group (V).

Pain assessment was done using Randall LO and Selitto JA paw withdrawal method which showed decrease in pain threshold in all the groups on Day 28; but this change in pain threshold was less in P.santalinus gel group (158±8.1) on day 0, (117±9.8) on day 28 [Table/Fig-4].

CFA induced inflammation of the rat hindpaw has been extensively used as model of acute and chronic inflammation and arthritis [10]. Studies have shown that when CFA is injected subcutaneously at a relatively high dose, initially it leads to unilateral paw inflammation and acute arthritis, followed by contralateral paw swelling and a rheumatoid-arthritis like disease in later stages. But when a low dose of CFA is injected, both the hind paw oedema and arthritis remain strictly unilateral [14,15]. This was the model chosen for developing chronic inflammation in our study rats.

The standard drug used in this study, diclofenac was applied in the form of a gel preparation. Diclofenac, a non-steroidal anti-inflammatory agent is known for its prominent anti-inflammatory and analgesic properties even in arthritic conditions.

Two formulations of the study drug, Pterocarpus santalinus were tested for their anti-inflammatory and analgesic activities in this model. P. santalinus in the form of paste and gel were used for this purpose. Gels generally have a high rate of acceptance amongst consumers due to cosmetic elegance. Gels are generally thixotropic materials, exhibiting a stable form at rest but becoming fluid when agitated hence it shows better performance effect in terms of stability and therapeutic applicability [7,16]. Hence, for topical application of P. santalinus, gel was selected compared to other semisolid dosage form.

Our study revealed that topical treatment with P.santalinus gel in chronic inflammation model of rats, significantly reduced the paw volume in the rats [Table/Fig-2,3]. This significant reduction in paw volume might also have contributed to the decrease in body weight in these animals as seen in [Table/Fig-1]. Rats treated with topical P.santalinus gel also have demonstrated mild analgesic effect of this drug as can be seen from the minimal reduction in pain threshold in this group [Table/Fig-4] when compared to its baseline readings. The mild analgesic property of P.santalinus gel might correlate with the reduction in inflammation due to this drug which in turn reduced the inflammation associated pain.

It is well known that cell injury leads to activation of the arachidonic acid pathway and thereby production of prostanoids and leukotrienes and recruitment of immune cells at this site further releases mediators including cytokines and growth factors. Some of these inflammatory mediators activate peripheral nociceptors directly [17]. In the groups I, II, III and V, as there was no significant reduction in paw volume (representing inflammation), pain threshold was significantly lower than baseline values. This indicates that inflammation associated pain was still persisting in the other groups due to the release of inflammatory mediators from damaged cells like bradykinin, histamine, 5-hydroxytryptamine (5-HT), ATP and nitric oxide.

The qualitative phytochemical analysis of P. santalinus has confirmed the presence of various components, such as carbohydrates, steroids, anthocyanins, saponins, tannins, phenols, triterpenoids, flavonoids, glycosides, and glycerides [18]. Savinin, a lignan from Pterocarpus santalinus has been shown to inhibit TNF-α production and T cell proliferation which may act as an active principle in the anti-inflammatory effect [19]. A previous study has shown that anti-inflammatory effect of methanolic extract of P. santalinus as gel formulation was better than that of diclofenac gel in Carrageenan induced rat paw oedema method (acute inflammation) [7]. Our study has revealed its efficacy (gel formulation) even in chronic inflammatory model in which P. santalinus gel significantly reduced oedema and there was only slight reduction in pain threshold.

It is known that ROS participate in the process of inflammation in various tissues and authors have demonstrated in vitro free radical scavenging activities of P. santalinus methanolic wood extract [5]. This property might provide the basis of anti-inflammatory activity of P. santalinus in this model of chronic inflammation.

Our study has shown that the anti-inflammatory and analgesic activities of P.santalinus gel were better than the standard diclofenac gel applied topically in chronic inflammation model of rats. Previous studies have used extract preparations of P.santalinus for evaluating its anti-inflammatory and analgesic activities and the authors have observed that methanolic extract of this drug given orally or applied topically had comparable efficacy to standard drug diclofenac intraperitoneally or topically respectively [6,7]. In a clinical study which compared diclofenac concentrations in the soft tissues and blood on topical and oral use, topical application of diclofenac has shown higher concentrations in adjacent adipose tissue and skeletal muscle than oral preparations, but synovial concentrations were lower than with oral use [20]. It has been shown that diclofenac achieves higher concentration in synovial fluid on oral and intramuscular administration which peaks within 2-4 hours. But gastric irritation is known to be the most common adverse effects of NSAIDs including diclofenac. An added advantage of P.santalinus is its gastroprotective action proven in various studies [21,22]. Further studies should be done to test the anti-inflammatory activity of oral P.santalinus bark wood powder as mentioned in ayurvedic literature.

This study demonstrates a significant anti-inflammatory and mild analgesic activity of topical P.santalinus gel in CFA induced chronic inflammation in rats. P.santalinus/Raktachandan in the form of a gel formulation can be a promising and safe alternative to topical NSAIDs in chronic inflammatory conditions.

Limitations of our study were that we were not able to perform the histopathological examination of the inflammed area which could have revealed the effects of the drugs at the tissue level.

Pterocarpus santalinus bark wood powder gel showed significant anti-inflammatory and mild analgesic activity on topical application in rat model of chronic inflammation. Future studies can be planned to study the mechanism of action of our study drug as anti-inflammatory agent in acute and chronic inflammation models.