antinociceptive and anti-inflammatory activities

International Journal of Applied Research in Natural Products Vol. 2(1), pp. 1-8, March-April 2009 Available online http://www.healthy-synergies.com 2009 Healthy Synergies Publications

_____________________ *Corresponding Author: Tel: +66-53-945353 Fax: +66-53-945355 E-mail: [email protected]

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Original Article Antinociceptive and Anti-inflammatory Activities of a Chinese Herbal Recipe (DJW) in Animal Models Kunanusorn P*, Teekachunhatean S, Sangdee C, Panthong A Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Thailand. Summary: Since in our previous study, Duhuo Jisheng Wan (DJW), which means pill of pubescent angelica root and mulberry mistletoe combination, demonstrates clinically comparable efficacy to diclofenac in the symptomatic treatment of osteoarthritis (OA) of the knee after 4 weeks of treatment. Therefore, in order to verify its mechanisms of action, this study was performed to investigate the antinociceptive and anti-inflammatory activities of DJW in various animal models. The antinociceptive activity of DJW was investigated by using the formalin test in mice model. The acute inflammatory model using the carrageenin-induced hind paw edema in rats and the chronic inflammatory model using the cotton pellet-induced granuloma formation in rats were utilized. Results showed that DJW possessed a marked antinociceptive activity in both phases of the formalin test in mice. However, in the carrageenin-induced hind paw edema model, which is known to be sensitive to cyclooxygenase (COX) inhibitors, DJW showed an insignificant anti-inflammatory effect, and in the cotton pellet-induced granuloma model, it had no antigranuloma formation and showed no effect on the transudate weight. In addition, DJW showed no suppressive effects on weight gain and the thymus weight of the rats. In conclusion, the overall results demonstrate that DJW possess both central and peripheral antinociceptive activities. However, its anti-inflammatory activity, if any, could not be demonstrated in these two inflammatory models in the present study and remains to be elucidate. Industrial relevance: Since drug therapy in OA patients, such as paracetamol, non-steroidal anti-inflammatory drugs (NSAIDs), and topical analgesics may prove ineffective in some patients, and long-term therapy with NSAIDs often have been associated with serious adverse effects. Such patients are turning increasingly to herbal medicines and DJW may be an alternative since it demonstrates clinically comparable efficacy to diclofenac in the symptomatic treatment of OA of the knee after 4 weeks of treatment in our previous study. The present study would help clarifying its mechanism(s) of action. With this additional information, it would be helpful for the industry to produce herbal formulation with known mechanism of action, less side effects, and has clinically comparative efficacy to conventional medicine in the symptomatic treatment of OA of the knee. Keywords: DJW; antinociceptive; anti-inflammatory; formalin test; animal models.

Background

Osteoarthritis (OA) is a progressive rheumatic disease characterized by the degeneration of articular cartilage. It is the most common of all rheumatic disorders (Howell, 1986) and OA of the knee is a frequent cause of pain and disability (Felson, 1990). Drug therapy, such as paracetamol, non-steroidal anti-inflammatory drugs (NSAIDs), topical analgesics, opioid analgesics and intra-articular steroid injection, may prove ineffective in some patients, and long-term therapy with NSAIDs often have been associated with serious adverse effects (Buchanan, 1990; Tramer et al., 2000). Hence, there appears to be a need for drugs with good efficacy and low toxicity in the treatment of OA, specifically for patients who do not respond well to conventional medical therapy. Such patients are turning increasingly to herbal medicines.

Duhuo Jisheng Wan (DJW), which means pill of pubescent angelica root and mulberry mistletoe combination, is perhaps the best known and most widely used Chinese herbal recipe since the Tang Dynasty (652 A.D.) (Xu et al., 1994; Geng et al., 1997) and also sold as a patent remedy for arthralgia (Angelica Combination Tea Extract, Web page; Du Huo Jisheng Wan, Web page). Its ingredients include 15 plants (Xu et al., 1994). Some plants are indicated to relief joint pain in reports of traditional and folk use (Du huo, Web page; Ledebouriella, Web page; Qin jiao, Web page) and some have antinociceptive and/or anti-inflammatory effects in scientific literatures (Ozaki, 1992; Chen et al., 1995; Kubo et al., 1996; Yasukawa et al., 1998; Wei et al., 1999; Giner-Larza et el., 2000). In addition, in our previous study DJW demonstrates clinically comparable efficacy to diclofenac in the symptomatic treatment of OA of the knee after 4 weeks of treatment

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(Teekachunhatean et al., 2004). However, studies to verify the antinociceptive and anti-inflammatory effects of this herbal recipe are still lacking. Therefore, the objectives of this study were to investigate and evaluate the antinociceptive activity of DJW in a model of the formalin test in mice; the anti-inflammatory activity in the carrageenin-induced hind paw edema of acute inflammation model and the cotton pellet-induced granuloma formation of chronic inflammation model in rats.

Materials and methods Experimental animals: Male Sprague-Dawley rats weighing 100-120 g and 180-200 g as well as male Swiss albino mice weighing

20-40 g were purchased from the National Laboratory Animal Center, Nakorn Pathom, Thailand. All animals were kept in a room maintained under environmentally controlled conditions of 24 + 1 oC and a 12 h light-dark cycle. The animals had free access to water and food and were acclimatized at least one week before starting the experiments. All experiments were conducted in accordance with the ethical principles and guidelines for the use of animals prepared by the National Research Council of Thailand.

DJW The modified DJW ingredients were 7.75% each of Radix Angelicae Pubescentis (Duhuo), Radix Gentianae

Macrophyllae (Qinjiao), Cortex Eucommiae (Duzhong), Radix Achyranthis Bidentatae (Niuxi), Radix Angelicae Sinensis (Danggui), Herba Taxilli (Sangjisheng), Radix Rehmanniae Preparata (Shudihuang), Rhizoma Chuanxiong (Chuanxiong), Cortex Cinnamomi (Rougui) and Radix Ledebouriellae (Fangfeng), and 5% each of Radix Paeoniae Alba (Baishao), Radix Codonopsis (Dangshen), Radix Glycyrrhizae (Gancao) and Poria (Fuling), and 2.50% of Herba Asari (Xixin). All raw materials were imported from the Peoples Republic of China. Xixin, Niuxi, Shudihuang and Rougui were imported from Shantou Traditional Chinese Medicine Factory, whereas the other ingredients were imported from Qixin Co., Ltd. (Hebei Province).

Preparation of the ethanol extract of DJW Proportions of the fine powder of all raw materials used as ingredients were mixed thoroughly. The mixture

was then macerated in 95% ethanol, allowed to stand for 24 h and filtered through a filter paper using a vacuum pump. The maceration was repeated 2 times. All the filtrates were pooled and evaporated under reduced pressure and controlled temperature (50-60 oC) by using a vacuum rotary evaporator. The extract was then lyophilized and its yield was used in all experiments by suspending in 5% Tween 80.

Drug administration All reference drugs and the extract were suspended in 5% Tween 80 and orally administered in an equivalent

volume of 0.5 ml/100 g body weight of the animal. The control group received the same volume of vehicle by the same route.

Antinociceptive study The antinociceptive activity of DJW was studied using the formalin test and compared with reference drugs

(Hunskaar and Hole, 1987). Briefly, male Swiss albino mice weighing 20-40 g were divided into 6 groups of 6 mice per group. Three doses of DJW extract (250, 1,000, 4,000 mg/kg), 10 mg/kg of diclofenac and 50 mg/kg of codeine (serving as the reference groups) were given orally. The control group received 5% Tween 80.

In the early phase assessment, 20 l of 1% formalin in NSS was injected subcutaneously into the right dorsal hind paw of the mouse 1 h after the administration of the reference drugs or various doses of the extract. Then, between 0-5 min after the formalin injection, the number of seconds the mice spent intensively licking the right dorsal hind paw was recorded.

In the late phase assessment, another set of 6 groups of mice each was used. Formalin was injected 40 min after drug treatment and the licking time was recorded between 20-30 min after the formalin injection. Percent inhibition of licking response was calculated.

Anti-inflammatory study Carrageenin-induced hind paw edema in rats (Winter et al., 1962) Male rats of 100-120 g body weight were divided into 3 groups of 6 animals per group. Since the anti-

inflammatory dose of the extract was expected to be higher than its antinociceptive dose, DJW extract of 8,000 mg/kg dose was then used. A 10 mg/kg dose of diclofenac as reference drug or vehicle (5% Tween 80 in the control group) and the extract were given orally 1 h prior to the carrageenin injection. Lambda carrageenin was prepared as 1% suspension in sterile normal saline solution (NSS). A volume of 0.05 ml of 1% carrageenin was injected intradermally into the plantar side of the right hind paw of an unanesthetized rat restrained in a plastic cage.

Foot volume of the animal was determined by means of a volume displacement technique using a plethysmometer (model 7150, Ugo Basile, Italy). The right hind paw was immersed into the measuring chamber containing 0.05% sodium chloride in distilled water, exactly to an ink mark at the anatomical hair line. Each paw volume was obtained from the average of 3 readings. The paw volume was measured prior to and at 1, 3 and 5 h after the carrageenin injection.

Cotton pellet-induced granuloma formation in rats (Swingle and Shideman, 1972)

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Male rats of 180-200 g body weight were divided into 4 groups of 6 rats per group. The first group was a control group that received 5% Tween 80 only. The second and the third group received 5 mg/kg dose of prednisolone and 10 mg/kg dose of diclofenac, respectively. The last group received 8,000 mg/kg dose of DJW extract. Two sterilized, 20 mg adsorbent cotton pellets were implanted subcutaneously, one on each side of the abdomen of the animal under light ether anesthesia and the sterile technique. A suture was then made and the animal was allowed to recover. All groups of animals were administered vehicle, the reference drugs, and the extract orally in a once daily dosage regimen throughout the experimental period of 7 days. On the eighth day after cotton pellet implantation, each rat was anesthetized with pentobarbital sodium. The heart blood was collected and the serum was separated for determination of the amount of alkaline phosphatase and total protein. The rat was then sacrificed and the implanted pellets were dissected out, carefully removed from the surrounding tissues and weighed immediately for wet weight. The thymus was also dissected out. Both cotton pellets and the thymuses were dried at 60 oC for 18 h and their dry weights were determined. The granuloma and transudative weights and the percent inhibition of granuloma formulation of the test compounds were calculated. The change in body weight from the first and last day of the experiment was also recorded.

Statistical analysis The data from the experiments were expressed as mean + SD. Statistical comparisons between the groups

were analyzed by using one-way analysis of variance and the post hoc least-significant difference test. The p values of less than 0.05 were considered significant.

Results The results of the licking response in the early phase of the formalin test are shown in Table 1. DJW, at

doses of 250, 1,000, and 4,000 mg/kg, significantly inhibited the licking response compared with the control group, with percentages of 56, 45 and 55% respectively. Codeine (50 mg/kg) and diclofenac (10 mg/kg) also showed significant licking response inhibition of 54% and 73%, respectively. The inhibition of licking response to the test drugs in the late phase of the formalin test is shown in Table 2. Similar to the early phase, codeine and diclofenac exerted significant inhibitory effects on the licking response (67% and 95%, respectively). At doses of 250, 1,000, and 4,000 mg/kg, DJW exhibited significant antinociceptive activity in a dose dependent manner, with the percentages of inhibition being 40, 74 and 88%, respectively.

DJW at a dose of 8,000 mg/kg showed an insignificant anti-inflammatory effect in the carrageenin-induced hind paw edema in rat model (Table 3). However, diclofenac, at a dose of 10 mg/kg significantly inhibited the paw edema formation at 1, 3 and 5 h after the carrageenin injection, with 57, 65 and 43% inhibition, respectively. In order to test possible delayed effects of DJW, it was also given 2 and 4 h prior to the carrageenin injection. Again, DJW still exerted insignificant anti-inflammatory effect (data not shown).

Table 4 shows the results of cotton pellet-induced granuloma formation in rats. Diclofenac at 10 mg/kg dose produced significant inhibition of the granuloma formation (21%), as did 5 mg/kg dose of prednisolone (39%). On the contrary, DJW at 8,000 mg/kg had no inhibitory effect on granuloma formation. The transudative weight of the control group was found to be 165.33 + 10.67 mg. Diclofenac (10 mg/kg) and prednisolone (5 mg/kg) significantly reduced the weight of the transudate to 138.33 + 9.33 and 125.18 + 10.15 mg, respectively. In contrast, an 8,000 mg/kg dose of DJW showed no effect on the transudative weight.

The body weight gain in the control group was 29.00 + 7.87 g, and in the diclofenac and DJW groups were 25.33 + 3.93 and 32.67 + 11.78 g, respectively and not significantly different from the control group (Table 5). Prednisolone, at a dose of 5 mg/kg, markedly and significantly reduced the body weight gain to 13.33 + 6.28 g. The dry thymus weight of the rats in the control group was 43.55 + 9.12 mg/100g body weight (Table 5). Both diclofenac and DJW showed no suppressive effect on the thymus weight of the rats (47.84 + 12.27 and 40.99 + 6.93 mg/100g body weight, respectively) when compared with the control group, whereas prednisolone significantly reduced the thymus weight to 27.19 + 7.54 mg/100g body weight (Table 5).

Significant elevation of alkaline phosphatase level in the serum of rats in the control group (28.45 x 10 -4U of enzyme/mg of serum protein) was observed when compared with that of the normal/non-implanted rats (20.83 x 10 -4U of enzyme/mg of serum protein) (Table 6). The increase in serum alkaline phosphatase caused by the cotton pellet implantation was significantly reduced from the control to normal level by prednisolone at a dose of


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Table 1. Effects of DJW, codeine and diclofenac in the early phase of the formalin test in mice.

Group Dose (mg/kg)

Licking time (min)

Inhibition of licking response (%)

Control - 2.55+0.40 - Codeine 50 1.16+0.11* 55 Diclofenac 10 0.68+0.31* 73 DJW 250 1.13+0.06* 56 DJW 1,000 1.41+0.35* 45 DJW 4,000 1.14+0.30* 55

Data represent mean+S.D. (n=6). *Significantly different from the control group (p

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Table 6. Effects of DJW, prednisolone and diclofenac on serum alkaline phosphatase in the cotton pellet-induced granuloma model.

Group Dose (mg/kg)

Serum alkaline phosphatase activity (U of enzyme/mg of serum protein x 10-4)

Normal - 20.83+1.28*

Control - 28.45+4.73 Prednisolone 5 21.00+1.66*

Diclofenac 10 22.26+5.02 DJW 8,000 21.98+10.12

Data represent mean+S.D. (n = 6). Normal = the nonimplanted group. Control = the implanted group that received 5% tween 80 only. *Significantly different from the control group (p < 0.05). Diclofenac at a dose of 10 mg/kg and DJW at a dose of 8,000 mg/kg also reduced the serum levels of alkaline phosphatase (22.26 x 10 -4 and 21.98 x 10 -4U of enzyme/mg of serum protein, respectively) but these reductions did not reach the level of significance.

DJW did not cause any noticeable adverse effects even at the highest dose tested. In addition, the necropsy after each experimental setting also showed no abnormalities of internal organs, such as heart, lungs, liver, spleen, stomach, and kidneys.

Discussion The results of the present study reveal that DJW possesses antinociceptive activity in formalin model but it

has minimal anti-inflammatory effects in carageenin-induced paw edema and cotton pellet-induced granuloma models.

Carrageenin-induced rat hind paw edema is considered as a model of acute inflammatory process. Paw edema is mediated by the initial release of histamine, serotonin and bradykinin during the 1st hour after carrageenin injection. This is followed by the release of prostaglandins at around the 3rd hour and lasts for about 6 h after carrageenin injection (Di Rosa and Willoughby, 1971). This model is known to be sensitive to COX inhibitors and has been used to evaluate the effect of NSAIDs that primarily inhibit the COX involved in the prostaglandins synthesis (Di Rosa and Willoughby, 1971). The insignificant inhibitory effect of DJW on carrageenin-induced paw edema at the 3rd hour suggests that the DJW action may not involve the inhibition of the synthesis or the release of prostaglandins. Since DJW showed no inhibitory effect at the 1st hour, it may not possess any influence on other mediators, e.g., histamine and serotonin, which are also released during this hour. In addition, the finding that oral DJW, at 2 and 4 h prior to the carrageenin injection, did not yield significant effects, suggests that its ineffectiveness in this model is not due to its delayed absorption.

The cotton pellet granuloma method has been widely used to assess the transudative and proliferative components of chronic inflammation. It consists of 3 phases, which are 1) a transudative phase, defined as the increase in the wet weight of the pellet that occurs during the first 3 h, 2) an exudative phase, defined as plasma leaking from the bloodstream around the granuloma that occurs between 3 and 72 h after the implantation of pellet, and 3) a proliferative phase, measured as the increase in the dry weight of the granuloma that occurs between 3 and 6 days after the implantation (Swingle and Shideman, 1972). Although the anti-inflammatory drugs can inhibit both the transudative and proliferative phases, NSAIDs exert only slight inhibition, whereas steroidal anti-inflammatory agents strongly inhibit both phases (Swingle and Shideman, 1972). In this study, DJW showed no effect on both transudative and proliferative effect. On the other hand, diclofenac, an NSAID, showed slight inhibitory activity on the formation of transudate and granuloma, whereas prednisolone, a steroidal drug, exerted a marked effect. In the present study, only prednisolone markedly reduced the body weight gain and the thymus weight. Although steroids, particularly corticosteroids such as prednisolone, stimulate protein synthesis in the liver, they have pronounced catabolic effects on lymphoid and connective tissue, muscle, fat and skin. These results indicate that DJW has no anti-inflammatory or steroid-like activity in this chronic inflammatory model.

Arachidonic acid metabolites can mediate or modulate leukocyte influx into inflammatory sites, leading to tissue damage by releasing lysosomal enzymes and toxic oxygen radicals (Salmon and Higgs, 1987). The activity of lysosomal enzymes, such as alkaline phosphatase, raised in serum during the inflammatory process, results in damage to the tissue and cartilage that can lead to perpetuate the inflammation further. The serum alkaline phosphatase, which is elevated during cotton pellet granuloma formation, peaks on the 7th day and decreases by day 14 when healing occurs (Bessey et al., 1946; Nishikaze et al., 1980). Elevated lysosomal enzyme activity in serum and exudate during inflammation can be normalized by steroidal drugs, such as hydrocortisone, via the stabilization of lysosomal membrane (Ismail et al., 1997). In this study, elevated serum


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alkaline phosphatase activity was normalized by prednisolone only. This means that DJW and diclofenac may not be able to stabilize the lysosomal membrane during chronic inflammation.

Although some plants in DJW may have exhibited anti-inflammatory effects, this study could not reveal these effects in either carrageenin-induced rat paw edema or cotton pellet-induced granuloma models. This may be due to the relatively low dose (only 2.5-8%) of each ingredient used. In addition, since DJW consists of many plants, the antagonism between the ingredients may have led to an insignificant anti-inflammatory effect in the present study. Alternatively, methanol, chloroform and ethyl acetate extracts of Radix Angelicae Pubescentis, one of the major ingredients of DJW have been demonstrated to possess anti-inflammatory effects in the model of carrageenin induced paw edema (Chen et al., 1995). In addition, the anti-inflammatory action of DJW may be very slow in onset since it has been reported that the crude water extract of Radix Angelicae Pubescentis only exerts anti-inflammatory effect against hind paw edema induced by injecting complete Freunds adjuvant (CFA) at 24, 72 and 168 h (Wei et al., 1999). Our study used ethanol extract of DJW, did not use CFA to induce paw edema and did not extend the observation longer than 5 h since the edema induced by carrageenin usually and markedly subsides after 5 h. These discrepancies between our results and that of Chen et al. (1995) and Wei et al. (1999) may then be due to the different methodologies and time course of observation and may be the reason of our failure to observe the anti-inflammatory action of DJW in the present study.

The formalin test is different from most models of pain, as it can assess the way animals respond to moderate, continuous pain generated in injured tissue. Because of this connection to tissue injury, it is believed that this test provides a more valid and reliable model for clinical pain than other tests of nociception (Dubuisson and Dennis, 1977; Abbott et al., 1981, 1982; Alreja et al., 1984). It is a very useful method not only for assessing antinociceptives, but also for elucidating the mechanism of pain and analgesia, whether the site of action is central and/or peripheral (Shibata et al., 1989). The formalin test consists of two distinct phases, possibly reflecting different types of pain (Dubuisson and Dennis, 1977; Hunskaar et al., 1985; Hunskaar and Hole, 1987; Tjolsen et al., 1992). The early phase starts immediately after an injection of formalin and lasts for 3-5 min. It is probably due to direct chemical stimulation of nociceptors (Dubuisson and Dennis, 1977; Hunskaar et al., 1985; Tjolsen et al., 1992). This phase can be inhibited by centrally acting antinociceptives (Hunskaar et al., 1985; Hunskaar and Hole, 1987). The late phase starts approximately 15-20 min after the formalin injection and lasts for 20-40 min. This phase seems to be due to the combination of an inflammatory response in the peripheral tissue, partly mediated by prostaglandins, and functional changes in the dorsal horn of the spinal cord that are initiated by a C fiber barrage during the early phase (Tjolsen et al., 1992). This phase can be inhibited by NSAIDs and steroids, as well as centrally acting drugs (Hunskaar and Hole, 1987; Chen et al., 1995). Experimental results have indicated that histamine, serotonin and bradykinin are also involved in the late phase (Shibata et al., 1989).

In this study, codeine, diclofenac and all doses of DJW produced antinociceptive effect in both phases of the formalin test, but all drugs exerted more marked effect in the late phase. Concerning the early phase, the reference drug, codeine, exhibits its central analgesic effect by affecting the pain transmission and modulation pathway (Schumacher et al., 2007), whereas diclofenac may mediate its effect via various possible mechanisms. The central action of diclofenac may be the result of a depression of C fiber-evoked activity or an inhibitory effect on central prostaglandin synthesis (Cashman, 1996). Alternatively, it may be mediated in part by the endogenous opioid peptides or activation of descending serotonin pathways or even by a mechanism mediated by the inhibition of excitatory N-methyl-D-aspartate (NMDA) receptors (Cashman, 1996).

It is interesting to note that DJW modulated both the early and late phases of pain induced by formalin injection and that its antinociceptive effect was dose dependent and was more pronounce in the late phase. It is generally regarded that this late phase represents an ongoing inflammatory process mediated through the increase of the synthesis and release of prostaglandins and other mediators. Since DJW was proved to possess minimal anti-inflammatory effect in carageenin-induced paw edema and cotton pellet implantation, its aninociceptive effect in the late phase is difficult to explain. Alternatively, if DJW had anti-inflammatory effects, it could not be detected by the two models tested in the present study.

In our previous study that demonstrates comparable efficacy between DJW and diclofenac in the treatment of patients with OA of knee albeit the slower onset of action of DJW than diclofenac. The beneficial effects of DJW on these patients may be due to different mechanism from diclofenac of which its primary action is anti-inflammatory. The present study also emphasizes this notion that DJW did not demonstrate any effect on acute (carageenin-induced paw edema) or chronic inflammatory (cotton pellet granuloma) models. Since inflammatory component of OA is usually minimal (Brooks et al., 1982; Bradley et al., 1991; Wegman et al., 2004), and that DJW exerted significant antinociceptive effect in the present study, therefore it is probable that the beneficial effect of DJW in patients with OA of the knee in our previous study may be due mainly to its antinociceptive effect in the same fashion as paracetamol, a drug with no significant anti-inflammatory effect.

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In conclusion, the overall results demonstrate that DJW possess both central and peripheral antinociceptive activities. However, its anti-inflammatory activity, if any, could not be demonstrated in these two inflammatory models in the present study and remains to be elucidate.

Acknowledgements This work was supported by the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. References

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