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

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
_____________________
*Corresponding Author:
Tel: +66-53-945353
Fax: +66-53-945355
E-mail: pkunanus@mail.med.cmu.ac.th
1

Antinociceptive and Anti-inflammatory Activities of DJW

(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 antiinflammatory 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)
2

Kunanusorn et al

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

Antinociceptive and Anti-inflammatory Activities of DJW

Table 1. Effects of DJW, codeine and diclofenac in the early phase of the formalin test in mice.
Group
Dose
Licking time Inhibition of licking response (%)
(mg/kg)
(min)
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<0.05).
Table 2. Effects of DJW, codeine and diclofenac in the late phase of the formalin test in mice.
Group
Control
Codeine
Diclofenac
DJW
DJW
DJW

Dose
(mg/kg)
50
10
250
1,000
4,000

Licking time
(min)
2.78+0.38
0.92+0.41*
0.15+0.20*
1.66+0.29*
0.71+0.32*
0.33+0.21*

Inhibition of licking response (%)

67
95
40
74
88

Table 3. Effects of DJW and diclofenac in the carrageenin-induced paw edema model.
Drug

Dose
(mg/kg)

Time after carrageenin injection

1h
3h
5h
EV (ml)
EI (%)
EV (ml)
EI (%)
EV (ml)
EI (%)
Control
0.14+0.03
0.52+0.08
0.37+0.07
Diclofenac
10
0.06+0.03*
57
0.18+0.06*
65
0.21+0.09*
43
DJW
8,000
0.15+0.06
0
0.41+0.12
21
0.34+0.15
8
Data represent mean+S.D. (n = 6). The control group received 5% tween 80 only. *Significantly different from the control
group (p < 0.05). EV = edema volume, EI = edema inhibition.
Table 4. Effects of DJW, prednisolone and diclofenac on granuloma formation and transudation in the cotton pellet-induced
granuloma model.
Granuloma
Granuloma
Inhibition
weight
(%)
(mg/mg
cotton)
Control
211.89+13.49
46.56+3.13
165.33+10.67
1.35+0.17
Prednisolone
5
161.27+10.92*
36.08+3.72*
125.18+4.14*
0.83+0.20*
39
Diclofenac
10
178.88+12.03*
40.56+4.14*
138.33+9.33*
1.06+0.22*
21
DJW
8,000
210.43+27.18
45.81+3.97
164.63+23.34
1.30+0.18
4
Data represent mean+S.D. (n = 6). The control group received 5% tween 80 only. *Significantly different from the control
group (p < 0.05).
Group

Dose
(mg/kg)

Granuloma wet
weight
(mg)

Granuloma dry
weight
(mg)

Transudative
weight
(mg)

Table 5. Effects of DJW, prednisolone and diclofenac on body weight and dry thymus weight in the cotton pellet-induced
granuloma model.
Group

Dose
Body weight (g)
(mg/kg)
Initial
Final
Control
193.00+5.62 222.00+4.56
Prednisolone
5
195.67+6.98 209.00+9.44*
Diclofenac
10
194.67+6.41 220.00+6.32
DJW
8,000
191.33+5.01 224.00+9.72
Data represent mean+S.D. (n=6). *Significantly different
enzyme/mg of serum protein).

Dry thymus weight

(mg/100g)
Gain
29.00+7.87
43.55+9.12
13.33+6.28*
27.19+7.54*
25.33+3.93
47.84+12.27
32.67+11.78
40.99+6.93
from the control group (p <0.05).5 mg/kg (21.00 x 10 -4U of

Kunanusorn et al

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
5

Antinociceptive and Anti-inflammatory Activities of DJW

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 antiinflammatory. 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.

Kunanusorn et al

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
Abbott FV, Franklin KB, Ludwick RJ, Melzack R. 1981. Apparent lack of tolerance in the formalin test suggests
different mechanisms for morphine analgesia in different types of pain. Pharmacol Biochem Behav 15:637640.
Abbott FV, Melzack R, Samuel C. 1982. Morphine analgesia in tail-flick and formalin pain tests is mediated by
different neural systems. Exp Neurol 75:644-651.
Alreja M, Mutalik P, Nayar U, Manchanda SK. 1984. The formalin test: a tonic pain model in the primate. Pain
20:97-105.
Angelica Combination Tea Extract : Du Huo Jisheng [Internet]. [cited 2008 Mar 10]. Available from:
http://store.orientalpharmacy.com/ancoteaexduh1.html
Bessey OA, Lowry OH, Brock MJ. 1946. Method for the determination of alkaline phosphatase with five cubic
millimeters of serum. J Biol Chem 164:321-329.
Bradley JD, Brandt KD, Katz BP, Kalasinski LA, Ryan SI. 1991. Comparison of an antiinflammatory dose of
ibuprofen, an analgesic dose of ibuprofen, and acetaminophen in the treatment of patients with osteoarthritis
of the knee. N Engl J Med 325(2):87-91.
Brooks PM, Potter SR, Buchanan WW. 1982. NSAIDs and osteoarthritis - help or hindrance? J Rheumatol
9(1):3-5.
Buchanan W. 1990. Implications of NSAID therapy in elderly in-patients. J Rheumatol 4:29-32.
Cashman JN. 1996. The mechanisms of action of NSAIDs in analgesia. Drugs 52 (Suppl 5):13-23.
Chen YF, Tsai HY, Wu TS. 1995. Anti-inflammatory and analgesic activities from roots of Angelica pubescens.
Planta Med 61:2-8.
Di Rosa M, Willoughby DA. 1971. Screens for anti-inflammatory drugs. J Pharm Pharmacol 23:297-298.
Dubuisson D, Dennis SG. 1977. The formalin test: A quantitative study of the analgesic effects of morphine,
meperidine, and brain stem stimulation in rats and cats. Pain 4:161-174.
Du huo [Internet]. [cited 2008 Mar 10]. Available from:
http://www.holisticonline.com/HerbalMed/_Herbs/h381.htm
Du Huo Jisheng Wan [Internet]. [cited 2008 Mar 10]. Available from: http://herbs4cure.com/angelica.htm
Felson DT. 1990. Osteoarthritis. Rheum Dis Clin North Am 16:499-512.
Geng JY, Huang WQ, Ren TC, Ma XF. 1997. Practical traditional Chinese medicine & pharmacology: Herbal
Formulas. Beijing: New World Press.
Giner-Larza EM, Manez S, Giner-Pons RM, Carmen Recio M, Rios JL. 2000. On the anti-inflammatory and
anti-phospholipase A2 activity of extracts from lanostane-rich species. J Ethnopharmacol 73(1-2):61-69.
Howell DS. 1986. Pathogenesis of osteoarthritis. Am J Med 80(4B):24-28.
Hunskaar S, Fasmer OB, Hole K. 1985. Formalin test in mice, a useful technique in evaluating mild analgesics. J
Neurosci Methods 14:69-76.
Hunskaar S, Hole K. 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory
pain. Pain 30:103-114.
Ismail TS, Gopalakrishnan S, Begum VH, Elango V. 1997. Anti-inflammatory activity of Salacia oblonga Wall.
and Azima tetracantha Lam. J Ethnopharmacol 56(2):145-152.
Kubo M, Ma S, Wu J, Matsuda H. 1996. Anti-inflammatory activities of 70% methanolic extract from
Cinnamomi Cortex. Biol Pharm Bull 19(8):1041-1045.
Ledebouriella [Internet]. [cited 2008 Mar 10]. Available from: http://www.holisticonline.com/HerbalMed/_Herbs/h335.htm
Nishikaze O, Takita H, Takase T. 1980. Activity of newly discovered protease in carrageenan-induced
inflammation in rats. IRCS 8:725.
Ozaki Y. 1992. Antiinflammatory effect of tetramethylpyrazine and ferulic acid. Chem Pharm Bull (Tokyo)
40(4):954-956.
Qin jiao [Internet]. [cited 2008 Mar 10]. Available from: http://www.holisticonline.com/HerbalMed/_Herbs/h383.htm
Salmon JA, Higgs GA. 1987. Prostaglandins and leukotrienes as inflammatory mediators. Br Med Bull
43(2):285-296.
Schumacher MA, Basbaum MI, Way WL. 2007. Opioid analgesics & antagonists. In: Katzung BG, editor. Basic
& Clinical Pharmacology. 10th ed. New York: McGraw-Hill; p 489-510.

Antinociceptive and Anti-inflammatory Activities of DJW

Shibata M, Ohkubo T, Takahashi H, Inoki R. 1989. Modified formalin test: characteristic biphasic pain response.
Pain 38:347-352.
Swingle KF, Shideman FE. 1972. Phases of the inflammatory response to subcutaneous implantation of cotton
pellet and their modification by certain anti-inflammatory agent. J Pharmacol Exp Ther 183:226-234.
Teekachunhatean S, Kunanusorn P, Rojanastein N, Sananpanich K, Pojchamarnwiputh S, Lhieochaiphunt S,
Pruksakorn S. Chinese herbal recipe versus diclofenac in symptomatic treatment of osteoarthritis of knee: a
randomized controlled trial. BMC Complement Altern Med [Internet]. 2004 [cited 2008 Mar 1]; 4:19.
Available from: http://www.biomedcentral.com/1472-6882/4/19
Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K. 1992. The formalin test: an evaluation of the method.
Pain 51:5-17.
Tramer MR, Moore RA, Raynolds DJ, McQuay HJ. 2000. Quantitative estimation of rare adverse events which
follow a biological progression: a new model applied to chronic NSAID use. Pain 85:169-182.
Wegman A, van der Windt D, van Tulder M, Stalman W, de Vries T. 2004. Nonsteroidal antiinflammatory drugs
or acetaminophen for osteoarthritis of the hip or knee? A systematic review of evidence and guidelines. J
Rheumatol 31(2):344-354.
Wei F, Zou S, Young A, Dubner R, Ren K. 1999. Effects of four herbal extracts on adjuvant-induced
inflammation and hyperalgesia in rats. J Altern Complement Med 5(5):429-436.
Winter CA, Risley EA, Nuss GW. 1962. Carrageenin-induced edema in hindpaw of the rat as an assay for antiinflammatory drug. Proc Soc Exp Biol Med 11:544-547.
Xu XC, Yuan JR, Tian JZ, Chen SM. 1994. Commonly used Chinese patent medicines. Beijing: Higher
Education Press.
Yasukawa K, Kaminaka T, Kitanaka S, Tai T, Nunoura Y, Natori S, Takido M. 1998. 3 Beta-phydroxybenzoyldehydrotumulosic acid from Poria cocos, and its anti-inflammatory effect. Phytochemistry
48(8):1357-1360.