Tes formalin dilakukan untuk mengkonfirmasi mekanisme analgesik kemungkinan aksi EJ-01 yang dapat

membedakan rasa sakit di pusat (awal fase) dan perifer (fase akhir) komponen (Tjolsen et al., 1992). Sebuah obat
yang bertindak terutama pada sistem saraf pusat seperti morfin dapat menghambat kedua fase (Martindale et al.,
2001). Nyeri inflamasi secara selektif dihambat oleh NSAID karena agen ini mengurangi peradangan. Pada uji
formalin, fase awal (0-5 menit) adalah fase neurogenic yang juga dikenal sebagai nyeri non-inflamasi dan
dimediasi oleh efek sentral melalui aktivasi langsung dari serat C menyebabkan substansi P dan bradikinin rilis
sementara akhir fase, juga disebut sebagai nyeri inflamasi, adalah karena inflamasi infor dan dimediasi oleh efek
perifer melalui dins prostaglandin dan pelepasan sitokin (Granados-Soto et al., 2001). Hal ini juga diketahui bahwa
intraplantar suntikan TNFa,IL-1b,IL-6, and chemokines induce hyperalgesia and nociception in the rat hind-paw
(Cunha et al., 1992). Local treatment with specific antisera against these cytokines reduced the second phase of the
formalin nociception which would appear to onfirm that cytokines (TNF- , IL-1 , etc.) produced peripherally are
involved in this process. It is also reported that there is participation of bradykinin, cytokines, prosta- glandins, and
sympathetic amines in the formalin-induced orofacial nociception in rats (Chichorro et al., 2004).
EJ-01 failed to reduce the licking time in the early phase of formalin test; this might indicate that it has
failed to cause the blockade of nociceptive fiber or the release of substance P and bradykinin. Since the EJ-01
could reduce the licking time in the late phase only, it might be due to the inhibition of the inflammatory mediator.
Thus, it might be concluded that the analgesic effect of EJ-01 at the late phase is due to the inhibition of the
synthesis or release of inflammatory mediators such as cytokines and prostaglandins.
The hot plate and the tail flick models are considered as the specific tests for the evaluation of the central pain
(Marchioro et al., 2005) at the supraspinal and spinal levels (Wong et al., 1994), respectively. The EJ-01 did not
consid- erably increase the mean reaction time to the heat stimulus in both hot plate and tail flick tests at any time
period of the experiment except at 30 mg kg 1 dose at 5 h of its adminis- tration when compared with the vehicle
control. The hot plate method is one of the most common tests for evaluating the analgesic efficacy of drugs in
rodents (Somchit et al., 2004). However, care must be taken for drugs that produce false- positive results by
modifying the behavior of the rodents (Tjolsen et al., 1991). The observed effect at 30 mg kg 1 dose at 5 h in both
the tests indicates a false-positive observation as we did not find antinociceptive effect in the early phase of
formalin-induced pain. The central antinociceptive activity is related to activation of the endogenous inhibitory
control of pain (Saade & Jabbur, 2008). In the central pain models, the antinociceptive activity was not observed
probably due to the difficulty in crossing the bloodbrain barrier (BBB) by the EJ-01, revealing a probable
supraspinal antinociceptive effect if the compound was administered by intracerebroven- tricular route (Grisel &
Mogil, 2000). Our result shows that EJ-01 does not possess the opioid-like drug activity as it did not show the
analgesic activity in any of the central pain model (early phase of formalin-nociception, tail flick, and hot plate
test) used in our study which may be due to inability of EJ-01 to cross BBB or lack of intrinsic opioid-like activity
which needs to be explored by further investigation. The decreased analgesic activity of morphine in central pain
models at the 5th hour of the study may be due to characteristic property of opioid agonists (eg morphine, fentanyl,
and etorphine) which shows rapid onset with an early maximum effect, which remains for very short period after
drug administration, that mediate analgesia via central mechanisms under both normal and inflammatory
conditions (Aceto et al., 1997; Millan et al., 1987).
Cytotoxicity was determined by the MTT assay. In this test, EJ-01 did not affect the cell viability (RAW 264.7)
at 25, 50, and 100 mg mL1 concentrations as compared with the control and therefore these concentrations were
employed in the present in vitro anti-inflammatory study.
Among the inflammatory mediators, NO, prostaglandin E2 (PGE2), TNF-a, and IL-1b have crucial roles
during autoimmune diseases, infections, pain, edema, and fever (Perkins & Kelly, 1994). Pain and the immune
system influence each other, making it difficult to determine whether blocking nociception contributes for a
reduction in the production of pro-inflammatory cytokines or vice-versa, with the reduction in the formation of
pro-inflammatory cytokines resulting in less severe pain (Shavit et al., 2006).
In murine macrophage RAW 264.7 cells, LPS induces iNOS transcription and transduction and then the NO
production (Xie & Nathan, 1994). Furthermore, LPS stimu- lation is well known to induce NF- kB nuclear
translocation (Freeman & Natanson, 2000), a transcription factor necessary for iNOS, COX-2, TNF-a, and IL1b transcription. Therefore, RAW 264.7 cells provide an excellent model for drug screening and for subsequent
evaluation of potential inhibitors of the pathway leading NO production and cytokine release by NF-kB
pathway. Based on this information, efforts have been made to reveal the anti-inflammatory activities of EJ-01
on LPS-induced NO, IL-1b, and TNF-a production in murine macrophage RAW 264.7 cells.
NO synthesized by iNOS plays a critical role in pain conditions with an inflammatory component and the
sup- pression of NO production can be a very important target in the development of anti-nociceptive and anti-

inflammatory agents (De Alba et al., 2006). One needs careful attention while estimating NO in biological
systems (Feldman et al., 1993) as NO is rapidly oxidized to nitrite and/or nitrate. NO x content in biological
samples is a good indicator of amount of NO synthesis, because quantitatively these compounds are considered
to be the most stable metabolite of NO metabolism over the other metabolites (Blum et al., 2000) and
measurement of NOx levels is routinely used as an index of NO production (Moshage et al., 1995). In this study,
EJ-01 has been demonstrated to inhibit LPS-induced NOx produc- tion in RAW 264.7 macrophages (Figure 4A).
Similar effects have been demonstrated with the other kaempferol com- pounds of plant origin (Aggarwal et al.,
2009; Hamalainen et al., 2007).
To clarify the anti-inflammatory effect of EJ-01, we analyzed the levels of TNF-a and IL-1b in LPSstimulated macrophages. The cytokines, TNF-a and IL-1b are released by damaged tissue resident
macrophages. Bradykinin, TNF-a, IL-1, and IL-8 are particularly important in eliciting the inflammatory pain.
These agents liberate prostaglandins, other inflammatory pain mediators and are suggested to be correlated with
pain (Burke et al., 2006). Our results are further supported by a related in vivo study in rat formalin- induced
nociception where TNF-a and IL-1b levels are increased in paw and reduced by ketoprofen (Ahmad et al.,
2012) suggesting that EJ-01 might also produce its anti- nociceptive effect in a similar way in this study. IL-1 b
can produce hyperalgesia following either intraperitoneal, intra- cerebroventricular, or intraplantar injection
(Perkins & Kelly, 1994; Watkins et al., 1994). Moreover, IL-1b was found to increase the production of
substance P and PGE2 in a number of neuronal and glial cells (Jeanjean et al., 1995). Administrations of IL-10
and other anti-inflammatory cyto- kines have been demonstrated to prevent cytokine mediated inflammatory
hyperalgesia (Maier et al., 1993). In the in vitro model, we noted that reduced levels of IL-1b by EJ-01 (Figure
4C) which can suggest that EJ-01 may reduce inflammatory pain.
One more cytokine, TNF-a has been shown to play important role in both inflammatory and neuropathic
hyper- algesia. Intraplantar injection of complete Freund's adjuvant (CFA) in adult rats resulted in a significant
elevation in the levels of TNF-a, IL-1b, and nerve growth factor (NGF) in the inflamed paw. A single injection of
anti-TNF-a antiserum before the CFA significantly delayed the onset of the inflammatory hyperalgesia and
reduced IL-1b but not NGF levels (Woolf et al., 1997). Intraplantar injection of TNF-a also produces mechanical
(Cunha et al., 1992) and thermal hyperalgesia (Jeanjean et al., 1995).
Apart from the anticytokine antibodies therapies, other anticytokine therapies may also provide benefit in
relieving inflammation. Thalidomide and chlorpromazine are able to block TNF activity in vitro (Moreira et al.,
1993) and in vivo (Aarestrup et al., 1995; Ghezzi et al., 1996). These com- pounds which inhibit cytokine
production were also shown to be antihyperalgesic both in animals (George et al., 2000; Sommer et al., 1998) and
in man (Mehl-Madrona, 1999; Peuckmann et al., 2003). It was shown that thalidomide also has an antinociceptive.
effect in inflammatory pain. It inhibited carrageenan and LPS-induced mechanical hyper- algesia in rats and
inhibited zymosan and acetic acid induced writhing responses in mice. These effects were associated with the
inhibition of TNF-a production (Ribeiro et al., 2000b) suggesting that EJ-01 might also produce its antinociceptive
effect in a similar way in this study.
In conclusion, the EJ-01 isolated from E. jambolana exhibited antinociceptive effect and produced a decrease in
NOx, IL-1b, and TNF-a levels in stimulated RAW 264.7 cells, suggesting its anti-inflammatory effect and
attenuating inflammatory pain. Our in vivo results contributed to a better knowledge of this medicinal plant and the
pharmaco- logical profile of EJ-01. The results suggested that EJ-01 is a valuable analgesic constituent of leaves of
E. jambolana and support the pharmacological basis for the use this plant as traditional herbal medicine for
treatment of inflammatory pain.