Background
Pain is one of the most common manifestations of many diseases afflicting millions of people worldwide [
1,
2]. It is major symptom of various ailments that incessantly producing severe physical and psychological distress for many patients at the same time disrupting their quality of life [
3,
4]. In such situation, analgesics drugs are useful due to these agents ability to relieve pain without producing a loss of consciousness. Currently, three major classes of drugs are used in the pharmacological therapy of pain, namely non-steroidal anti-inflammatory drugs (NSAIDs), opioids and analgesic adjuvants, which target different components of the peripheral and central nervous system [
5‐
7]. Unfortunately, the undesirable adverse effects such as gastrointestinal damage, renal toxicity, sedation, tolerance and respiratory depression, always overshadowed those drugs effectiveness and limited their uses [
8,
9]. Due to this issue, peoples living in many developing countries in particular have been turning to natural products, particularly, those derived from medicinal plants/herbal medicines, as an alternative sources of pain-relieving agents [
10,
11]. The application of plant-based natural products in the treatment of various ailments have been associated with the assumption that natural products are at least safe for consumption and cheaper than synthetically-developed drugs [
12,
13].
One of the plants that have been traditionally used to treat pain is
Melastoma malabathricum L. (family Melastomaceae) [
14]. Locally known to the Malay as “
Senduduk”,
M. malabathricum has been used in the Malay traditional medicines to treat ailments such as stomach ulcers, dysentery and diarrhoea, those associated with pain (i.e., toothache and stomachache), to accelerate wound healing, for post-natal care and prevention of scars from small pox infection, and postpartum remedy [
15‐
19]. Scientifically, the leaves of
M. malabathricum have been reported to exert no acute toxicity [
20] and, antibacterial [
20,
21], antiviral [
20], antioxidant [
22], cytotoxic [
22], anti-inflammatory [
23,
24], anticoagulant [
25], antiulcer [
26], antidiarrheal [
20], antinociceptive [
16,
24] and antipyretic [
24] activities.
In order to justify the present antinociceptive study, it is important to highlight on the previous reports related to the antinociceptive activity of
M. malabathricum. In the two previous reports, the aqueous [
24] and ethanol [
16] extracts of the leaves were used and only the role of opioid receptors in modulating those extracts antinociceptive activity was investigated. In addition, we have recently published on the central and peripheral antinociceptive potential of methanol extract of
M. malabathricum (MEMM), and reported on the involvement of vanilloid receptors, glutamatergic system and NO-mediated/cGMP-independent pathway, but not opioid receptors, in the modulation of MEMM antinociceptive activity [
27]. Taking these facts into account, the present study was designed to determine the antinociceptive potential of several fractions derived from MEMM, namely petroleum ether- (PEMM), ethyl acetate- (EAMM,) and aqueous- (AQMM) extract, and to determine the mechanisms of antinociception exhibited by the most effective partition using various animal models. Briefly, the fractions were screened using the acetic acid-induced abdominal constriction test to select the most potent fraction. The most potent fraction (in this case PEMM) were then tested against the hot plate- and formalin-induced paw licking-test to establish its antinociceptive profile and subjected to further investigation on the possible mechanisms of action involving the role of opioid and vanilloid receptors, glutamate system and nitric oxide/cyclic guanosine phosphate (NO/cGMP) pathway.
Discussion
In an attempt to contribute to the discovery of alternative/new pain relieving agents with lack of unwanted side effects from medicinal plants, further antinociceptive investigations were performed on
Melastoma malabathricum leaves. The present study was a continuation to our recently published report on the antinociceptive activity of methanol extract of
M. malabathricum (MEMM) [
27]. The justification for performing this experiment can be related to the phytoconstituents of methanol extract. Methanol is classified as a polar solvent due to the presence of hydroxyl (−OH) group. However, there is also methyl group presence in methanol, which is sort of non-polar. Due to the presence of both polar and non-polar components in methanol, compounds that dissolve in water and oils equally well including their intermediates dissolves well in methanol. According to Ahmad et al. [
36], the ability to extract various types of compounds using a solvent like methanol is parallel to the ability to increase extract’s yield. Moreover, Caunii et al. [
37] reported that methanol: i) can give higher concentrations of bioactive molecules from plants, and; ii) is the best solvents for extraction of different classes of phenolic compounds. These reports might be used to explain the high total phenolic content (TPC) value of MEMM [
31] and supported the UHPLC analysis of MEMM, which demonstrated the presence of various flavonoids-based bioactive compounds (e.g. gallocatechin, epigallocatechin, catechin, chlorogenic acid, caffeic acid, quercetin, quercetin-3-O-glucoside, p-coumatic and hesperidin). In addition to these, the phytochemicals screening of MEMM also demonstrated the high presence of triterpenes, saponins and tannins [
31]. Taking into account the presence of various classes of bioactive compounds with different polarity [
27,
31], further investigations need to be carried out before the bioactive compound(s) responsible for the observed antinociceptive activity could be determined. Thus, the present study was designed to separate the bioactive compounds into non-polar, moderate polar (intermediate) and polar compounds by successive partitioning of MEMM using several solvents with different polarity to obtain the respective PEMM, EAMM and AQMM. It is worthmentioning that this attempt was performed based on suggestion made by Caunii et al. [
37] that if the methanol extract expressed remarkable biological activity, further analysis towards isolation and purification of the responsible bioactive compounds should be carried out.
In the present study, the fractions (PEMM, EAMM and AQMM) were first subjected to the antinociceptive study using the abdominal constriction test to assess the antinociceptive efficacy of each extract and the results obtained show that PEMM and EAMM exerted the antinociceptive activity in almost similar intensity. However, PEMM was chosen for further antinociceptive study based on its lower ED50 value in comparison to the EAMM. PEMM was also found to exert antinociceptive activity against the hot plate test and both phases (early and late) of the formalin-induced paw licking test. With regards to the mechanisms of antinociception of PEMM: i) PEMM inhibited the capsaicin- and glutamate-induced paw licking test suggesting the involvement of vanilloid receptors and glutamatergic system, respectively; ii) naloxone (a non-selective opioid antagonist) failed to reverse PEMM antinociceptive activity indicating the non-involvement of opioid receptor system, and; iii) L-arginine (a nitric oxide precursor), but not L-NAME (an inhibitor of NO synthase), MB (an inhibitor of cGMP), or their respective combination, reversed the antinociceptive activity of PEMM suggesting the involvement of NO-mediated/cGMP-independent pathway.
The acetic acid-induced abdominal constriction test has been associated with the activation of peripheral nociceptive processes [
38‐
40] and induction of acute peritoneal inflammation (localized inflammatory response) by the phlogistic agent. Moreover, the latter process occurs via the action of cyclooxygenase (COX) and increase in prostaglandins (PGE
2 and PGF
2α) biosynthesis [
35,
40‐
42]. Hence, any agents capable of inhibiting the action of COX or restricting the synthesis of PGEs could be good antinociceptive agents as seen with the peripherally acting non-steroidal anti-inflamamtory drugs (NSAIDs), ASA [
42,
43]. Interestingly, PEMM also attenuated the acetic-acid-induced peripheral nociception indicating the presence of analgesic principles with ability to attenuate inflammatory-mediated pain [
44] by acting, partly, to inhibit the action of COX and/or synthesis of PGEs as described earlier. Unfortunately, the abdominal constriction test is considered: i) a non-specific test due to its inability to provide information on the peripheral and/or central nociceptive level inhibited by PEMM [
45] and ii) to have poor specificity as it can give false positive results when use to test certain non-analgesic drugs such as muscle relaxants [
46]. Thus, the applications of other nociceptive models are necessary before the final conclusion on the possible mechanisms of action adopted by PEMM could be drawn. In the present study, the hot plate test and formalin-induced paw licking test were adopted to further determine the antinociceptive activity of PEMM.
The hot plate test is selective toward the centrally-acting analgesic drugs such as opioid analgesics (such as morphine) [
47] and measures the complex feedback to a non-inflammatory, acute nociceptive input resulting from a brief exposure to a noxious thermal stimulus. In the present study, PEMM successfully attenuated the thermal-induced nociceptive effect suggesting the extract ability to inhibit the central nociceptive center.
Another model of nociception, the formalin-induced paw licking test, is widely used to evaluate the ability of extracts/compounds to affect the peripheral and/or central nociceptive pathways. Being a model of persistent/continuing pain, the formalin test displays a biphasic response following the administration of formalin known as the early and late phases, which represents the respective centrally- and peripherally-mediated nociception [
48]. Drugs acting at the central level (such as morphine) inhibit both phases of the formalin test in comparison to drugs acting at the peripheral level (such as ASA), which inhibit only the late phase. In the present study, PEMM was also found to inhibit both phases of the formalin-induced nociception, thus, further suggesting its ability to block the central nociceptive center. Overall, findings obtained from the three nociceptive assays implied that PEMM contains non-polar bioactive compound(s) with ability to modulate the central and peripheral nociceptive mechanisms. PEMM was also able to attenuate both the non-inflammatory- and inflammatory-mediated pain.
The roles of opioid receptors in the regulation of modulation of nociceptive processing have been demonstrated in many previous studies [
49,
50]. However, the effectiveness of opioid analgesics (such as morphine) has been overshadowed by many adverse side effects (e.g. respiratory depression, vomiting, nausea, constipation, tolerance, and dependence). This is further worsening by the fact that prolongs use of morphine leads to the development of analgesic tolerance, which requires dosage increases to maintain its analgesic effect. This is problematic since dosage increases also amplify the frequency and severity of its side effects. Therefore, searching or developing new analgesics without these side effects is imperative. The non-opioid activity exerted by PEMM despite its ability to act centrally and peripherally as seen with morphine seems to be an added advantage when discussed in contact of finding new and alternative analgesics with unwanted side effects.
Furthermore, the roles of vanilloid receptors, also known as transient receptor potential cation channel subfamily V member 1 (TRPV1), and glutamatergic system in the regulation of nociceptive transmission have been reported elsewhere [
51,
52]. Vanilloid receptors, are activated by capsaicin, an active ingredient in hot chili peppers, and selectively acting on neurones within the peripheral and central nervous systems [
53,
54]. The present study shows that PEMM possessed antagonistic effect against the vanilloid receptors based on its ability to inhibit nociceptive transmission modulated via the vanilloid receptors. This observation was supported by earlier researches that reported on the ability of antagonists of TRPV1 receptors to exert antinociceptive activity and, to attenuate inflammatory- and neuropathic-pain [
51,
52].
On the other hand, the glutamatergic system (glutamate and glutamatergic receptors) has also been acknowledged to be vital in the peripheral, spinal, and supraspinal nociceptive neurotransmission [
55]. Activation of glutamatergic receptors, to a great extent, is interceded by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors, and the presence or absence of NO and NO-related substances [
56]. Furthermore, earlier report by Dickenson and Sullivan [
57] shows that the antagonists of NMDA receptor block the spread of pain sensation and lessen the hyperexcitability of spinal cord neurons generated by C-fiber stimulation. In line with the above-mentioned reports, the present study revealed the ability of PEMM to attenuate the glutamate-induced nociceptive effect, which could possibly be achieved by acting as an NMDA receptor antagonist or by modulating the NO-mediated pathway. Interestingly, the next findings did support the latter claim that PEMM attenuated glutamate-induced nociception via the NO-mediated pathway.
The role of NO/cGMP pathway in the modulation of nociceptive transmission at the PNS and CNS levels has been well documented [
58,
59]. In the earlier discussion we have highlighted that PEMM exerts a characteristic of morphine by acting at the central and peripheral nociceptive levels. Since morphine also exhibits antinociceptive activity via the NO/cGMP pathway activation [
60,
61], there is a need to also evaluate the involvement of NO/cGMP pathway in the antinociceptive activity of PEMM. In the present study, PEMM antinociception was reversed by high level of NO (due to presence of L-arginine alone) but was not affected by low level of NO (due to the presence of L-NAME alone). This observation is concurrent with suggestion that the effect of NO on nociceptive response depends on dosage levels and the rate and timing of its release [
58,
59]. As mentioned earlier, the presence of NO activates soluble guanylyl cyclase (sGC) leading to increase in cGMP levels, which in turn affects pain and analgesia. The role of cGMP pathway in the modulation of nociceptive process was observed when MB, an inhibitor of cGMP pathway, exerted antinociceptive activity when given alone. However, MB failed to affect the antinociceptive activity of PEMM suggesting that the antinociceptive activity of PEMM did not involved modulation of cGMP pathway. This finding also indicates that PEMM might trigger antinociceptive activity via the NO-dependent/cGMP-independent pathway. Moreover, since opioids like morphine induced antinociceptive activity via the NO/cGMP pathway, it seems reasonable to suggest that the non-opioid-acting PEMM triggered antinociceptive activity via a different pathway mediated by NO, but independent of cGMP activity (NO-mediated/cGMP-independent pathway). The role of NO-dependent/cGMP-independent pathway in the modulation of antinociceptive activity has been reported by Morioka et al. [
62] and could be used to support the present observations.
Previous phytochemical screening of MEMM demonstrated the strong presence of flavonoids, triterpenes, tannins, saponins and steroids, but no alkaloids in the leaves of
M. malabathricum [
31]. On the other hand, the phytochemical screening of PEMM demonstrated the strong presence of only triterpenes with low presence of flavonoids, tannins and saponins. The low presence of flavonoids in PEMM particularly was further supported by the HPLC and UHPLC-ESI analyses. In the former analysis, only two peaks were detected at 366 nm, which upon comparison of retention time and chromatogram against 10 pure flavonoids did not match to any of them, thus, suggesting their absence in PEMM. In the latter analysis, only two small peaks were detected and identified as gallocatechin and epigallocatechin, respectively. In support of the phytochemical screenining of PEMM, Nurestri et. al. [
63] isolated three pentacyclic triterpenoids, namely ursolic acid, 2-hydroxyursolic acid and asiatic acid in addition to glycerol-1,2-dilinolenyl-3-O-β-D-galactopyranoside and glycerol 1,2-dilinolenyl- 3-O-(4,6-di-O-isopropylidene)-β-D-galactopyranoside from MEMM while partitioning of MEMM using hexane (HEMM), which is also grouped as a non-polar solvent like petroleum ether, lead to the isolation of a triterpene (α-amyrin) and two amides (patriscabatrine and auranamide) [
25]. Based on the presence of triterpene-based compounds as described above, further explanation with regards to the possible mechanisms of antinociception of PEMM could be plausibly suggested. For examples; i) 3β, 6β, 16β-trihydroxylup-20(29)-ene inhibited glutamate- and formalin-induced nociceptive models, and exert antinociceptive activity that is dependent on the opioid and serotonergic systems [
64]; ii) siaresinolic acid reduced the acetic acid-induced nociceptive response via the activation of non-opioid system and ATP–dependent potassium channels [
65]; iii) 24-hydroxytormentic acid inhibited the acetic acid- and formalin-, but not thermal-induced nociception via mechanism that did not involve modulation of the opioid, nitric oxide or serotonin systems [
66], and; iv) α-amyrin and β-amyrin, in mixture, exerted significant antinociception only against the acetic acid-, capsaicin-, glutamate- and formalin-, but not thermal-induced nociceptive models in mice. Interestingly, this mixture also exhibited a non-opioid-mediated antinociceptive activity [
67]. As described above, two reports [
66,
67] on the failure of triterpenes to attenuate thermal-induced nocicpetion seems to contradict our findings with triterpene-rich PEMM. This discrepancy could be attributed to the low dose (30 mg/kg) of pure triterpenes used by those authors whereas in the present study PEMM fraction was effective only at the doses of 250 and 500 mg/kg.
Further analysis using the GCMS method on the presence of volatile compounds in PEMM revealed the presence of 29 volatile compounds of which five are the major compounds, namely, oleoamide (9-octadecenamide) (11.54%), 23-ethyl- (3β-23S)-cholest-5-en-3-ol (10.06%), palmitic acid (6.95%), phthalic acid (5.90%) and linolenic acid methyl ester (5.69%). Of these, at least, oleoamide and palmitic acid has been reported to show some pharmacological activities related to the antinociceptive activity of PEMM. Oleamide, for example, has been reported to exert anti-inflammatory activity when assessed using the lipopolysaccharide (LPS)-induced BV2 microglial [
68]. The inhibition of inflammation occurs via inhibition of NO and PGE
2 production. Moreover, oleamide also inhibits the activation of NFκB and PI 3-kinase, as well as the phosphorylation of inhibitor κB kinase, Akt, p38 MAPK, and ERK, and accumulation of reactive oxygen species (ROS) induced by LPS on BV2 microglial. On the other hand, Déciga-Campos et al. [
69] have earlier reported on the antinociceptive activity of several palmitic acid derivatives, namely N-(4-Methoxy-2-nitrophenyl)hexadecanamide, 2-amino-3-(palmitoylamino)benzoic acid or 4-amino-3-(palmi-toylamino)benzoic acid when assessed using the abdominal constriction and hot plate tests.