Polymethoxyflavones from Nicotiana plumbaginifolia (Solanaceae) Exert Antinociceptive and Neuropharmacological Effects in Mice

Polymethoxylavones (PMFs) are known to exhibit significant anti-inflammatory and neuroprotective properties. Nicotiana plumbaginifolia, an annual Bangladeshi herb, is rich in polymethoxyflavones that possess significant analgesic and anxiolytic activities. The present study aimed to determine the antinociceptive and neuropharmacological activities of polyoxygenated flavonoids namely- 3,3′,5,6,7,8-hexamethoxy-4′,5′-methylenedioxyflavone (1), 3,3′,4′,5′,5,6,7,8-octamethoxyflavone (exoticin) (2), 6,7,4′,5′-dimethylenedioxy-3,5,3′-trimethoxyflavone (3), and 3,3′,4′,5,5′,8-hexamethoxy-6,7-methylenedioxyflavone (4), isolated and identified from N. plumbaginifolia. Antinociceptive activity was assessed using the acetic-acid induced writhing, hot plate, tail immersion, formalin and carrageenan-induced paw edema tests, whereas neuropharmacological effects were evaluated in the hole cross, open field and elevated plus maze test. Oral treatment of compounds 1, 3, and 4 (12.5–25 mg/kg b.w.) exhibited dose-dependent and significant (p < 0.01) antinociceptive activity in the acetic-acid, formalin, carrageenan, and thermal (hot plate)-induced pain models. The association of ATP-sensitive K+ channel and opioid systems in their antinociceptive effect was obvious from the antagonist effect of glibenclamide and naloxone, respectively. These findings suggested central and peripheral antinociceptive activities of the compounds. Compound 1, 3, and 4 (12.5 mg/kg b.w.) demonstrated significant (p < 0.05) anxiolytic-like activity in the elevated plus-maze test, while the involvement of GABAA receptor in the action of compound 3 and 4 was evident from the reversal effects of flumazenil. In addition, compounds 1 and 4 (12.5–25 mg/kg b.w) exhibited anxiolytic activity without altering the locomotor responses. The present study suggested that the polymethoxyflavones (1–4) from N. Plumbaginifolia could be considered as suitable candidates for the development of analgesic and anxiolytic agents.


INTRODUCTION
Pain is an unpleasant sensory perception accompanied by physiological damage comprising actual or potential tissue injury (de Sousa, 2011). Such sensation also relies on individual's emotional state and can be exacerbated by the psychological disorders like anxiety and depression. It adversely affects the quality of life and is one of the common reasons for visiting physicians and taking medications (de Santana et al., 2015). However, conventional medicines such as non-steroidal antiinflammatory drugs (NSAIDs), steroids, and opioid analgesics are associated with significant side effects including gastric ulcers, dependence and depression, making drug therapies more complex and difficult (Poetker and Reh, 2010;Tiwari and Singh, 2014).
oral toxicity was determined according to the recommendation (420-fixed dose procedure) of Organization for Economic Cooperation and Development (OECD). After experiments, the animals were subjected to euthanasia using pentobarbital following AVMA Guidelines for the Euthanasia of Animals of 2013 edition. All experimental methods and principles were endorsed by the Ethics Committee of Stamford University Bangladesh (SUB/IAEC/15.03). Every effort was taken to alleviate the potential sufferings of the animals.

Animals
Pharmacological investigations were conducted on 20-25 g, 8-10 weeks old healthy male Swiss albino mice. The animals were obtained from the Animal Resources Branch of the International Center for Diarrhoeal Disease Research, Bangladesh (icddr,b). The procured animals were rehabilitated in 120 × 30 × 30 cm cages with wood shavings bedding, kept under controlled laboratory condition of 24 ± 2 • C temperature, 55-60% relative humidity and 12-h light-dark cycle (light on 7:00 a.m. to 7:00 p.m.). They had free access to standard diet and water ad libitum. They were acclimatized in the laboratory environment for 14 days prior to the investigations. The healthcondition of the animals was checked regularly. The animals were randomly chosen and allocated into negative control, positive control, and experimental group. Each group had six animals (n = 6). Investigators, who were responsible for recording data, were unaware of the assigned group to avoid any experimental bias. The test animals did not have any access to food 3-4 h prior to the commencement of the experiments and experimentations were carried out between 9:00 a.m. and 5:00 p.m.

Treatments
Animals of experimental groups were treated with single oral dose 12.5 or 25 mg/kg b.w. of the isolated compounds. The doses were selected based on pilot experiment and previously reported oral effective doses of PMFs (Jang et al., 2013). All experimental doses were formulated with physiological saline containing 0.5% Na-CMC. The control group was treated per oral (p.o) with 0.5% Na CMC as vehicle at the dose of 10 mL/kg b.w. The vehicle and experimental PMFs were administered 60 min before the experiments. PMFs have been reported to possess excellent absorption and oral bioavailability properties (Li et al., 2009), and are effective after 1 h of oral administration (Jin et al., 2012). The pharmacokinetics of drugs from oral treatment is considered to be similar to that of intraperitoneal (i.p.) treatment (Turner et al., 2011). Therefore, the oral effect of experimental PMFs has been compared with the intraperitoneally (i.e., parenteral) administered standard drugs. For the antinociceptive tests, morphine sulfate, and diclofenac sodium were used as positive control drugs at the dose of 5-and 10-mg/kg b.w., respectively. Since parenteral drugs have quicker onset of action compared to oral administration of (Turner et al., 2011) standard drugs, diclofenac (i.p.) or morphine (i.p.) were administered 30 min before the experiments. In the hot plate and tail immersion tests, naloxone was employed (i.p.) at the dose of 2 mg/kg b.w., 15 min before the administration of morphine sulfate or isolated compound to verify the possible involvement of opioid system. To evaluate the association of ATP-sensitive K + channel in the analgesic effect, glibenclamide (Gbc), an ATP-sensitive K + channel blocker, was administered at the dose of 10-mg/kg b.w. (i.p.), 15 min prior to administration of the isolated compound in the acetic acid-induced writhing test. Diazepam was used as the positive control in the hole cross, open field, and elevated plus maze test at 1 mg/kg (i.p.) b.w. 30 min prior to the commencement of the experiments. The participation of benzodiazepine receptor in the anxiolytic effect was justified using flumazenil, a selective benzodiazepine antagonist of GABA A receptor, at the dose of 2.5 mg/kg (i.p.), 15 min before the administration of the plant isolates or diazepam in elevated plus maze test.

Safety Evaluation (Acute Toxicity Test)
The selected doses (12.5-25 mg/kg, b.w.) of the experimental compounds were administered into the animals following the guidelines (420 fixed dose procedure) recommended by Organization for Economic Cooperation and Development (OECD) for acute toxicity study. Experimental animals were allocated into a control and eight test groups. Six animals were selected for each group (n = 6). Control and experimental group animals received single oral dose of vehicle (1% Na-CMC, 10 mL/kg b.w.) and test compound (12.5 or 25 mg/kg b.w.) respectively. The animals abstained from feed and water 2 h prior to treatment with vehicle or test compounds. After oral gavage, each group of mice was separately housed and had access to feed and water ad libitum. The animals were then observed occasionally for the next 24 h and daily for a total of 14 days. During this period, animals were observed for mortality, aberrant behavior, salivation, tremors, convulsion, and any abnormalities regarding food consumption, eyes (irritation or discharges), hair, skin (irritation, rashes, swelling or itching), and feces (physical appearance and bowel movements). At the end of the observational session, the body weight of the body weight of the animals that survived were recorded animals was recorded. Then they were sacrificed following euthanasia and vital organs of the body were isolated, examined for any abnormalities and weighed to determine any significant changes (Sehar et al., 2008;Talwar et al., 2013).

Antinociceptive Assays
Acetic Acid-Induced Writhing Test The experiment acetic acid-induced pain, characterized by writhing syndrome was performed to evaluate the central and peripheral analgesic effect of the plant isolates. Experimental mice were acclimatized in the individual experimental chamber for 60 min before the test. Each mouse received intraperitoneally (i.p.) 1% w/v acetic acid (10 mL/kg) 60 min after vehicle or isolates and 30 min after diclofenac treatment. The nociceptive characteristics such as twisting of the trunk, elongation of the hind limb, stretching of the abdomen induced by the noxious stimulus were marked as writhing episodes. The time taken to begin writhing response after acetic acid administration was recorded as the onset of writhing. Beginning with the first writing response, the number of writing episodes were noted in each 10 min in the 60 min of the observational period (Bagdas et al., 2016). Analgesic activity was defined as the diminishing of writhing episodes and the percentage of inhibition of writhing was calculated as follows: Writhing of control group − writhing of treatment group Writhing of control group × 100

Hot Plate Test
The experiment was carried out to measure the analgesic activity of the compounds 1-4 against thermal pain threshold. Mice were gently handled and placed on the heated surface of hot plate apparatus (Eddy's hot plate, Kshitij Innovations, Haryana, India). The hot plate surface was covered by cylindrical glass and temperature was kept stable at 55 ± 1 • C. The time taken to react to the thermal nociception by responses such as licking of the paw(s), jumping was recorded as latency time (Eddy and Leimbach, 1953). Animals which exhibited pre-treatment latency of 5-20 s were selected for the experiment and maximum latency time was fixed at 20 s (cut-off time) to avoid any tissue injury. A latency of each mouse was recorded just before treatment (0 min) and they served as a control of their own. Then latency was recorded at 30, 45, 60, 90, and 120 min following the administration of vehicle, isolates (p.o.) or morphine (i.p.). An increase in latency time was considered analgesic against thermal pain. The maximal possible analgesic effect was calculated as a percent (% MPE) from the following formula: Post-treatment reaction time − pre-treatment reaction time 20 − pre-treatment reaction time × 100 The % MPE was plotted against time and area under the curve (AUC 0-120 min ) was determined by trapezoidal rule (Bhargava et al., 1989).

Tail Immersion Test
The tail immersion test was performed to evaluate the centrally mediated analgesia of the isolated compounds 1-4 against the thermal nociceptive stimulus. Briefly, mice were immobilized gently by using "chux" and 1-2 cm of the tail of each mouse was immersed into warm water thermostatically maintained at 52 ± 1 • C (Janseen et al., 1963). The rapid flick of the tail was regarded as the end-point of nociception and time taken to flick tail was noted (latency time). Mice that flicked their tail between 1.5 and 3.5 s before treatment were considered for the experiment. Then the mice were treated with vehicle, isolates or morphine and the latency was counted after 30, 45, 60, 90, and 120 min of treatment. A 20 s cut-off time was fixed in order to prevent tissue injury of mice. The pre-treatment latency of each mouse served as baseline. The maximal percent of analgesia at each observation time and (AUC 0-120 min ) was determined as described in hot plate test.

Formalin-Induced Nociception and Paw Edema Test
The effect of isolates against chemical-induced pain was modeled by formalin test as the experiment is valid and frequently used for the evaluation of analgesic as well as anti-inflammatory agents (Hunskaar and Hole, 1987). Mice were adapted in an individual observational cage for 60 min. The experimental mice were gently held and pre-treated with vehicle, the plant isolates or morphine before formalin injection. The nociception was elicited by the treatment of 20 µL solution of 5% formalin into the right hind paw of mice. The licking and biting of the right hind paw by the mice were considered as responses of nociception and time spent for showing the responses was recorded in every 5 min for total 60 min of observation. The first 10 min of observation was defined as phase I or neurogenic phase and the next 50 min was Phase II or inflammatory phase. The vertical paw thickness of each mouse was measured before and 5 h after the treatment of formalin using a digital fine caliper (M: 091552; Shanghai Shenhan Measuring Tools Co. Ltd., Shanghai, China). Then edematogenic inflammatory response was calculated from difference between the paw thickness ( ) of post and preformalin treatment (Wheeler-Aceto and Cowan, 1991;Xiao et al., 2008).

Carrageenan-Induced Paw Edema Test
Anti-inflammatory effect of the isolates was studied by the carrageenan-induced paw edema test. The experimental mice received vehicle, experimental compounds or diclofenac and were subjected to measure their left hind paw thickness by a Vernier caliper before carrageenan treatment. A 25 µL solution of 1% w/v lambda carrageenan was injected deliberately under the subplantar aponeurosis of the right hind paw of the mice in order to induce edema (Morris, 2003). The degree of inflammation was defined by the thickness of edema. The paw edema thickness ( in mm) was measured from the difference between left hind paw thickness of before and 0, 1, 2, 3, 4, 5, and 6 h after carrageenan administration. After the observing session, experimental mice were euthanized. The experimental paws were dissected and soaked overnight in 2.5 mL of physiological saline at the temperature of 0-4 • C. The solution was centrifuged for 15 min at 3,000 rpm to obtain as upernatant and pellet. A volume of 0.5 mL supernatant was mixed with 2 mL solution of 0.5 M KOH (dissolved in MeOH) and incubated at 50 • C for 20 min. Upon cooling, the mixture was supplemented with MeOH up to 5 mL volume and thoroughly mixed. Five minutes later the absorbance of the mixture was read at 278 nm by UV spectrophotometer (Chopade and Sayyad, 2015). The PGE2 content was represented by optical density (OD) value of the mixture.

Hole Cross Test
The effect of isolated compounds on the motor activity of the mice was evaluated by the hole cross test. The experiment was conducted following the method described by Takagi et al. (1971). Briefly, experimental mice were pre-treated with vehicle, compounds 1-4 or diazepam. They were individually placed in a box (13 × 14 × 20 cm 3 ), having two compartments separated by a fixed partition. The partition contained a hole of 3 cm to facilitate the passage for mice. The mice were placed gently facing toward the hole in one of the compartments of the box. The transitions of each mouse from one compartment to another were documented for 3 min. The observation was made at 0, 30, 60, 90, and 120 min following treatment and % inhibition was determined from the transitions.

Open Field Test
Effect of the compounds on general locomotor performance was evaluated by the open field test. The procedure was carried out as described by Frye and Walf (2002). The open field apparatus (76 × 57 × 35 cm 3 ) consisted a floor divided into 48 black and white colored square grids. Each square was colored alternatively. The 24 perimeter squares were considered as peripheral squares while others were central. Experimental subjects were pre-treated with compounds 1-4, vehicle or diazepam. They were then individually placed at the center of the floor and the number of peripheral and central square crossed was documented for 5 min. The experimentation was performed in an isolated and sound-attenuated area. The total number of square crossed (ambulation) was calculated and percent of central square crossed was determined. The number of ambulation was used to determine the % inhibition of locomotion using following formula: The increase in the number of central square crossing and percentage increase of central square crossing of mice were regarded as anxiolytic whereas reduction of such explorations was considered as anxiogenic (Prut and Belzung, 2003).

Elevated Plus Maze Test
The anxiolytic action of the compounds was evaluated on elevated plus maze apparatus. The apparatus was elevated at a height of 50 cm from the floor and consisted of two elongated closed and open arms of equal dimensions (50 × 10 cm). The closed arms were enclosed by side-wall of 40 cm height. Each type of arms was arranged perpendicularly to each other. Mice were pre-treated with vehicle, isolates, or diazepam and each mouse was individually placed in the center of the maze facing their head toward one of the closed arms. The number of entries and time spent in close and open arms of the maze was recorded for the period of 5 min. The entrance of four paws of mice into an arm was regarded as an entry. The increase of the entries and percent entries, as well as exploration time in open arms of mice was considered as anxiolytic, whereas the opposite was regarded as anxiogenic (Pellow et al., 1985).

Involvement of Opioid Receptor
To investigate the role of opioid system in the antinociceptive action of the isolated PMFs, mice were treated with a nonselective opioid antagonist, naloxone (2 mg/kg b.w., i.p.), 15 min before administration of the compound (25 mg/kg b.w., p.o.), or morphine (5 mg/kg b.w., i.p.) in the tail immersion and hot plate test. The thermal latency time of the experimental mice was documented before the treatment of standard drug or experimental compound and at 30, 45, 60, 90, 120 min of investigation period (Khan et al., 2011). The cut-off period was maintained for 20 s to avoid tissue injury as described in hot plate and tail immersion test. The documented data were assembled with the hot plate and tail immersion test result for comparison.

Association of the ATP-Sensitive K + Channel Pathway
The association of ATP-sensitive K + channel in the pain inhibition action of the isolates in mice was evaluated as previously described Perimal et al., 2011). Animals were pre-treated with glibenclamide (10 mg/kg b.w., i.p.), an ATP-sensitive K + channel inhibitor 15 min before the administration diclofenac or effective dose (25 mg/kg b.w., p.o.) of the plant isolates. Mice were treated with 1 % w/v acetic acid (i.p.) after 60 min of the isolated compounds 1-4 administration and 30 min of diclofenac. Then, time taken to start writhing (onset time) was recorded and writhing episodes were counted in every 10 min for 60 min as described in the acetic acid-induced writhing test. These data were assembled with acid-induced writhing test result for comparison.

Involvement of Benzodiazepine Receptor
To verify the role of benzodiazepine system in the anxiolytic action of the isolated PMFs, flumazenil, a selective antagonist of GABA A receptor was administered at the dose of 2.5 mg/kg (i.p.), 15 min before the administration of isolates or diazepam in elevated plus maze test (Aragão et al., 2006). After 60 min of isolated PMFs and 30 min of diazepam employment, the experimental mice were placed on the central square of maze facing to the close arm. Thenumber of entries and time spent in close and open arms was recorded for 5 min and % open arm time and entries were calculated as stated in elevated plus maze test.

Data Analysis
All results have been shown as median (n = 6) with range (minmax). The area under the curve (AUC) response was calculated by trapezoidal rule as an expression of the intensity of the effect. Data analysis was performed using Kruskal Wallis followed by Mann-Whitney test at the levels of significance ranging from p < 0.05 to 0.001 by SPSS 22 (IBM, USA).

Acute Toxicity
Oral treatment of selected doses for compounds 1-4 did not cause any abnormal effects or mortality during 14 days of observation. In addition, administration of PMFs 1-4 did not produce any injuries, sign of abnormalities and significant differences to the gross weight of the vital organs or body ( Table 1) in animals.
Naloxone pre-treatment reverted the antinociceptive action of compounds 1-4 and morphine significantly (p < 0.05), where it itself could not induce any significant differences in thermal protection with respect to control group. The effect of compounds 1-4, morphine and naloxone pre-treatment on the latency time was also significant (p < 0.05) at different experimental periods as shown in Supplementary  Table 3.
only compound 3 and morphine could approach the statistical significance (p < 0.01). Compound 3 showed dose-dependent protection in thermally induced pain and the effect was statistically significant (p < 0.01) from 45 min onwards for all experimental doses. Morphine (5 mg/kg b.w., i.p.) exhibited significant reduction in thermal nociception for all observational sessions while the maximal effect was seen for compound 3 at the dosage of 25 mg/kg b.w. by 20.35 (14.80-25.45)% on 120 min than it [13.30 (6.65-21.34)%] (Figure 4A). The AUC 0−120 min response demonstrated that thermal nociceptive protection by morphine and compound 3 at 12.5 and 25 mg/kg b.w. was 11.91, 3.00, and 4.87 times greater and statistically significant (p < 0.01) with respect to control group respectively ( Figure 4B). Naloxone treated animals could not produce any significant effect compared to control animals. Naloxone pre-treatment reversed the percent analgesic as well as AUC 0−120 min effect of morphine and compound 3 (Figures 4A,B). Morphine and compound 3 and pre-treatment of naloxone also caused significant differences (p < 0.01) in thermal latency time at multiple observational periods compared to control group as shown in Supplementary  Table 4.

Formalin-Induced Nociception and Paw Edema
The treatment of experimental PMFs 1-4 (p.o.) and morphine (i.p.) diminished the formalin induced early and late phase nociceptive responses as shown in

Carrageenan-Induced Paw Edema
The subplantar injection of carrageenan to the control group mice caused gradual increase in paw edema from 1 to 5 h, which decreased at 6 h as shown in Table 3. Mice treated with experimental PMF 1, 3 and 4 at all test doses caused significant (p < 0.05) reduction of paw edema from 3 to 6 h compared to control group. Compound 3 at the dose of 25 mg/kg b.w., p.o. and diclofenac (10 mg/kg b.w., i.p.) exhibited significant reduction as well as percent inhibition of paw edema from 1 h to end of the experimental period. PGE2 content (equivalent to OD) of diclofenac and PMF 1, 3 and 4 treated mice were significantly (p < 0.01) less than thecontrol group. Exoticin (2) could neither inhibit edematogenic syndrome nor PGE2 content by any of the experimental dose. The inhibition of paw edema throughout the entire experimental time and reduction of PGE2 content was maximum by diclofenac where compound 3 showed highest at 25 mg/kg b.w. compared to other tested PMFs ( Table 3). The result also showed that there was dose effect for all tested compounds.

Hole Cross
The treatment of experimental compounds 2, 3 (p.o) and diazepam (i.p.) caused the decrease of movement of mice in the hole cross test compared to control group mice (Table 4). However, the effect was significant (p < 0.05) for compound 2 and 3 at the dose of 25 mg/kg b.w. Diazepam (1 mg/kg b.w.) and compound 2 (25 mg/kg b.w.) administration produced significant inhibition of movement at all experimental periods where the effect was maximum by exoticin at 30 min and from
The PMFs of N. plumbaginifolia significantly (p < 0.01) delayed the onset as well as caused the inhibition of writhing episodes (Figures 2A,B) in the acetic acid-induced writhing test. This test is a reliable method for the evaluation of central and peripheral analgesic effect of new agents (Le Bars et al., 2001). Administration of acetic acid (i.p.) causes liberation of cyclooxygenase (COX), prostaglandins (PGs), lipoxygenase (LOX), histamine, serotonin, bradykinin, and cytokinin (TNF-α, IL-8, IL-1β), in the tissue of visceral fluid. These inflammatory mediators disrupt as well as increase the permeability of blood brain barrier (BBB) and excite primary afferent nociceptors by entering into the dorsal horn of central nervous system (CNS). This results in a pathological condition of pain, which is characterized as writhing (Ikeda et al., 2001;Radu et al., 2013). Therefore, it could be suggested that the PMFs diminished the release of the acid-induced endogenous inflammatory mediators. The delayed writhing onset by the PMFs indicates their interruption in nociceptive signals transduction to primary afferent nociceptors. In addition, attenuation in the numbers of acetic writhing episodes, delayed onset time of writhing, might be attributed to the down-regulation of the inflammatory cytokines TNF-α, IL-1β, and IL-6 proteins (Yin et al., 2016).
Carrageenan-induced paw edema test is widely used in the evaluation of the anti-edematogenic effect of the experimental compound. Subplantar injection of carrageenan produces a biphasic edematogenic response where serotonin, histamine, and kinins are released in the first phase (0-1 h), whereas the final phase (1-6 h) is associated with the release of prostaglandins (PGs), particularly of their E series by the activation of cyclooxygenase (COX) in tissues. Kinins provide the continuity between these inflammatory phases (Morris, 2003;Mothana, 2011). Inducible nitrogen oxide synthase (iNOS) might also be involved in the formation of carrageenan-induced paw edema (Salvemini et al., 1996;Mortada et al., 2017). The findings of the present study revealed that PMFs 1, 3, and 4 could significantly reduce carrageenan-induced paw edema (Table 3). Therefore, a significant decrease of both first and second phase edematogenic response by compound 3 could be due to the inhibition of release of serotonin, histamine, and PGs or downregulation of iNOS production. On the other hand, compound 3 and 4 could significantly inhibit the second phase edema suggesting their suppression of release of COX products as well as PGs. It has been found that PGE2 plays a major role in hyperalgesia, IL-6 production and tissue edema at sites of inflammation (Portanova et al., 1996). The significant (p < 0.01) reduction of PGE2 content by the plant PMF 1, 3, and 4 ( Table 3) could be attributed to their anti-edematogenic as well as anti-inflammatory effect.
Previous studies reported that mono and polymethoxyflavones (PMFs) exert opioid mediated antinociceptive effect (Thirugnanasambantham et al., 1993;Pandurangan et al., 2014;Nadipelly et al., 2016). Structure activity relationships study revealed that the presence of methoxy group (-OCH 3 ) at 5 or 7 positions on flavone moiety could induce opioid mediated analgesia (Thirugnanasambantham et al., 1993). The experimental PMFs of present study also comprises -OCH 3 group at 5 and/or 7 positions. Moreover, results of the present investigation demonstrated that naloxone pre-treatment diminished the thermal analgesia of PMFs 1-4 in the hotplate or tail immersion test (Figures 3, 4). This confirmed the association of opioid system in the antinociceptive effect of the plant PMFs. The results also suggested that the effect of PMF 1 and 4 involved supraspinal, where PMF 3 involved both spinal and supraspinal opioid receptors.
Drugs acting on the spinal and supraspinal systems (e.g., morphine, diazoxide) may involve the ATP sensitive K + channel (K ATP ) for their antinociceptive effect. In addition, antinociceptive effect of diclofenac, ketorolac involves opening of K ATP by increasing the intracellular cyclic guanosine monophosphate (cGMP) level in tissues. Activation of NO-cGMP pathway causes the opening of K ATP followed by an effluxof K + ion, membrane re-or hyper-polarization and reduces the membrane as well as cellular excitability (Lawson, 1996;Ocaña et al., 2004). Glibenclamide has been reported to selectively block the ATP sensitive K+ channels without affecting voltage gated and Ca 2+ activated K + channels (Alves and Duarte, 2002;Jesse et al., 2007). Results of the current study exhibited that the effects of PMFs 1-4 were significantly reversed by glibenclamide (Figures 2A,B). Thus, it can be suggested that the PMFs 1-4 might involve opening of K ATP system followed by reduction of membrane and cellular excitability for their antinociceptive effect.
Oral treatment of experimental doses did not exert any toxicity, abnormalities of the organ or cause the death of animals, which indicated that the selected test doses were safe for the study. The neurobehavioral study showed that increase in the dose of the experimental PMFs caused a reduction in locomotor activity, whereas compounds 2 and 3 produced a significant (p < 0.05) reduction at a maximal experimental dose (25 mg/kg b.w.) in the hole cross test ( Table 4). The effect was also reflected by diazepam and in the open field test (Figures 5A-C). However, compound 3 increased the number as well as the percent of central squares entries in the open field. As shown by the results, compounds 1 and 4 also increased the number as well as the percent of central squares entries in the open field but did not cause any significant alteration of the locomotor activity. It has been reported that anxiolytic drugs such as benzodiazepines and 5-HT 1A agonists increase central and percent of squares entries of mice on open field (Prut and Belzung, 2003). The results indicated that compounds 1, 3, and 4 could possess anxiolytic effect. Therefore, the plant isolates were subjected to evaluate anxiolytic activity using elevated plus maze test.
Elevated plus maze is a commonly applied method for the evaluation of anxiolytic activity of new agents (Dawson and Tricklebank, 1995) and it is validated for both rodents and mice (Pellow et al., 1985;Lister, 1987). The apparatus contains two open and closed arms. Animals show extreme aversion to exploring in the open arms. The indexes of anxiety-exploration time and number of entries in open arms are sensitive to the drugs that act on benzodiazepine as well as GABA A receptor sites. These drugs increase the percent of open arm frequencies as well as exploration time and decreases total arm entries (Pellow et al., 1985;Griebel et al., 1996). The results of the experiment showed that PMFs 1, 3, 4 and diazepam significantly increased the percent of exploration time and frequency of entries in open arms (Figures 6B,C). These effects could be attributed to their anxiolytic activity. However, compounds 1 and 4 did not cause any significant reduction of total number of entries at the experimental doses, where compound 3 at the maximal dose (25 mg/kg) and diazepam produced significant (p < 0.05) reductions ( Figure 6D). Flumazenil, a selective benzodiazepine antagonist of GABA A receptor, significantly (p < 0.05) diminished the effects of compounds 3 and 4. This confirmed the involvement of GABA A receptor in their anxiolytic activity. The results showed that flumazenil could not attenuate the anxiolytic effect of PMF 1, suggesting a different mechanism of action. Experimental results also demonstrated that an increase of dose reduced the anxiolytic effect of the PMFs. Drugs acting on benzodiazepine system has been reported to significantly decrease the locomotor function (Prut and Belzung, 2003). The effects could be due to the reduction of locomotor activity which are evident in the hole cross (Table 4) and open field test (Figure 5) results.
Overall, applications of N. plumbaginifolia in the treatment of different painful conditions are evident in traditional systems of medicine and analgesic action along with anxiolytic effects of its crude extract have already been justified. This investigation determined that bioactive compounds of the plant could be related to its actions and justify the ethnopharmacological importance of the plant. The results of the present investigation revealed the antinociceptive potential of the experimental PMFs of N. plumbaginifolia, where 3,3 ′ ,5,6,7,8-hexamethoxy-4 ′ ,5 ′ -methylenedioxyflavone (1) 6,7,4 ′ ,5 ′ -dimethylenedioxy-3,5,3 ′ -trimethoxyflavone (3) and 3,3 ′ ,4 ′ ,5,5 ′ ,8-hexamethoxy-6,7-methylenedioxyflavone (4) produced significant, dose-dependent effect. The antinociceptive action of the test compounds involved opioid receptor, ATP-sensitive K + channel as well as suppression of inflammatory mediators such as PGs, COX, LOX. Although the PMFs have been administered intragastrically and standard drugs intraperitoneally, the effect of compound 3 was greater than the respective standard drug in hot plate and acetic acid tests to some extent (% MPE and onset of writhing, respectively) for the positive response which could be considered a significant finding. Evaluation of neuropharmacological activities aided the elucidation of their anxiolytic activity. The study also demonstrated anxiolytic-like action involving benzodiazepine receptors without altering the locomotor responses at experimental doses. However, direct modulation of the receptors and nociceptive mediators by the experimental PMFs will be a greater part of interest. Considering the present findings into account, it may suggest that the PMFs of N. plumabginifolia could be suitable candidates for the development of analgesics as well as anxiolytic agents.

AUTHOR CONTRIBUTIONS
The study was conceived and designed by BKD, MAR, and MSS. The experiments were carried out by MSS and SI. MSS and RBR performed data analysis. MSS, MAR, and RBR drafted the manuscript. SDS, LN, LCM, and MMRS have gone through the manuscript to edit and improved the quality of the manuscript significantly. The final content of the manuscript was revised and approved by all the authors.

ACKNOWLEDGMENTS
The experiments were partly conducted in the Molecular Pharmacology and Herbal Drug Research Laboratory, established at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka under the Higher Education Quality Enhancement Project (HEQEP), AIF, Round-III, Window-II for sub-project (CP-3258), University Grants Commission (UGC) of Bangladesh.