Ficus hispida Bark Extract Prevents Nociception, Inflammation, and CNS Stimulation in Experimental Animal Model

Background. Ficus hispida is traditionally used in the ailment of pain, inflammation, and neurological disorders. The present study set out to evaluate the in vivo antinociceptive, anti-inflammatory, and sedative activity of the ethanol extract of Ficus hispida bark (EFHB). Methods. The antinociceptive activity of EFHB was evaluated by using acetic acid induced writhing, formalin, hot plate, and tail immersion methods in Swiss albino mice. Its anti-inflammatory activity was assessed by using carrageenan and histamine induced rat paw oedema test in Wister rats. The central stimulating activity was studied by using pentobarbital induced hypnosis, hole cross, and open field tests in Swiss albino mice. Results. EFHB demonstrated antinociceptive activity both centrally and peripherally. It showed 62.24% of writhing inhibition. It significantly inhibited licking responses in early (59.29%) and late phase (71.61%). It increased the reaction time to the thermal stimulus in both hot plate and tail immersion. It inhibited the inflammation to the extent of 59.49%. A substantial increase in duration of sleep up to 60.80 min and decrease of locomotion up to 21.70 at 400 mg/kg were also observed. Conclusion. We found significant dose dependent antinociceptive, anti-inflammatory, and sedative properties of EFHB in experimental animal models.


Introduction
Ficus hispida (Linn.) (Moraceae) is a medium sized tree generally known as Dumoor in Bangladesh. It is a popular plant which is widely distributed throughout subcontinent including India and Bangladesh [1]. Recent publications revealed the presence of several new alkaloids in the twigs, steam-bark, and the leaves of the F. hispida. Twigs contain ficushispimines A and B (pyrrolidine alkaloid), ficushispimine C ( -(dimethylamino)caprophenone alkaloid), and ficushispidine (indolizidine alkaloid) whereas hispiloscine (phenanthroindolizidine alkaloid) was isolated from the stem-bark and leaves [2,3]. Moreover, the barks of F. hispida contain -amyrin acetate, N-triacontanyl acetate, lupeol acetate, and 10-ketotetracosyl arachidate [4]. Pharmacological properties of the whole plant, fruit, root, and leaves of F. hispida have already been reported. The whole plant has astringent, antidysenteric, antipsoriasis, antianemic, and antihemorrhagic properties [5,6]. The fruit is edible and acts as a coolant and tonic. A mixture of honey and its juice is a good antihemorrhagic [7]. The roots and leaves are reported for their antidiarrhoeal [8], antidiabetic [9], antibacterial [10], hepatoprotective [11], antioxidant [12], and cardioprotective [13] properties. The leaves and fruits have been also reported for their analgesic activity in acetic acid-induced writhing model in mice [14].
Traditionally in Bangladesh, F. hispida bark is used against pain, inflammation, and neurological disorders such as epilepsy and depression. However, very limited pharmacological investigation was carried out on F. hispida bark till date. Therefore, the present study was designed to explore the in vivo antinociceptive, anti-inflammatory, and sedative activity of F. hispida bark.

Plant Collection and
Extraction. F. hispida barks were collected from the fresh plants from Chittagong district, Bangladesh. The samples were deposited at Bangladesh National Herbarium (BNH) and identified by the experts. A voucher specimen (DACB: 35921) was submitted there for further reference. The barks were shade dried and crushed into powder with the help of Capacitor Start Motor, China. Five hundred grams (500 g) of bark powder was produced which was transferred into a clean glass vessel and soaked in 1.5 L of ethanol. The glass vessel was airtight and saved for 7 days at room temperature with random shaking. After 1 week, the mixture was filtered initially by a piece of sterilized cotton material followed by Whatman 24 cm filter paper (Bibby RE200, Sterilin Ltd., UK) and revised several times to get a very clear filtrate. After that, the ethanol was recovered by using rotary vacuum evaporator (R-205, Buchi, Switzerland) at reduced pressure. The ethanol extract was then dried with the help of SpeedVac (RVT4104, Thermo Scientific). It yielded a 66 g of gummy concentrate (13.20%) crude ethanol extract.

Test Animals.
For laboratory animal studies, Swiss albino mice (20-25 g) and Wister rats (175-202 g) of both sexes were used. These animals were collected from the animal research branch of International Center for Diarrheal Disease Research, Bangladesh (ICDDR, B). They were kept under standard laboratory environments maintained at 25 ± 2 ∘ C and under 12/12 h light/dark cycle and fed with standard diet and water ad libitum during adaptation period. The animals were put away from any food/water overnight before the experiments. During the experimental period, these animals were treated according to the "Ethical Principles and Guidelines for Scientific Experiments on Animals (1995)" formulated by the Swiss Academy of Medical Sciences and the Swiss Academy of Sciences. The experimental procedure was approved by the Bangladesh Council of Scientific and Industrial Research (BCSIR) ethics committee (BCSIR/IAEC/08/11-12).

Phytochemical
Screening. Phytochemical screening of EFHB was carried out by using standard quantitative procedures to investigate the presence of alkaloid, steroid, reducing sugar, saponin, tannin, and flavonoid [15,16].

Determination of Total Phenolic Content.
Total phenolic content of EFHB was determined by modified Folin-Ciocalteu method [17]. 1 mg/ml of EFHB was added with the mixture of Folin-Ciocalteu reagent (5 ml) and 75 g/l of Nacarbonate solution (4 ml). The combination was kept at 37 ∘ C for half an hour and the absorbance was measured by UV spectrophotometer at 765 nm (analytikjena, Model 205, Germany). Finally, total phenolic content was calculated (mg of gallic acid/g of dry extract) by using the standard gallic acid calibration curve equation: = 6.2548 − 0.0925 and 2 = 0.9962.

Open Field Test.
Open field experimental method is routinely used to evaluate emotional and locomotors activities in rodents [31]. A specially designed experimental box was used for this test which had a series of alternatively colored black and white square floor. Five groups containing five mice/group (total twenty-five mice) were treated with 1% Tween 80 in normal saline (10 ml/kg), EFHB (100, 200, and 400 mg/kg), and diazepam (1 mg/kg). After the treatment, the number of squares traveled by the mice was monitored for 3 min. Percentage inhibition of movements was determined by using the following formula:

Hole Cross Test.
Hole cross experimental method was run in a cage ( × × = 30 × 20 × 14 cm) containing a fix steel partition in the middle [32]. A 3 cm diameter hole was made at a height of 7.5 cm in the center of the cage. 25 mice were divided into five groups equally. The different groups of animals were administered with 1% Tween 80 in normal saline (10 ml/kg), EFHB (100, 200, and 400 mg/kg), and diazepam (1 mg/kg). Pain stimulus was produced by placing the animals on hot plate maintained at the temperature of 55 ± 0.5 ∘ C. Paw licking or jumping off the plate was considered as response to pain stimulus. Reaction time for each group was recorded at 0, 30, 60, 90, and 120 min during the observation period. The number of movements of mice through the hole from one chamber to another was counted after oral administration of tested materials. Percentage inhibition of movements was determined by using (1).

Statistical Analysis.
All of the experimental results are reported as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test [33] using SPSS 11.5 software. Differences between groups were considered significant at a level of < 0.05.    (Table 7).

Pentobarbital Induced Hypnosis Test.
A substantial increase in duration of sleep was observed in the dosages of 200 and 400 mg/kg (53.70 ± 1.77 and 60.80 ± 2.01 min, resp., as compared to 73.50 ± 2.20 min in the control group at < 0.001) in pentobarbital induced hypnosis test. A notable decrease in onset of action was also showed at the doses of 200 and 400 mg/kg as compared to the control group. The results were dose dependent and statistically significant ( < 0.001 and < 0.01) ( Table 8).

Open Field
Test. EFHB showed a noticeable decrease in locomotion in the test animals with increasing time. At 240 min, the number of movements was gradually decreased up to 37.50 ( < 0.01), 30.40 ( < 0.01), and 21.70 ( < 0.001) for the dose of 100, 200, and 400 mg/kg, respectively, whereas movement decreased up to 5.90 ( < 0.001) in case of standard drug diazepam (Table 9).

Hole Cross Test.
The EFHB displayed suppression of motor activity and exploratory behavior in the test animal. The locomotor activity lowering effect was evident from the 2nd observation period (30 min) and continued up to 7th observation period (240 min). At 240 min, number of holes crossed was gradually decreased up to 6.10 ( < 0.01), 5.20 ( < 0.01), and 4.30 ( < 0.001) for the dose of 100, 200, and 400 mg/kg, respectively. The standard drug, diazepam, decreased the number of holes crossed throughout the observation period (Table 10).

Discussion
We did not observe any mortality up to the dose of 4 g/kg b.w. of EFHB (p.o.) in the test animals which ensured the safety of the experimental dose.
Acetic acid generally produces pain by increasing the level of PGE 2 and PGF 2 . It also induces sympathetic nervous system mediators, which were naturally observed at higher level at first 30 min of injection [34]. It excites pain in nerve endings and activates the production of noxious substances within the peritoneum which generates the writhing response. EFHB exhibited significant dose dependent writhing reduction. This result indicated the peripheral antinociceptive effect of EFHB which may occur due to the inhibition of the synthesis or/and action of prostaglandin. Moreover, as the acetic acid mimics the inflammatory local pain, it may also suggest the probable association of EFHB with the peripheral inhibition of bradykinins and prostanoids [35]. This may be due to the presence of -amyrin acetate which is previously reported for its antinociceptive activity [36].
Formalin test was performed since the results of the writhing test alone could not ascertain the source of antinociception. It evaluates antinociceptive property in two distinct phases. In the early/first phase, neurogenic pain is induced by direct chemical stimulation of the sensory afferent fibers; particularly c-fibers. The involvement of substance P and bradykinin has also been reported. Pain is induced by prostaglandins, histamine, bradykinin, and serotonin in the late phase [37]. Drugs that act on the central nervous system inhibit both phases while peripherally acting drugs inhibit the late phase only [38]. EFHB suppressed the licking time in both phases and appeared effective on both tonic inflammatory and central pain.
The hot plate and tail immersion tests are widely used for assessing central antinociceptive activity. They are distinguished by their tendency to respond to the pain stimuli, as the hot plate demonstrates supraspinal reflex mediated by 1 and 2 opioid receptors while the tail immersion monitors a spinal reflex involving 2 and opioid receptors [39,40]. Opioid agents exhibit their analgesic effects both via supraspinal ( 3 , 1 , 1 , and 2 ) and spinal ( 2 , 2 , and 1 ) receptors [39]. In our experiments, EFHB exhibited significant dose dependent but lesser antinociceptive activity than morphine. To verify possible centrally acting antinociceptive mechanism of EFHB, the inhibitory effect of naloxone was carried out. opioid receptor is the major target site for morphine, while naloxone reversed the antinociceptive effect of morphine. Likewise, naloxone also reversed the analgesic response of EFHB against thermal stimuli. This can be explained by the presence of -amyrin acetate which is responsible for analgesic effect by vanilloid receptor (TRPV 1 ) [41] and also by reducing neuropathic hyperalgesia produced by the direct activation of the cannabinoid receptors [42].
The inflammatory response induced by carrageenan is characterized by a biphasic response with marked oedema formation due to the rapid production of inflammatory mediators. These mediators are subsequently sustained by the release of prostaglandins and nitric oxide which is produced by cyclooxygenase (COX-2) and nitric oxide synthase (iNOS), respectively [43]. EFHB was effective in reducing the oedematogenic response between the 3rd and 5th h

12
Evidence-Based Complementary and Alternative Medicine after carrageenan administration signifying that EFHB is able to inhibit one or more intracellular inflammatory signaling pathways. On the other hand, histamine increases the vascular permeability and is known as an important inflammatory mediator [42]. When histamine is subcutaneously injected, it forms a wheal around the injected place due to increase of vascular permeability of the host capillary venules. Substances that antagonize this activity reduce the area. Since EFHB effectively suppressed the oedema in a dose dependent manner, it can be suggested that EFHB exhibited antiinflammatory actions by inhibiting the synthesis or action of histamine. The bark of F. hispida contains -amyrin acetate which is reported for its anti-inflammatory activity by decreasing IL-1 , TNF-, KC, and IL-6 levels and by inhibiting expression of NF-B and COX-2 [44]. It also contains lupeol acetate, which produces anti-inflammatory activity by regulating TNF-and IL-2 [45]. Moreover, plant derived phenolic and flavonoids often exhibit antioxidant potential [46]. Thus they protect the cells from ROS and generate antiinflammatory activity [47]. In this study, EFHB showed significant amount of phenolic and flavonoid levels and may have been responsible for the potent anti-inflammatory activity recorded (Table 1).
A CNS depressant works either by decreasing the onset or by increasing the duration of sleep or both. EFHB increased the pentobarbitone-induced sedative effect in a dose dependent manner. Barbiturates naturally work on the cerebral cortex to generate their action [48]. EFHB improved sleeping time that can be attributed to its action on the central sleeping mechanism [49]. Moreover, EFHB decreased the locomotor activity which is a parameter of the level of excitability of the CNS [50]. Decrease of locomotor activity is closely related to the depression of the CNS [51]. Sedativehypnotic drugs generate their bioactivity through GABA A receptor [52]. EFHB may act by potentiating GABAergic inhibition via membrane hyperpolarization. The sedative activity of EFHB can also be correlated with the presence of -amyrin acetate in the F. hispida bark. -Amyrin acetate is already reported for its sedative and anxiolytic effects [53]. Furthermore, flavonoids were found to be ligands for the GABA A receptor [54]. Tannin, saponin, and glycoside are reported for their sedative effects [55].

Conclusion
Present study showed potent antinociceptive, anti-inflammatory, and sedative activities of EFHB. All activities were dose dependent and statistically significant. Presence of -amyrin acetate, lupeol acetate, and phenolic and flavonoid constituents may play an important role in these bioactivities. We are hopeful that these research findings will definitely provide a rationale for further chemical and biological study on EFHB in the near future.

Disclosure
The authors are responsible for the writing and contents of the paper.

Conflicts of Interest
The authors declare no conflicts of interest.