Pharmacological studies on the antinociceptive, anxiolytic and antidepressant activity of Tinospora crispa

Pharmacological studies were performed in mice on the methanol extract of Tinospora crispa (TC), and of its hexane (HF) and chloroform (CF) fractions. Significant antinociceptive activity was observed for TC, HF, and CF in the acetic acid‐induced writhing and formalin‐induced paw licking tests. Anxiolytic and antidepressant activities were assessed using the open field, hole board, and elevated plus maze (EPM) tests. TC, HF, and CF demonstrated a significant decrease in spontaneous locomotor activity. They also showed an increase in the number of head‐dippings in the hole‐board test, suggesting decreased fearfulness. TC, and most of its fractions, showed a significant increase of the time spent in the opened arm of the EPM, indicating reduced anxiety. This study provides some support to explain the traditional use of T. crispa as a remedy for pain.

Pharmacological studies were performed in mice on the methanol extract of Tinospora crispa (TC), and of its hexane (HF) and chloroform (CF) fractions. Significant antinociceptive activity was observed for TC, HF, and CF in the acetic acid-induced writhing and formalin-induced paw licking tests. Anxiolytic and antidepressant activities were assessed using the open field, hole board, and elevated plus maze (EPM) tests. TC, HF, and CF demonstrated a significant decrease in spontaneous locomotor activity. They also showed an increase in the number of head-dippings in the holeboard test, suggesting decreased fearfulness. TC, and most of its fractions, showed a significant increase of the time spent in the opened arm of the EPM, indicating reduced anxiety. This study provides some support to explain the traditional use of T.
Pain is an unpleasant feeling-usually associated with tissue damage caused by noxious stimuli, inflammation, and disease processesthat can often be chronic (Hylands-White, Duarte, & Raphael, 2017;May et al., 2017). The management of pain mainly relies on the use of analgesics such as paracetamol, non-steroidal anti-inflammatory drugs (NSAIDs), and opioids, all of which present some significant adverse side effects (Carter et al., 2014). Other drugs, that can be prescribed, include adjuvants, such as anxiolytics that help reduce pain-related anxiety, and antidepressants that help to manage the depressive state encountered in chronic pain conditions and provide analgesia by interfering with the endogenous pain control mechanisms (Hylands-White et al., 2017). Unfortunately, many of the current anxiolytics and antidepressants exhibit some undesirable side effects too, which contribute to poor patient compliance (David & Gourion, 2016;Slee et al., 2019). There is a need to explore other sources, such as medicinal plants, for the discovery of new potential drug leads (Fajemiroye, Da Silva, De Oliveira, & Costa, 2016). This study was undertaken to assess the antinociceptive, anxiolytic, and antidepressant activity of the methanol extract of T. crispa (TC) and two of its fractions, namely the n-hexane fraction (NH) and the chloroform fraction (CF), in order to better understand the use of this plant in traditional medicine.

| Plant collection
The whole plant of T. crispa was collected at the mature stage from the Lawachara National Park, Moulavi Bazar, Bangladesh, in January 2018. The parts of the plant were cut into small pieces that were washed under tap water and then dried in the dark at 21-30 C for 15 days. The whole plant material was ground by a mechanical grinder and passed through a size of 60 mesh sieve to obtain a fine powder that was stored in an air-tight container.

| Preparation of extracts
The dried T. crispa plant powder (600 g) was macerated in methanol (4 L) for 15 days at room temperature with occasional shaking and stirring. Following filtration, first with a cotton plug then with a Whatman no. 1 filter paper, the filtrate was evaporated to dryness under vacuum at 40 C to obtain a concentrated extract (30.55 g dry weight, 5.09% w/w). The methanolic extract of T. crispa whole plant (TC) was further fractionated using n-hexane and chloroform to obtain two fractions of (HF) and (CF), respectively. All extracts, fractions, and standards used in the in vivo tests were suspended in normal saline using dimethyl sulfoxide (DMSO) at the highest concentration of 1 and 1% Tween-80.

| Experimental animals
Swiss albino mice (weighing 25-30 g, aged 4-5 weeks), of either sex, were used throughout the study. They were collected from the animal laboratory at Jahangirnagar University, Dhaka-1342, Bangladesh. The animals were kept in groups of five in a controlled laboratory environment (12-hr dark/12-hr light cycle; temperature 25 ± 2 C) for 7 days for acclimatization. The animals were given standard feed and water ad libitium. The animals fasted overnight and were weighed before the experiment. The design and performance of the research study involving mice were permitted by the Ethical Review Committee of the Faculty of Biological Science, University of Chittagong through the submission of a research protocol before the study, and ethical permission (Pharmacol/DPH/UC/01, 2018) was obtained.

| Acute toxicity studies
This was accomplished according to the OECD guidelines (OECD Guidelines, 2002). The test animals were abstained overnight prior to the experiment and continued under standard laboratory conditions.
The animals were randomly chosen and divided into groups (n = 5) which were orally administered different increasing the doses (up to 2,000 mg/kg) of TC, HF, and CF. The control group received 1% Tween 80 in water (p.o.). The animals were kept under observation for 24 hr to record the general signs and symptoms of toxicity. The mortality rate was recorded for each group at the end of this period.
2.6 | Antinociceptive activity 2.6.1 | Acetic acid-induced writhing test Peripheral analgesic activity was studied by observing the writhing response (i.e., contractions of the abdominal muscles and stretching of the hind limbs) of mice following the intraperitoneal administration of acetic acid (Koster, 1959). Forty experimental mice were randomly selected and divided into eight groups (n = 5). Normal saline with 1% DMSO (0.1 ml/10 g body weight, negative control), diclofenac sodium comparison to the negative control group was taken as an index of analgesia and was calculated using the following formula: where W c is the average number of writhing reflex in the control group, and W t is the average number of writhing in the test groups.

| Formalin-induced paw licking test
This was carried out according to a previous methodology (Okokon & Nwafor, 2010). Animals were divided into eight groups (five mice per group). A 2.5% formalin solution in saline (20 μl where TL c is the average time spent licking in the control group, and TL c is the average time spent licking in the test groups.
where M c is the average number of movements in the control group, and M t is the average number of movements in the test groups.

| Hole board test
The study was conducted using a wooden board, measuring 20 cm by

| Statistical analysis
The results obtained were expressed as mean ± SEM of five animals.

| Elevated plus maze (EPM) test
Diazepam significantly increased the time spent in the opened arm.
also showed a trend toward increased time spent in these arms, although this did not reach significance for all treatments. The highest, and significant, time increase vs. the negative control was observed for CF at 200 mg/kg in the closed arm (p < .05) and TC at 400 mg/kg (p < .001) in the opened arms (Table 5).

| DISCUSSION
Many medicinal plants are employed in traditional medicine worldwide to relieve pain (Wirth, Hudgins, & Paice, 2005) and treat depression and anxiety (Sarris, Panossian, Schweitzer, Stough, & Scholey, 2011). The present study was conducted to investigate the antinociceptive, anxiolytic, and antidepressant effects of a methanolic extract of Tinospora crispa (TC), and of its hexane (HF) and chloroform (CF) fractions, using in vivo and in silico approaches. In order to support the traditional use of T. crispa for the relief of inflammation and pain, peripheral antinociceptive activity was first evaluated using the acetic acid-induced writhing test. The latter measures abdominal muscle contractions (writhing) that are induced following the release of inflammatory mediators, such as prostaglandins and bradykinins in peripheral tissues (Sakiyama, Sujaku, & Furuta, 2008). The formalininduced paw licking test was also used to assess the analgesic activity.
In this test, formalin induces pain in two distinct phases that relate to different nociceptive mechanisms (Dubuisson & Dennis, 1977). In the early phase, formalin directly acts on the nociceptors and the pain can be controlled with centrally acting analgesics. The late phase, on the other hand, involves an inflammatory pain response that can be inhibited by non-steroidal anti-inflammatory drugs such as diclofenac (Clavelou, Dallel, Orliaguet, Woda, & Raboisson, 1995). We observed that TC, HF, and CF showed significant antinociceptive activity in both tests.

T A B L E 4
Activity of TC, HF, and CF on the exploratory behavior in the hole-board test in mice a
The rationale for screening TC and its fractions for their activity on the central nervous system (CNS) was based on the knowledge that some sedative and anxiolytic effects had previously been reported for another species of Tinospora (Barua et al., 2019;Mishra et al., 2016). The open field test was used to measure the behavioral and locomotor activity of mice (Tatem et al., 2014). Locomotor activity is considered an indicator of alertness, and any decrease in the locomotor performance indicates a CNS-depressant effect (Gahlot, Lal, & Jha, 2013;Hunskaar & Hole, 1987;Sousa et al., 2004). TC, and its fractions, demonstrated a significant decrease in the locomotor activity of animals, which may be as a result of interfering with the neural mechanisms underlying locomotion (Côté, Murray, & Knikou, 2018). In the hole-board test, which evaluates exploratory behavior independently from locomotor activity, TC-treated mice showed a significant increase in the number of head-dippings, suggesting a decreased fearfulness (Brown & Nemes, 2008;Crawley, 1985;Takeda, Tsuji, & Matsumiya, 1998). Anxiety disorders are thought to originate from a dysregulation of a range of neuronal systems, involving gamma-aminobutyric acid (GABA), serotonin, melatonin, adrenalin, dopamine, glutamate, neuropeptides, and the endocannabinoids (Murrough, Yaqubi, Sayed, & Charney, 2015) Anxietylike behavior was assessed in the elevated-plus maze (EPM) test (Rodgers, 1997). TC and most of its fractions showed a significant reduction in the time spent in the closed arm and increase of the time spent in the opened arm, indicating reduced anxiety (Gagan, Richa, Avninder, Sandeep, & Vivek, 2010).
It remains to be seen if the observed pharmacological effects are attributable to the presence of any specific phytochemicals, although it is worth mentioning that some previously isolated compounds in T.

| CONCLUSION
We observed that the whole plant of TC exerted significant antinociceptive, anxiolytic, and antidepressant activity on mice. This study provides some support for the observed in vivo antinociceptive, anxiolytic, and antidepressant effects of this plant.