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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 64  |  Page : 335-345  

Evaluation of the effectiveness of Acmella uliginosa (Sw.) Cass. flower methanolic extract in pain amelioration and memory impairment in the experimental rat models: Search for an alternative remedy over opioid painkillers


1 Department of Zoology, Cell and Molecular Biology Laboratory, University of North Bengal, Darjeeling, West Bengal, India
2 Department of Botany, Molecular Genetics Laboratory, University of North Bengal, Darjeeling, West Bengal, India
3 Department of Zoology, Bodoland University, Kokrajhar, Assam, India

Date of Submission16-Feb-2019
Date of Decision29-Mar-2019
Date of Web Publication23-Aug-2019

Correspondence Address:
Soumen Bhattacharjee
Department of Zoology, Cell and Molecular Biology Laboratory, University of North Bengal, Raja Rammohunpur, Darjeeling - 734 013, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_71_19

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   Abstract 


Background: The flower of Acmella uliginosa (AU) (Sw.) Cass., a naturally grown herb in India, is consumed as a natural painkiller for its notable analgesic properties. Objective: The objective of the study was to establish the role of AU flower methanolic extract in antinociception and its neuromodulatory activities to assess any disadvantage of the drug akin to opioids. Materials and Methods: In experimental rats, plant flower extracts were fed at a dose of 100 mg and 200 mg/kg body weight (BW) for 14 days. Analgesic activity was evaluated through formalin-induced paw licking test. T-maze, novel object recognition (NOR), and rotarod tests were done to assess the role of the extract in memory alteration and neuromotor coordination, respectively. Acetylcholinesterase (AChE), reduced glutathione (GSH), and superoxide dismutase (SOD) activity from the brain homogenates were done to assess the induced oxidative stress. Results: The plant proved to be a promising analgesic when fed orally up to 200 mg/kg BW dose. No acute toxicity was seen up to 1000 mg/kg. In the T-maze test, extract-fed animals showed a reduction in food searching time. In NOR test, the discrimination index between new and familiar objects was high in extract-fed animals compared to standard group. In rotarod test, the extract did not alter the neuromotor coordination. AChE, GSH, and SOD activities were normal in extract-treated animals. Conclusion: Memory alteration and oxidative stress are two major drawbacks associated with opioid drugs. Our results indicate that the AU flower methanolic extract qualifies as a potent painkiller and overcomes the disadvantages of opioid analgesics.

Keywords: Acetylcholinesterase, Acmella uliginosa, antinociception, cognitive memory, oxidative stress, spatial memory


How to cite this article:
Paul S, Modak D, Dutta S, Chaudhuri TK, Bhattacharjee S. Evaluation of the effectiveness of Acmella uliginosa (Sw.) Cass. flower methanolic extract in pain amelioration and memory impairment in the experimental rat models: Search for an alternative remedy over opioid painkillers. Phcog Mag 2019;15, Suppl S2:335-45

How to cite this URL:
Paul S, Modak D, Dutta S, Chaudhuri TK, Bhattacharjee S. Evaluation of the effectiveness of Acmella uliginosa (Sw.) Cass. flower methanolic extract in pain amelioration and memory impairment in the experimental rat models: Search for an alternative remedy over opioid painkillers. Phcog Mag [serial online] 2019 [cited 2019 Sep 23];15, Suppl S2:335-45. Available from: http://www.phcog.com/text.asp?2019/15/64/335/265036



SUMMARY

  • Acmella uliginosa is a traditionally used painkilling herb found in the Indian subcontinent
  • The plant flower methanolic extract shows potential pain ameliorating activity in formalin-induced pain model of experimental Wistar rats
  • The extract overcomes the memory deteriorating activities which are associated with opioid-like painkiller drugs
  • The plant extract could be used as an alternative herbal remedy against pain without any possible side effects and might be consumed for a longer time without causing harm in the patient's body.




Abbreviations used: Ach: Acetylcholine; AChE: Acetylcholinesterase; ANOVA: Analysis of variance; ATChI: Acetylthiocholine iodide; AU: Acmella uliginosa; AUM: Acmella uliginosa methanolic; BW: Body weight; CNS: Central nervous system; Cox: Cyclooxygenase: CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; DI: Discrimination index; DTNB: Dithiobisnitrobenzoic acid; FCA: Freund's complete adjuvant; GSH: Reduced glutathione; IAEC: Institutional Animal Ethical Committee; NOR: Novel object recognition; NSAID: Non-steroidal anti-inflammatory drug; OECD: Organization for Economic Cooperation and Development; PBS: Phosphate-buffered saline; ROS: Reactive oxygen species; SD: Standard deviation; SEM: Standard error mean; SOD: Superoxide dismutase; WHO: World Health Organization.


   Introduction Top


Pain is any unpleasant sensory and emotional experience associated with actual or potential tissue damage. Three major classes of pain have been classified depending on the receptors involved and the response pathways. The types are nociceptive pain, inflammatory pain, and pathological pain.[1]

Patients suffering from both inflammatory disorders and autoimmune diseases consume a high dose of painkiller during disease severity. However, the consumption of the aforesaid conventional painkillers leads to a lot of disadvantages. These include high cost, continuous long-term consumptions, opioid-induced increased sensitivity to pain,[2] tolerance, addiction, and cases of allodynia (central pain sensitization) on the cessation of painkillers such as morphine.[3] It also includes the damage of some important body systems, including kidney and liver. Learning and memory in different animal models have been reported to be affected by conventional use of opioids.[4],[5] The mu (μ), delta (δ), and kappa (κ) receptors have been observed to be directly involved with learning and memory process.[6] There is strong evidence which suggests that opioids can modulate and alter the glutamatergic transmission, neurogenesis, dendritic stability, and long-term potentiation in experimental animal models.[7],[8],[9] It has been suggested that the systemic administration of morphine impairs the memory process in animal models.[10],[11],[12] Furthermore, the continued consumption of oral pain ameliorating medicines indicated impairment of working memory compared to those patients who have not received oral opioids drugs.[13]

Two major classes of painkillers are commercially available that act in two different ways to reduce pain. The first type is the nonsteroidal anti-inflammatory drugs which inhibit the cyclooxygenase activity. This particular group of drugs also reduces inflammation and are known as a potent remedy against inflammation.[14] On the other hand, opioids or opiates are another group of potent painkillers which produce their principal effects by binding to at least three different receptors (μ, δ, and κ) on the nerve cell.[15],[16] For example, morphine, an antinociceptive drug, when applied to a patient body, interacts with endorphin-sensitive synapses to reduce pain.[17],[18]

Herbal medicinal systems are world's oldest traditional medicine systems that include Ayurveda, Unani, and Chinese herbal medicinal systems. These systems strictly rely on the use of crude or semi-purified plant parts as oral or topical remedies to different diseases and symptoms. These medicines have been traditionally used, and generally, lack any side effect and do not seriously harm the patient after medicine withdrawal or accidental medicine overdose. Furthermore, synergistic activity (the combined activity of more than one compounds present in a natural proportion in any herbal products) is an important key factor in traditional medicine systems which add more benefit to the patient's recovery on consumption. However, experiment-based efficacy study of most of such plants is lacking till date. Even after such drawbacks, according to the World Health Organization (WHO), herbal medicines form roughly 80% of the total medications used throughout the world today. Of the 252 essential chemicals that have been selected by the WHO, around 11% comes from a plant, and 9% comes from animal origin.[19],[20]

Acmella uliginosa (AU) (Sw.) Cass. (occasionally termed as AU in this article) is an indigenous herb belonging to the family Asteraceae. The plant is also known as Akarkara or Gorokhbon in Bengali, Pirazha in Assamese, Maanja-lei in Manipur, and Maratitige in Telugu. The plant is naturalized in India and shows a very widespread distribution worldwide. The plant is grown as an annual herb and widely found throughout the tropical and subtropical parts of world including India, distributed in waste and wet places and open moist fields. It has conical small yellow flowers, and the whole plant is claimed to possess medicinal properties.[21] The plant grows up to 1 m height, generally, creep or sometimes stand erect [Figure 1]. It has been consumed as food by many ethnic populations, including Malay people and Rajbanshi people from Northeast India.[22] The flower shows a pungent taste followed by tingling and numbness of the tongue and often used to cure mouth ulcer-related pain.[23] This plant, commonly known as “toothache plant,” is very popular and used as folk medicine among the tribal communities.
Figure 1: The naturally grown Acmella uliginosa collected from University of North Bengal adjacent region; located in the sub-Himalayan West Bengal, India (Photo courtesy Subhashis Paul)

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In earlier works, the plant leaf showed potential immunomodulatory activity in experimental rats;[24] anti-oxidant activities of the aerial parts, leaves, and stems of AU have also been described efficiently in different solvent extracts.[25],[26] Anti-inflammatory activity of the aqueous extract of the aerial parts of the plant has also been monitored in Wistar albino rats and mice.[27],[28] Apart from these, the plant flower has been potentially used as a sexual stimulating agent in male rats.[29] Other activities including diuretic,[30],[31] larvicidal,[32],[33] and insecticidal activities [34] of the plant have been reported. The plant also showed potent antinociceptive activity against different rat models of pain. It has been suggested by Ong et al.[23] that the pain amelioration may occur through the modulation of the opioid system as a result of AU methanolic (AUM) extract consumption. Therefore, the central nervous system (CNS)-mediated action of the AUM extract can be thought to have control on the opioid system by a similar pathway. In an earlier work by Paul et al.,[35] the synergistic role of AU flower crude homogenate in combination with equal proportion with crude Aloe vera gel has been studied. The pain ameliorating activity of AU in Freund's complete adjuvant (FCA)-induced arthritic rats along with the Aloe gel produced a strikingly better result compared to an experiment, where only Aloe gel was used.[35] An in vitro study of the AUM showed substantial RBC membrane stabilizing activity and inhibition of protein denaturation which also suggests a probable inhibition of inflammatory condition.[36]

In the present work, in addition to validation of antinociceptive properties of AUM extract, we have assessed its opioid-like side effects vis-a-vis memory and neuromotor coordination impairment in the appropriate animal models. All results are correlated together to explore the pain ameliorating properties of AUM extract along with its possible side effects on memory.


   Materials and Methods Top


Collection of plant specimens

Wild AU(Sw.) Cass. (Class Magnoliopsida, Order Asterales, Family Asteraceae) plants were collected [Figure 1] from the North Bengal University campus which is located in the sub-Himalayan Terai regions of Northern West Bengal. All the plants were naturally grown, and the plants were identified by the taxonomy laboratory in the Department of Botany, University of North Bengal (Accession Number of the voucher specimen-NBU09884).

Preparation of extract

Fresh flowers of AU were collected and properly cleaned and washed with distilled water to remove the dust particles. The flowers were then shade dried at room temperature for 2 weeks, and then the dried flowers were grinded into the fine powdery material using an electrical grinder. This fine powder was mixed with methanol and kept in a shaking incubator (Riviera, India) at room temperature for 2 days. Then, the supernatant liquid was collected, and it was filtered by running through the Whatman No. 1 filter paper. The resultant methanolic extract was concentrated and condensed into a thick end product by inert nitrogen gas flow. Obtained extracts were stored at 4°C up to the commencement of experiments. The plant extracts were fed orally with the help of a sterile gavage in all the animals in all the experiments.

Animal maintenance and caretaking

Healthy mature Wistar albino rats 8 ± 1 weeks (140 ± 15 g) of either sex were used as the experimental animals. All the animals were purchased from the authorized animal vendor Ghosh Enterprise, Kolkata. They were maintained in the animal house of the Department of Zoology, University of North Bengal. The animals were maintained under standard laboratory conditions (temperature 24°C ± 2°C) with normal day/night cycle (12 h light/12 h dark) and were provided with bedding made of paddy husk, standard pellet food, and drinking water ad libitum. Three animals were kept in each polyester cage. The rats were acclimatized to laboratory conditions for 10 days prior to the commencement of experiments. The experimental procedures were carried out during 2015–2017 in strict compliance with the ethical guidelines approved by the Institutional Animal Ethical Committee of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Registration No 840/ac/04/CPCSEA; registration dated on January 01, 2004) of University of North Bengal, West Bengal, India. The animals were orally fed with the plant extract for all the experiments.

Drugs and chemicals

Molecular biology grade formalin was purchased from Merck, India; Diazepam was procured as valium 10 manufactured by the Piramal Enterprises Ltd., India; Morphine was purchased as morphitroy 10, produced by Troikaa Pharmaceuticals Ltd. India; and scopolamine was purchased from Sigma-Aldrich, India. Phosphate-buffered saline (PBS) was procured from HiMedia, India. All the other chemicals used in experiments were of molecular biology grade. In the formalin-induced paw licking test, morphine was used as the standard drug; scopolamine was used as the standard drug in the novel object recognition (NOR) test, and diazepam was used in rotarod test. In all the experiments, the normal animal group was treated in a placebo condition.

Acute toxicity test for AUM extract on rat models

Toxicity test for AUM flower extract was done as per the Organization for Economic Co-operation and Development guidelines with minor modifications. The doses considered for the tests were 200, 500, and 1000 mg/kg body weight (BW). Six rats were taken in each group to observe the lethality induced by the sample. The mortality of the animals was observed for 24 h following AUM administration. Any behavioral abnormalities were observed for the next 7 days. In a separate setup, kidney and liver were isolated from the animals of each group after sacrificing on the 28th day. In these groups, the feeding doses were considered to be 100 mg/kg BW and 200 mg/kg BW, respectively, after continuous 28-day long AUM feeding once every day. The isolated organs were fixed in 4% formalin followed by histological sectioning. Samples were stained using hematoxylin and eosin. These histological sections were compared with that of the untreated normal animals.

Experimental design

Each experimental group contained six rats (n = 6). Both the sexes were distributed in all groups. The mean values of the BW were considered to be similar in all groups. In every experiment, the feeding doses were the same, and the respective groups were designated by the same name. In the standard group, different and appropriate standard drugs were used in different experiments. A timeline diagram has [Figure 2] described the entire experimental design in a lucid way.
Figure 2: A timeline diagram to illustrate the duration and time of conduct of different experiments along with the groups for the respective experiments

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For formalin-induced paw licking test, four experimental groups were made. The first group was normal group which contained normal control rats. The second and third groups were dose groups designated as low-dose and high-dose groups. These animals were fed with AUM extract at a dose of 100 mg/kg BW and 200 mg/kg BW, respectively. The fourth group was designated as the standard group. In this group, morphine (5 mg/kg BW) was used as a standard drug to ameliorate the pain caused by formalin injection. Animals were fed with AUM for 14 days, and the experiment was done on the 15th day.

For the T-maze test, two different experimental sets were considered. The first set was used to perform the test from 1st to 7th day of the 14-day feeding schedule. The second set was used in the 8th–14th day duration. In each set, three rat groups were considered as experimental groups. The first group was the normal rat group. The second and third groups were low-dose and high-dose groups fed with AUM doses, respectively.

For NOR test, four experimental groups were taken into consideration. The first three groups were normal dose, low dose, and high dose. The fourth group was the standard group in which the animals received an intraperitoneal injection of scopolamine at a dose of 2 mg/kg BW. The experiments were done on the 8th and 15th day following the habituation period on 7th and 14th day, respectively.

In rotarod test, normal group and two-dose groups were considered. Along with these three groups, another group of animals fed with diazepam at a dose of 4 mg/kg BW was used as a standard control.

On the 15th day, acetylcholinesterase (AChE) activity was measured and compared between normal-dose, low-dose, and high-dose groups; reduced glutathione (GSH) and superoxide dismutase (SOD) activity were measured and compared between normal dose, low dose, high dose, and standard group of animals. Here, scopolamine (2 mg/kg BW) fed group of animals was considered to be standard.

In the laboratory, the animals were fed with plant extract orally every day in the afternoon by a gavage throughout the experimental days and were sacrificed in as per the experimental procedure following complete anesthesia as per the institutional ethical compliances. When completely anesthetized, the animals were sacrificed, and the blood was drawn directly from the heart. The brains were collected by opening the skull bones after sacrifice.

Antinociceptive activity screening by formalin-induced paw licking test

The formalin-induced paw licking test in rodents is a standard model for chronic pain. The test animals are sensitive to analgesic agents acting on CNS.[37] To perform the formalin-induced paw licking test, high-dose and low-dose group animals were fed with respective doses of AUM for 14 days. Normal group, however, was fed with an equal amount of distilled water. On the 15th day, 50 μl of 2.5% formalin solution was injected into the right hind paw of the rats. The animals were kept under observation in a glass cage for the next 30 min. The time (in seconds) used by the animal in paw licking in the first 5 min and the 15–30 min after the injection was recorded. The first 5 min were considered sensitive to neurogenic pain, and the 15–30 min were considered sensitive to inflammatory pain.[38],[39],[40] In the standard group, morphine was used as the standard drug to compare the pain ameliorating activity of the plant extract. Morphine was orally fed at a dose of 5 mg/kg BW 30 min before the formalin injection.

T-maze test for spatial memory assessment

The classical T-maze test was performed to access the possible effect of AUM extract on spatial memory in two different phases on experimental rat groups. The reduction in time (in seconds) needed for searching the food provided in a specific compartment of the maze as a part of spatial learning was measured. In the first phase, on the first experimental set, the experiment was conducted from 1st to 7th day after the initiation of feeding and then in the second experimental phase, again the test was conducted from the day 8th to 14th day after continuation of feeding from the 1st day. This phase was performed on the second experimental set. Metallic flat gray colored T-maze was placed in a fixed position in the laboratory room during the experimental periods [Figure 3]a. The stem of the maze contained an enclosed start box, where the animal was kept for acclimatization prior to each trial for 2 min (Dimension 5-inch × 5 inch). The two arms were 10 inch × 5 inch, and the food was kept in the extreme corner of the right arm (Goal arm). Rats fasted for 23 h before the onset of training, and food was supplied to them for 1 h and each day after the completion of daily experimental trials. All animals had received daily handling, and they were habituated for 3 successive days to the maze prior to the experiment initiation. During the habituation session of T-maze, each rat freely explored the apparatus for 5 min each day, and no reward was placed at the end of the arms of the T-maze. The animals were given a maximum time of 3 min to perform the behavior. Each session consisted of three trials with 15 min delay between the first and second trials and similarly between the second and third trials. Each rat was placed initially at the start box of the T-maze for 2 min. On the first trial, the rat was rewarded with a food pellet for entry into the correct goal arms. After eating the food pellet, the rat was again back to its home cage. If no response was found in the first trial, the animals were considered for a second trial after a considered period (15 min). After each trial, the entire maze was wiped clean with 70% ethanol solution to minimize odor cues.
Figure 3: (a) Description of T-maze apparatus and (b) novel object recognition test apparatus, respectively

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Novel object recognition test

The NOR test emphasizes on the innate tendency of an animal to preferentially explore new objects, compared to the familiar objects. The time (in seconds) to explore a new object compared to a known object is considered to assess the cognitive memory of the experimental animal. This experiment investigates the neural basis of cognitive memory. The test was performed as described by the published protocols [41],[42] with some minor modifications. Four groups of animals were used, namely normal dose, high dose, low dose, and standard group. The test apparatus was a gray-colored metal box (60 inch × 47 inch × 25 inch) [Figure 3]b. All the experiments were done at day time in the highest possible silent environment. The objects to be discriminated throughout the experimental studies were badminton feathers (two in numbers; colored white and green; used as the familiar object) and a square box made up of hard yellow-colored paper. The objects were chosen randomly following the extensive review.[43] The test was done in three steps, namely habituation (H), training (T1), and test (T2) phases. After 2 days of habituation (H) trials, 24 h of the interval was given between H and T1 phase, whereas 1 h interval was given between T1 and T2 phases. The test experiments were performed on the 8th and 15th day after continuous plant extract feeding schedule. Standard drug scopolamine was used for standard group animals for the experiment. During the H phase, the rats were allowed to freely explore the empty box for 10 min. After 24 h, the next day, T1 was conducted for every animal of all the experimental groups. In the T1 phase, each rat was placed in a specific marked region of the box [marked as × in [Figure 3]b with two identical objects (in this case, two badminton feathers) in two-specific regions located in the other two corners of the box [marked as Y1 and Y2 in [Figure 3]b. The training phase continued for 2 min, and the rats were left free to explore the similar objects in this time. Thirty min after T1, extract fed groups were given their regular oral doses of the plant extract, and standard group animals were injected with intraperitoneal scopolamine. After 1 h from T1, all the animal groups were exposed to the T2 phase individually. In this part, the new object (the paper box) was placed in the Y2 position replacing the previous object, whereas Y1 remained the same. Animals were left free in X and were observed for 5 min. Times taken by the animals to explore the two nonsimilar objects were measured separately. In the box, initially, all the animals were left free facing the same direction throughout the experimental process. Pointing the nose to an object at a distance of <2 cm, touching an object by nose or forepaws, pushing or pulling an object by the animal was considered as exploration behavior. Each animal was exposed to a single trial. Care was taken to avoid olfactory stimuli by cleaning the box with 70% ethanol between each trial.

Calculation

From all the trials, a series of variables were taken for further calculation: the total time was taken in exploring the two identical objects (badminton feathers) in the first phase trial (T1) and the total time taken in exploring the new object (N: the paper box) and the familiar object (F: badminton feather) in T2 phase trial.

The discrimination between F and N in T2 phase was measured by comparing the time spent each rat in exploring the F with that spent in exploring the N. Discrimination index (DI) represents the difference in exploration time expressed as a proportion of the total time spent exploring the two different objects (N and F) in T2 phase. DI was then calculated by the following formula:[44] DI = N − F/N + F. Positive values indicate a good discrimination performance, while negative values or those around zero indicate poor discrimination capacity.

Motor coordination study by rotarod test

The motor coordination of the animals was evaluated by performing rotarod test [45] on the 15th day of the experiment. The experimental apparatus was made up of a rotating rod of 3 cm diameter and 30 cm length, with a nonslippery surface. The time (in seconds) on the rotating rod taken by each individual animal was considered to be the cue to assess the neuromotor coordination of the animal. A rotarod treadmill device was used for this purpose. The rod was divided into four equal sections by three disks. The animals were preselected in a training session based on their ability to remain on the bar (at 20 rpm) for 2 min and then allowing four rats to walk on the rod at the speed of 20 rpm at the same time observed over a period of 5, 10, and 15 min. Experimental rat groups were fed with the appropriate amount of plant extracts (as given on previous 14 days), and the standard group was fed with diazepam (4 mg/kg BW) 30 min before the experiment on the experimental day. Movement of the animal on the rotating bar from the starting time to falling off from the bar was registered automatically as the performance time, and the videography was done. Time spent in the apparatus was observed for 2 min duration (120 s). Apparatus was cleaned thoroughly between each trial with 70% ethanol.

Enzymatic profiling of acetylcholinesterase activity

AChE activity was measured from all the three groups (normal dose, low dose, and high dose). The activity was measured as μM/min/ml of brain homogenate by the Ellman method with little modifications.[46] Brain homogenate was prepared from the experimental dose group and normal group animals in 0.1 M PBS (pH 7.4) at 1 g/5 ml w/v ratio. About 500 μl of the homogenate was used as the lysate. For the measurement of AChE activity, 5.5 ml of potassium phosphate buffer solution (pH 7.4) was taken in test tubes, and 80 μl of dithiobisnitrobenzoic acid (DTNB) was added to it. Forty microliters of brain homogenate was mixed to the respective tubes and was incubated in a 37°C incubator for 10 min. About 40 μl (75 mM) of acetyithiocholine iodide was then added to each of the test tubes, mixed well, and absorbance was measured by spectrophotometer (Systronics UV-VIS spectrophotometer 118) at 412 nm against blank up to 3 min.

Preparation of brain tissue homogenate for in vivo stress markers

Brains were separated out by cutting the skull bones and were washed with PBS to remove blood. The isolated brain was homogenized using PBS and centrifuged at 8000 rpm for 10 min. After centrifugation, the supernatant was collected and used for in vivo antioxidant enzymatic assays.

Estimation of brain reduced glutathione level

Reduced GSH activity was measured according to the standard protocol.[47] An aliquot of 1 ml brain tissue supernatant was treated with 0.5 of Ellman reagent (19.8 mg DTNB dissolved in 100 ml of 0.1% sodium nitrate). After the treatment with Elman reagent, 3 ml of phosphate buffer was added, and the absorbance was measured at 412 nm. The percentage of inhibition was calculated to assess the difference in GSH activity.

Determination of superoxide dismutase activity

For the estimation of SOD, the standard method was followed with minor modification.[48] The reaction mixture was prepared using 1 ml of 50 mM sodium carbonate, 0.4 ml of 25 μM nitroblue tetrazolium, and 0.2 ml of 0.1 mM freshly prepared hydroxylamine hydrochloride. Then, the reaction mixture was mixed with an addition of a clear supernatant of brain homogenate (0.1 ml, 1:10 w/v). The changes in absorbance of the sample were recorded at 560 nm. The percentage of inhibition was calculated to assess the difference in SOD activity from brain tissue homogenate.

Statistical analysis of the data

Quantitative data concerning the formalin-induced paw licking test, T-maze test, NOR, and rotarod tests were expressed as mean ± standard error mean. For remaining biochemical assays, the data are expressed as mean ± standard deviation. Comparison between more than two groups was done using one-way analysis of variance (ANOVA). In T-maze test, the food exploration time for the first and last experimental days for each group was compared using paired Student's t-test. The inter-group variation for the final day was analyzed through one-way ANOVA. In NOR test, when comparing within each group, the exploration times of the familiar object and new object in the T2 phase were analyzed by paired Student's t-test. Post hoc analysis with a Dunnett's multiple comparisons test was performed, where values of P ≤ 0.05 were taken to indicate a statistical difference. All the statistical analyses were performed using Graph Pad Prism Version 7.00 for Windows (Graphpad Software Inc., San Diego, USA).


   Results Top


Result of acute toxicity test

The AUM extract did not show any mortality or behavioral abnormalities in the immediate 24 h after AUM feeding as well as in the subsequent 7 days' posttreatment. Hence, the acute toxicity level of AUM up to a dose of 1000 mg/kg BW was “unclassified.” The histology of kidney and liver prepared from dose groups showed no difference with those of the normal rats [Figure 4]. No alteration in the organ integrity was noticed.
Figure 4: Comparison between the histological sections of kidney (upper panel) and liver (lower panel) of different experimental animal groups. (a-c) showing no difference between the kidney sections of normal group, low-dose groups, and high-dose groups, respectively; (d-f) showing the insignificant difference between the liver sections of normal group, low-dose group, and high-dose group, respectively. The figures showing histological sections at ×10 magnification. The inset at the lower right side of each photograph shows the sections at × 40 magnification

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Determination of antinociception by formalin-induced paw licking test

The AUM extract-treated animals showed a significant decrease in the paw licking time (139 ± 4.16 and 129 ± 13 s for low-dose and high-dose groups, respectively) compared to the pained animals of normal group (193 ± 6.09 s). As the paw licking time directly corresponds to the severity of the pain, the results show that there is a significant decrease in both neurogenic and inflammatory pain in extract-treated dose groups when compared to normal animal group [Figure 5]. However, the standard drug morphine showed the best amelioration of pain in both the types of pain (paw licking time 61.2 ± 2.05 s). The neurogenic pain was ameliorated in a dose-dependent manner resulting in the highest amelioration of pain seen in high dose (38.2 ± 3.46) compared to the low dose (59.9 ± 2.35). The inflammatory pain was better restored in low-dose group than high-dose group. However, statistically, the magnitude of pain amelioration was the same from the point of view of the significance level. In both the dose groups and standard group, the decrease in paw licking time was highly significant (P ≤ 0.001 or ≤ 0.01). The morphine-treated animals showed the best pain cessation in both the phases compared to normal animals.
Figure 5: Bar diagram showing the paw licking time of different rat groups after formalin injection. Paw licking time in neurogenic pain, inflammatory pain, and total pain is measured separately (grouped bars a-c, respectively). * indicates inter-group significance for each type of pain intensity compared to the normal animals of the same group. ***indicates P ≤ 0.001, **indicates P ≤ 0.01, and *indicates P ≤ 0.05

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T-maze test

In the T-maze test, time score was calculated, and the error score was not considered. Animals from all the three groups showed a significant decrease in food searching time with the progression of experimental days. In both short-time and long-time feeding schedule (1–7 days and 8–14 days, respectively), there was no alteration in the trend of food searching behavior observed from the decrease in the food searching time when the first experimental day and last experimental day for each group were compared [Figure 6]a and [Figure 6]b. The different groups showed no significant difference in food searching pattern on the final day. The results signify that there is no significant change in the spatial memory of the experimental animals when fed with AUM compared to the normal animals.
Figure 6: Bar diagram showing the decrement in time (in seconds) required by the animals in food searching behavior in T-maze test at 1st to 7th (a) and 8th to 14th (b) experimental days, respectively. *indicates the significant decrement in time between the first and final days of the experiment for each group. ***indicates P ≤ 0.001, **indicates P ≤ 0.01

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Novel object recognition test

In the NOR test, standard drug group scopolamine showed very less DI compared to experimental groups. The total time used in the exploration of both new and familiar objects increased in the second trial (T1) compared to the first trial (T2) in all the groups. However, the normal and both the dose groups showed an increased tendency in the exploration of new objects. On the 8th day, the mean DI for normal dose, low dose, high dose, and scopolamine groups were 0.56, 0.38, 0.49, and 0.16, respectively, and on the 15th day, the calculated DI values were 0.42, 0.45, 0.47, and 0.01, respectively. The difference in the exploration time between familiar and new objects is also shown in [Figure 7] and [Figure 8]. There is a marked increase in the exploration times seen in the normal-dose, low-dose, and high-dose groups for the new object compared to familiar object in T2, whereas scopolamine group reflected no difference in the exploration time between new and familiar object [Figure 8]a and [Figure 8]b.
Figure 7: The comparison of new and familiar object exploration time (in seconds) in different groups in novel object recognition test. The upper [a,b,c,d] and lower [e,f,g,h] panels show the results of the 8th and 15th day of the experiment, respectively. (a and e) represents normal group; (b and f) represents low-dose group; (c and g) represents high-dose group; and (d and h) represents standard scopolamine group. Scopolamine group on both the experimental days showed low DI compared to other groups. This is characterized by a nonsignificant difference between the exploration times of new and familiar objects. Other experimental groups showed a significant difference in the exploration time. The new objects were explored for more time compared to the familiar object. **indicates P ≤ 0.01, *indicates P ≤ 0.05

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Figure 8: The percentage of time used in the exploration of different objects in T1 and T2 by different animal groups in the novel object recognition test. In the scopolamine group, there is a marked decrease in the new object exploration time compared to familiar object. (a and b) stands for the 7th day trial and 15th day trial, respectively

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Neuromotor coordination in rotarod test

In the rotarod experiment, animals from both the dose groups showed no difference in performing the task on the apparatus when compared to the normal animals [Figure 9]. The standard group animals treated with diazepam showed a very significant decrease in motor control (4.06 ± 0.38 s at 5th min, 11.92 ± 2.28 s at 10th min, and 12.32 ± 2.04 s at 15th min at P ≤ 0.001). In all the three experimental repetitions (5 min, 10 min, and 15 min after a single 2 min trial on rotarod), normal group (117.48 ± 1.77, 116. ±2.82, and 112.66 ± 2.71 s at 5th, 10th, and 15th min, respectively) and dose groups (low-dose group: 116.73 ± 3.26, 115.55 ± 4.44, 116.59 ± 3.40 s at 5th, 10th and 15th min, respectively and high-dose group: 118.40 ± 1.59, 116.79 ± 3.20, and 116.03 ± 3.96 s at 5th, 10th, and 15th min, respectively) showed absolutely no difference in performing the task. Similarly, a contrasting difference was seen between the standard group and all other groups.
Figure 9: Bar diagram showing the difference in the performance on rotarod apparatus in different experimental animal groups. All groups are compared with normal animal group. ***indicates P ≤ 0.001

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Measurement of acetylcholinesterase activity

The supernatants from brain homogenate from all the groups showed no difference in their AChE activity. Normal group (356 ± 57 μM/min/ml of brain homogenate) and both the dose groups (363 ± 81 and 392 ± 36.8 μM/min/ml of brain homogenate, respectively, for low- and high-dose groups) had nonsignificant differences in their AChE activity [Figure 10]a.
Figure 10: Bar diagram showing the difference in the acetylcholinesterase activity (a); glutathione activity (b) and superoxide dismutase activity (c) assessed from the rat brain homogenate of different experimental groups compared to normal group. ***indicates significance at P ≤ 0.001; *indicates significance at P ≤ 0.05. Nonsignificance

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Measurement of glutathione activity

Brain tissue homogenate from all the experimental groups was experimented to measure their GSH activity [Figure 10]b. The percentage of inhibition from all the groups was compared. In this test, normal and both the dose groups showed no difference in the GSH (84.6% ± 0.96%, 84% ± 1.85% and 84.9% ± 1.88%, respectively, for normal-dose, low-dose, and high-dose groups, respectively). However, the standard group animals treated with scopolamine (78.8 ± 2.72) showed a significantly low level of GSH.

Measurement of superoxide dismutase activity

In the SOD activity test, standard group animals treated with scopolamine showed significant difference (14.20% ± 4.09%) in their values (P ≤ 0.05) compared to normal and two-dose groups. The percentage of inhibition was calculated among the groups. Normal group animals (24.22% ± 6.32%) and both the dose groups (24.70% ± 5.75% and 26.3% ± 3.94%, respectively, for low- and high-dose groups) did not show any significant difference among their SOD activity [Figure 10]c.


   Discussion Top


The traditional medicine systems provide remedies to a number of conventional symptoms. Synergistic effects of the plant components often show a better effect without any prime side effects after consumption. Moreover, the herbal remedies are of low cost, and the knowledge is traditionally distributed.[19] Prior to any other experiments done, the acute toxicity test has shown that there is no observed toxicity or lethality related to AUM extract consumption up to a dose of 1 g/kg BW. Histology of the kidney and liver of AUM-treated animals showed no difference with that of the normal animals. Kidney sections showed well-organized Bowman's capsule; all the ducts and blood vessels were well formed. In the histological examination of the liver, all animals showed no change in the integrity of the hepatocytes. All these observations conclude that no probable toxicity of AUM extracts up to a dose of 200 mg/kg BW when consumed regularly for a longer period.

The AU flower extract has previously been shown as an effective remedial product against nociceptive pain.[23] We also have found similar effectivity in the AU flowers collected from the sub-Himalayan West Bengal. In formalin-induced paw licking test, the propagation of neurogenic pain through nociceptive pain receptors is followed by a severe inflammatory pain. Our results showed that both the neurogenic and inflammatory pains are reduced after the feeding of the AUM extract. Neurogenic pain is the type of pain which is produced by the effect of the formalin on the sensory C fibers in the first few minutes (generally 4–5 min) of the formalin injection [Figure 5]. It is then gradually followed and replaced by inflammatory pains produced due to the inflammation of immune cells (monitored between 15 and 30 min) [Figure 5].[38],[39],[40] Thus, the experiment significantly emphasizes the role of AU flower in pain amelioration. This observation was made further significant by correlating the antinociceptive activity of the plant with its possible role in memory formation and alteration. This approach is very significant because it imposes importance to the side effects of AU, which is a major drawback of conventional painkillers.

The classical T-maze experiments were performed in AUM-treated animal groups and in normal animals [Figure 6]. The data clearly indicate that the AUM-treated animals have unaltered spatial memory following AUM feeding. In the T-maze test, animals from all the three groups memorized and followed the cues in the maze and reached the food source at a lesser time in every trial day compared to the previous day. The time score was maintained [Figure 6]a and [Figure 6]b, and number of directional errors were overlooked (data not shown) as the animals learned the path to the food source very perfectly with time, and the error scores were intended to be zero.

The NOR test is a very useful tool to assess the recognition capability of an animal. When an animal is exposed to a new and a familiar object, they show a natural tendency to explore the new object in a greater way. The difference in their exploration time can be well measured mathematically using the DI. Positive values close to 1 indicate a good discrimination performance; values close to zero or less than zero indicate poor discrimination capacity. In our experiment, scopolamine fed standard group showed a negative DI on the 8th day and DI value of 0.01 on the 15th day [Figure 7]. In normal and AUM dose groups, the DI was significantly higher than the standard group. The total exploration time at T1 was increased in T2 in every case [Figure 8]. When the percentage of the exploration time was compared, a marked similarity in the exploration time was observed between new and familiar objects at T2 in the scopolamine group compared to all other experimental groups [Figure 8]. Moreover, an increase in the exploration time in every group also indicates no probable inhibition of exploratory behavior of experimental animals. All these findings confirm that AUM does not alter the cognitive memory of experimental animals. These data were further confirmed by the AChE activity assessment done in all experimental groups. However, the neuromotor coordination was also assessed using the rotarod apparatus. The ability to walk on the rotarod denotes the synchronization of motor activities associated with nerve impulse propagation and transmission against a specific stimulus. Standard group animals fed with diazepam showed a drastic decrease in motor control when compared with normal group and dose groups [Figure 9]. Dose groups showed no difference in the neuromotor synchronization when compared with the normal group.

The effect on AUM extract on cholinergic transmission was assessed through AChE activity study in experimental animals. Patients suffering from Alzheimer's disease confront a loss in the acetylcholine (ACh) concentration at the synaptic cleft due to the high amount of AChE. This inhibition of signal transmission hampers the pathway leading to memory formation. In fact, a decrease in ACh or increase in AChE activity has been associated with a decrease in most memory forming processes. Hence, the inhibition of AChE has been a treatment strategy for such patients as well as patients suffering from dementia, ataxia, and Parkinson's disease.[49],[50],[51] AUM-treated dose groups, however, did not show any significant increase or decrease in the AChE activity when the extract was fed for 14 days [Figure 10]a. Animals from normal group as well as dose groups did not show any significant change in the AChE activity and thus confirming no probable hindrance of AUM extract on normal nerve impulse propagation. The propagation of an unaltered impulse along the nervous system can also be postulated through the results obtained from rotarod activity. Normalcy in neuromotor coordination is also a result of unaltered synaptic transmission leading to various motor and memory forming activities. The brain is an organ which is continuously exposed to reactive oxygen species (ROS). It is also well known that the organ has a very poor protection system against such stresses compared to other body tissues. The increased ROS also hinders the memory formation pathway in different ways.[52] There was no decrease in the GSH and SOD activity in the dose groups compared to scopolamine-treated stressed groups [Figure 10]b and [Figure 10]c. These evidences confirm that the consumption of AUM does not initiate any stressed condition in the brain which may lead to an alteration in memory.

The extent of the study was to establish AU flower as a potent remedy against the pain that would provide an alternative remedy for inflammatory diseases such as rheumatism. It was thought that the AU may effectively reduce the severity of the pain in patients suffering from diseases coupled with pain. A previous work showed the effectivity of AU flower homogenate in the amelioration of rheumatoid arthritis such as symptoms in FCA-induced arthritic rat models when used independently, and the result was improved when AU was supplemented with an equal amount of Aloe gel homogenate.[35] Aloe gel has also been documented as a potent anti-inflammatory herbal remedy.[22] However, experiments show that the use of both Aloe gel and AU may give rise to a better cure process against adjuvant-induced arthritis for the purpose when both are applied synergistically. Here, another note may also be added to the usefulness of the plant as a folklore medicine. As the plant did not show any hindrance in the common memory forming pathways and reduced the nociceptive pain significantly, this plant can hence be used as a common analgesic remedy devoid of any side effects. However, despite the interesting findings regarding the neuroprotective aspects of the AU, some more detailed observations on the bioingredients, histological studies of different brain portions, and real-time expression of memory associated mRNAs of the brain would produce a better view regarding the probable mode of action of AU. These will be the key areas for the future investigations.


   Conclusion Top


The present study demonstrated the role of the methanolic extract of AUflower in pain amelioration and its effect in different types of working memory. Our results have indicated that the plant flower possesses a very strong antinociceptive activity. On the other hand, AU consumption does not alter the spatial, cognitive, or working memory forming pathways. There is no change in the neuromotor coordination in the AUM extract-treated animals as well. Moreover, these behavioral experiments were supported by the unaltered AChE, GSH, and SOD activity from the brain homogenates of AUM flower extract-fed experimental animals. In brief, it can be stated that the AU may be used as a useful herbal resource in pain amelioration with notable advantages over the conventional opioid drugs and the regular consumption of this plant flower extract for an extended time does not contraindicate.

Acknowledgements

Authors would like to acknowledge Professor. A.P. Das (Retd.) of the Department of Botany, University of North Bengal for the identification of the plant sample. The authors also thank the Department of Zoology for providing the animal house and instrument facilities for the research purpose. Jalpaiguri Pharmacy College is acknowledged for providing the facility to conduct rotarod tests. The research was partially funded by the UGC-BSR fellowship scheme (file no 7-134 [2007]). Authors acknowledge UGC for the above-mentioned facility.

Financial support and sponsorship

The research was partially funded by the University Grants Commission under the Basic Scientific Research Fellowship Scheme for Meritorious Students in Science (Recipient-Subhashis Paul, UGC-BSR fellowship scheme file no 7-134 [2007]).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]



 

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