DOI: http://dx.doi.org/10.26510/2394-0859.pbe.2018.06

Research Article

Evaluation of analgesic activity of ethanolic and aqueous extracts of leaf of Plumeria rubra in albino rat

Dhanapal Venkatachalam*, Samuel Thavamani B., Muddukrishniah

Department of Pharmacognosy, Sanjo College of Pharmaceutical Studies, Velappara, Palakkad, Kerala, India

*For correspondence

Dr. Dhanapal Venkatachalam,

Department of Pharma-cognosy, Sanjo College of Pharmaceutical Studies, Velappara, Palakkad, Kerala, India.

Email: vddpaul@gmail.com

 

 

 

 

 

 

 

 

Received: 23 February 2018

Accepted: 18 March 2018

ABSTRACT

Objective: Plumeria rubra is an important traditional medicinal plant used in various ailments and the indigenous plant as a medicament for treatment of various inflammatory conditions is well documented in literature. The analgesic activity was evaluated by hot-plate and acetic acid induced writhing methods.

Methods: The leaves of P. rubra were collected from the local area in and around Palakkad, Kerala (India). About 1500 g of the powder was extracted using Soxhlet apparatus for 12 h using 5.0 L of Ethanol as solvent. The test extracts was administered intraperitoneally at a dose of 100 and 200 mg/kg while Pentazocine (5 mg/kg) and diclofenac sodium (10 mg/kg) served as standards.

Results: The ethanolic extract of 200 mg/kg having significant analgesic activity in Acetic acid induced abdominal writhing response and percentage of inhibition (PI) (p<0.01 )when compared to control.. The Ethanolic extract of 100 mg/kg and 200 mg/kg exhibited significant analgesic activity in the hot plate method (p<0.001) by increasing the pain reaction time (PAT) of the rats to  sec 7.25±0.38 at 45 min after treatment in comparison to control (4.08±13).

Conclusions: The results suggest that ethanolic extracts of P. rubra possesses potent analgesic properties, which support its use in traditional medicine and suggesting that the plant should be further investigated for its pharmacological active natural products.

Keywords: Analgesic activity, Acetic acid induced writhing method, Hot-plate, Plumeria rubra

Introduction

In recent years there has been a phenomenal rise in the interest of scientific community to explore the pharmacological actions of herbs or to confirm the claims made about them in the official books of Ayurveda.1 Plumeria rubra commonly known as temple tree a small fugitive artistic tree belongs to the family Apocynaceae. It is commonly known as 'graveyard flower' in English and 'Golainchi' in Hindi Kshirachampa in Sanskrit and Perungali in Tamil.2 It is native of Mexico and cultivated in gardens throughout India. It is also grown in sacred groves. Various parts of the plant are useful as medicine. In Ayurveda it is used in malarial fevers, antiseptic and stimulant.3 The leaves of P. rubra Linn. used in ulcer, leprosy, inflammation, analgesic, rheumatism, bronchitis, cholera, rubifacient, cold and cough.4 P. rubra Linn. plant traditionally used for the treatment of diarrhoea, dysentery, and typhoid.5 Analgesics are drugs that selectively relieve pain by acting in the CNS or on peripheral pain mechanisms, without significantly altering consciousness. Analgesics are divided into two groups, opioid analgesic and non-opioid analgesic.6 In most instances, these analgesic drugs, particularly opioid and nonsteroidal anti-inflammatory drugs (NSAIDs), can only relieve 50% of the pain in about 30% of patients.7 Studies have shown that opiates cause physical dependency, tolerance, and addiction while NSAIDs usually cause gastrointestinal disorders.8 As such, research to discover other alternatives to treat pain is crucial. Over 50% of all modern drugs are natural product origin and they play an important role in drug development programs of the pharmaceutical industry.9 Many of these herbs with analgesic activity had been used without any adverse effects. The present study aimed to evaluate the analgesic activity of ethanol and aqueous extracts of P. rubra in animal models.

Materials and Methods

Plant material

P. rubra leaves were collected from Palakkad, Kerala, India and authenticated by Dr. P. Jayraman, Director of plant Anatomy Research Centre Chennai. The P. rubra leaf was identified and deposited at the plant research centre, Chennai with the voucher number PRC/PR/2017. The fresh leaves were separated and kept for shade drying. Dried leaf material was powdered using mechanical grinder and passed through 60 mesh sieve to get the powder of desired coarseness. Powdered material was preserved in an air tight container.

Extraction of plant material

The leaves of P. rubra were washed with distilled water to remove dirt. The leaves were further air dried under shade and made into fine powder by using hand homogenizer and sieved through sieve No. 60 and the fine powder was for extraction procedure and evaluation. About 1000 g of the powder was extracted using Soxhlet apparatus for 12 h using 5.0 L of Ethanol and water as solvent. The extract was made free from solvent by keeping it on water bath at 50-60°C for about 6 h and gave a yield of 16.35%.

Animals

Swiss albino mice 90-170 gms maintained in the Animal house facility of the Department of Pharmacology, Sanjo College of pharmaceutical studies, were used in these experiments. The animals were maintained on standard small animal feeds (Excel feed, Ilorin) and water ad libitum. This research was carried out in accordance with the rules governing the use of laboratory animals as accepted internationally. The experiment was conducted between the hours of 900 h and 1600 h. The experimental groups consisted of six animals. They were maintained at constant room temperature (22°±1°C) and submitted to 12 h light/dark cycle with free access to food and water.

Experimental procedure

Acute oral toxicity study

Acute oral toxicity was conducted as per OECD guidelines (Organisation of Economic Cooperation and Development) 423 (acute toxic class method). The acute toxic class method is a step wise procedure of three animal of a single sex per step. Depending on the mortality and / or moribund status of animals, on the average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. This procedure results in the use of a minimal number of animals while allowing for acceptable data based scientific conclusion. The method uses defined doses, (5, 50, 300, 2000 mg/kg body weight) and the results allow a substance to be ranked and classified according to the globally harmonized system (GHS) for the classification of chemicals which causes acute toxicity. The method previously described by Lorke was adopted.10

Evaluation of analgesic effect11-14

Pain is not easily or satisfactorily defined and therefore is often interpreted as a suffering that results from the perception of painful stimuli. It's a common symptom and it indicates that something is wrong in the body and may give a clue to the nature of disease. Hence, "pain is a specific sensation with its own peripheral and central mechanisms independent of other five senses." Pain itself is not a disease; it is by far the most common medical complaint. It is usually perceived as an indication of ill health and most diseases have a component of pain. The control of pain is one of the most important uses to which drugs are put. Pain can be defined as the effect produced in consciousness by the arrival of nerve impulses generated by noxious stimuli in the brain. Drugs, which alter the pain sensitivity or remove pain, are called as painkiller or analgesics.

Acetic acid induced writhing response method15,16

Acetic acid induced writhing method was adopted for evaluation of analgesic activity. Writhing is defined as a stretch, tension to one side, extension of hind legs, contraction of the abdomen so that the abdomen of mice touches the floor, turning of trunk (twist). Any writhing is considered as a positive response. Analgesic activity of ethanolic and aqueous extract extracts of P. rubra was determined through acetic acid-induced pain in experimental animals. A total of thirty six Swiss albino mice were grouped into 6 groups of five animals each. Prior to pain induction and administration of the experimental doses, all the experimental animals were fasted for 12 hours but were allowed access to water ad libitium. Pain was induced by injecting 0.6% acetic acid solution at a dose of 10 ml/kg body weight into the left side of the abdomen intraperitoneally. Immediately, after injection with acetic acid abdominal muscle constriction in the abdomen and turning of body trunk of the laboratory animal was seen as an indication of pain. The different groups were treated as follows;

Group I (normal control) was administered with Co solvent (Propylene glycol: Tween 80: Water-12:3:12)

Group II (positive control) were induced with pain and administered with the standard drug

(Diclofenac sodium 10 mg/kg body eight)

Group III were induced with pain and administered with the 50 mg/kg of AEPR.

Group IV were induced with pain and administered with 100 mg/kg of AEPR.

Group V were induced with pain and administered with 50 mg/kg of EEPR.

Group VI were induced with pain and administered with 100 mg/kg of EEPR.

Each mouse was then placed in a transparent observation box and the number of abdominal constrictions (writhes) for each mouse was counted for 15 minutes commencing 5 minutes after intraperitoneal injection of acetic acid. The percentage writhing inhibition was then calculated and tabulated (Table 1).

Percentage of writhing response =

Where, C- The vehicle-treated control group; T - Treated group value.

Hot plate test 17

The paws of mice and rats are very sensitive to heat at temperatures which are not damaging the skin. The responses are jumping, withdrawal of the paws and licking of the paws. The hot plate, which is commercially available, consists of an electrically heated surface. The temperature is controlled for 55° to 56°C. This can be a copper plate or a heated glass surface. The animals are placed on the hot plate and the time until either licking or jumping occurs is recorded by a stop-watch. Evaluation of analgesic activity of the ethanolic and aqueous extract of P. rubra was also carried out using hot plate method. A total of thirty six Swiss albino mice were grouped into 6 groups of five animals each.

Group I (normal control) was administered with Co solvent (Propylene glycol: Tween 80: Water-12:3:12)

Group II (positive control) were induced with pain and administered with the standard drug

(Pentazocine 5 mg/kg body eight)

Group III were induced with pain and administered with the 5omg/kg of AEPR.

Group IV were induced with pain and administered with 100 mg/kg of AEPR.

Group V were induced with pain and administered with 50 mg/kg of EEPR.

Group VI were induced with pain and administered with 100 mg/kg of EEPR.

The rats were placed on a hot plate maintained at 55°C within the restrainer. The reaction time (in seconds) or latency period was determined as the time taken for the rats to react to the thermal pain by licking their paws or jumping. The reaction time was recorded before (0 min) and at 15, 30, 45, 60 and 75 min after the administration of the treatments. The maximum reaction time was fixed at 60 sec to prevent any injury to the tissues of the paws. If the reading exceeds 15 sec, it would be considered as maximum analgesia. Mean reaction time in seconds is recorded in (Table 2).

Statistical analysis

The results are analysed by one way ANOVA followed by Dunnet's test and p value <0.01 was considered significant.

Results and Discussion

Acute toxicity

The results showed no clinical signs and mortality of the animal therefore an LD50 >2000 mg/kg body weight may be assumed.

Acetic acid-induced writhing response

The effect of ethanolic and aqueous extracts of P. rubra on the acetic acid- induced abdominal constrictions in mice is presented in the Table 1. The result shows that ethanolic and Aqueous extracts (200 mg/kg), and the reference drug diclofenac sodium (10 mg/kg) more significantly (p<0.01) reduced abdominal writhing in mice when compared to the control group reducing the mean number of writhing from 12.5±2.7 in the control group to 5±0.37** at a dose of 100 mg/kg of ethanolic extract of P. rubra. The reduction was in a dose dependent manner. Also the extract caused a dose dependent increase in inhibition of abdominal writhing, increasing it from 0% in control group to 60% at the dose 200 mg/kg of ethanolic extract. Both the extracts at a dose of 100 mg/kg are also significant (p<0.05) when compared to control group. 200 mg/kg of ethanolic extracts was found more potent than all other extracts. The reference drug diclofenac sodium was found more potent than both the plant extracts at all of the dose level.

Table 1: Acetic acid induced writhing response of Plumeria rubra extracts.

Group no Drug treatment Dose mg/kg Mean writhing response Percentage inhibition
1 Control 27ml/kg 12.5±2.7 0
2 Diclofenac sodium 10 4±0.58** 68
3 AEPR 100 6.5±0.51* 52
4 AEPR 200 5.5±0.77** 58
5 EEPR 100 5.8±0.6* 55
6 EEPR 200 5±0.37** 64

One way ANOVA followed by Dunnet's test. Values are mean ± SEM. n=6, in each group *p<0.05, **p<0.01 when compared to control. AEPR –Aqueous extract of P. rubra EEPR-Ethanolic extracts of P. rubra

Hot plate method

The result of the effect of ethanolic and aqueous extracts of on the hot P. rubra plate method is presented in Table 2. The result shows that ethanolic extract 200 mg/kg having more significant in mean reaction time when compared to control (p<0.001). 100 mg/kg, 200 mg/kg of Aqueous extract of P. rubra and 100 mg/kg of ethanolic extracts shows significant in mean reaction time when compared to control (p<0.01).

Figure 1: Mean writhing response in acetic acid induced writhing in mice, standard – diclofenac (10 mg/kg), AEPR-Aqueous extract of P. rubra, EEPR- ethanolic extract of P. rubra.

Figure 2: % of inhibition in acetic acid induced writhing in mice standard – diclofenac (10 mg/kg), aepr-aqueous extract of P. rubra EEPR- ethanolic extract of P. rubra.

The present study was designed to evaluate the analgesic potential of ethanolic and aqueous extracts of leaf of Plumeria rubra in Swiss albino mice. Acetic acid-induced pain test has widely been used for screening new analgesic agents and it majorly involves cholinergic, histaminic peritoneal receptors, acetylcholine and histamine mediators. It is also used to asses peripherally acting analgesics.18,19 According to several chemicals can be used to induce writhing's in a laboratory animal for example acetic acid and phenylquinone.20 When acetic acid is intraperitoneally injected into the experimental animal the following characteristics are observed as indicators of pain; contraction of abdominal muscle, elongation of body part and extension of the hind limbs. Therefore, such presentation is thought to be mediated by peritoneal receptors.21 It has been proposed that acetic acid acts indirectly by releasing endogenous substances responsible for exciting the nerve endings and causing pain, but also excites neurons that are sensitive to drugs.22 To suppress pain conventionally, nonsteroidal anti-inflammatory drugs are prescribed.23 These drugs are used for treating various diseases such as arthritis, headache, pain and orthopaedic conditions. Analgesic drugs such as diclofenac relieve pain peripherally/ centrally by inhibiting cyclooxygenase enzyme (COX-1 and COX-2). Inhibition of cyclooxygenase enzyme reduces the production of pain mediators such as prostaglandins, substance P, histamine, serotonin, and bradykinin. Pain sensation is eventually reduced in the nociceptors.24 These findings strongly suggest that ethanolic and Aqueous extracts of leaf of Plumeria rubra possess peripherally or centrally analgesic property. Perhaps acting in a similar manner as conventionally used therapeutic drugs that reduce the pain perception in nociceptors by inhibiting production of prostaglandins. These results concur with other research studies on the evaluation analgesic activity of herbal plants extract using laboratory animals. Reduction in the number of abdominal writhing's in this study is in agreement with a study carried out by on analgesic properties of acetone leaf extracts of Carissa spinarum in mice.25 The findings are also in line with studies by on antinociceptive activity of Toddalia asiatica (L) Lam in models of central and peripheral pain.26 Studies conducted on herbal plants by many researchers have linked presence of secondary active metabolites such as flavonoids, saponins and alkaloids to analgesic activities among other properties.27-28 Flavonoids have the ability to disrupt synthesis of eicosanoids.29 Flavonoids also have the ability to reduce production of arachidonic acid through inhibition of neutrophils degranulation.30 Besides flavonoids, alkaloids also have been associated with the ability to inhibit pain perception.31

Table 2: Analgesic activity of P.rubra in hot plate method.

Group No. Treatment Dose (mg/kg) Mean reaction time in seconds
      0 Min 15 min 30 min 45 min 60 min 75 min
1 Control Cosolvent 1.95±0.16 2.50±0.15 2.9±0.13 3.46±0.11 4.08±13*** 4.60±0.1%
2 Pentazocine 5mg/kg 3.63±0.30 4.18±0.31 6.70±0.29** 7.5±0.29*** 8.4±0.19*** 9.3±0.12***
3 AEPR 100 mg/kg 2.85±0.30 3.48±0.6 4.2±0.13** 4.18±0.18** 5.45±0.16** 5.98±0.22**
4 AEPR 200 mg/kg 2.25±0.15 2.83±0.22 3.11±0.12 4.18±0.06* 5.25±0.07** 6.25±0.11**
5 EEPR 100 mg/kg 3.48±2.68 3.42±0.33 4095±0.17** 5.43±0.11** 5.27±0.48*** 7.36±0.11***
6 EEPR 200 mg/kg 2.68±0.21 3.35±0.29 3.9±0.29* 5.28±0.19** 7.25±0.38 8.28±0.3***

Conclusions

The lymphocytes were treated with 20, 40, 60 and 100 mM of dimethoate for 1 hour and the comet assay was performed. The cell damage was assessed by fluorescent microscopy and it was found that there was a progressive decrease in the cells with no damage and an increase in cells with greater damage as the concentration of the insecticide increased. For the MTT assay, the toxic effect of the same concentrations of dimethoate was studied after 2 hours exposure. The viability ranged from 96.08% at 20 mM to 85.08% at 100 mM. The viability fell further after an overnight incubation with crude root extract, but it showed a rise when incubated with 50% of root extract (rising to 98.5% which almost touches the value for control untreated cells). Thus the experiment shows that 50% of the root extract exhibits an ameliorative capacity to reverse the cellular damage caused by the insecticide.

Acknowledgements

The authors are very thankful to the Director and Principal of Sanjo College of Pharmaceutical studies, Vellapara, Palakkad for providing facilities to carry out the present research work.

Funding: No funding sources

Conflict of interest: None declared

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