Anti-Inflammatory activity of Ethanolic extract of Basella alba in acute and Sub-acute Model

 

Naidu Narapusetty¹*, O. Sivaiah¹, B. Balanasaraiah¹, M. Haranadhbabu, B. Prasad¹,

B. Hosanna Crown¹, Ch. M. M. Prasada Rao²

¹Department of Pharmacology, Bellamkonda Institute of Technology and Sciences, Podili

²Department of Pharm. Chemistry, QIS College of Pharmacy, Ongole

 

ABSTRACT:

The present study is carried out to investigate the anti-inflammatory potential of Ethanolic extract of Basella alba (BA). Anti-inflammatory activity was evaluated by using Egg Albumin, Turpentine Oil and Formaldehyde as phlogistic agents. The animals were treated with doses 250mg/kg and 500mg/kg of extract and Diclofenac Sodium at a dose of 10mg/kg is used as a standard drug. The BA showed a significant anti-inflammatory activity in a dose dependent manner in all the models when compared with the standard treatment. The extract (500mg/kg) exhibited maximum antiinflammatory activity i.e., 46.25%, 44.30%, 46.40% (P<0.001) like standard Diclofenac 47.02%, 46.50%, 48.57% in Egg albumin, Turpentine oil and Formaldehyde induced methods respectively. Based on the above results, we conclude that the BA has significant anti-inflammatory activity and might prove efficacious for further design and development of agents with significant biological activity.

 

 

 

INTRODUCTION:

Herbal therapy is also known as herbalism, which play a major role in treatment of so many diseases in so many countries and in traditions [1]. Inflammation is a complex process, which is frequently associated with pain and involves occurrences such as: the increase of vascular permeability, increase of protein denaturation and membrane alteration. When tissue cells become injured they release kinins, prostroglandins and histamine[2]. Rheumatoid arthritis a ravaging disease is a major public health burden in about 1% of the population worldwide.

 

As the currently used drugs are associated with severe side effects, the urge to develop new chemical entities with potent biological activity from natural sources with lesser side effects has become mandatory. Traditional medicine using plant extracts continue to provide health coverage for over 80% of the world’s population especially in developing countries [3]. Many medicinal plants have been investigated for novel drugs or templates for the development of new therapeutic agents⁴. Various species from the genus alba have been reported to possess anti-inflammatory activity. Basella alba (Telugu: Bachhali ; Family: Basellaceae) is a vine spinach, leaves are thick, rugose, succulent and green to purple colour. Basella Alba is advantageous for treating Ulcers and Abscesses, Rheumatic pain and Swellings [5]. Medicinal plants contain numerous biologically active compounds such as carbohydrates, proteins, enzymes, fats and oils, vitamins, alkaloids, quinines, terpenoids, flavonoids, carotenoids, sterols, simple phenolic glycosides, tannins, saponins, polyphenols etc. Basella alba contains Vitamin A, Vitamin E, Vitamin K, flavonoids, saponins and β- Carotene. The plant is reported to treat against laxative, rubefacient, skin diseases, burns, ulcers, diarrhoea, diuretic [6] and cancer. The present study was under taken to evaluate anti inflammatory activities of ethanolic leaf extract of Basella alba.

 

MATERIALS AND METHODS:

Collection of plant material and Extraction:

The leaves of Malabar spinach were collected from surrounding villages of Kakinada A.P. The plant authentication was done by Dr. A. Srinivasa Rao, Dept. of Botany, P.R Degree College, Kakinada, East Godavari District, Andhra Pradesh, India and the voucher was preserved. The plant material was thoroughly cleaned, shade dried at room temp. for 23 days and then pulverized to a coarse powder and shifted. 95% ethanol was added to coarsely powered (2kg) plant material and extracted by using soxhlet apparatus. The extract was concentrated by distillation under reduced pressure and evaporated to dryness.

 

Experimental animals:

Healthy adult albino rats of wistar strains weighing 150-250gm of either sex were used in this study. The animals were kept properly in polypropylene cages under standard laboratory conditions (12/12hr light/dark cycle at 25±5⁰c). The rats were fed a commercial diet and water ad libitum and were divided into 5 groups. The experimental protocol was approved by the Institutional Animal Ethical Committee (Approval no: 6/2016/CPCSEA).

 

Methodology:

Egg albumin induced rat paw edema: Five groups of adult rats (n=6) were used in this study. Animals were fasted over night with free access to water before the experiment. On the day of experiment, base line paw volume was recorded by using a plethysmometer (UGO Basile, 7140 Italy). Thereafter group-I (normal rats) received the vehicle (Distilled water 5ml/kg). Group-II (control rats) received the inducing agent and vehicle. Group-III (standard rats) received diclofenac sodium (10mg/kg) along with inducing agent. Group-IV and V received extract at doses of 250mg/kg and 500mg/kg respectively along with inducing agent. 1hr after administration of vehicle/drugs, edema was induced by administration of 0.1ml of fresh undiluted egg albumin solution into the subplantar region of right hind paw[7]. Paw volume of each rat from all groups was measured at 0, 1, 3, 6, 12 and 24hr after phlogistic agent administration. From the mean edema volume, the percent inhibition of edema was calculated by using following formula:

% Inhibition of edema = 100 (VC-VT/VC)

Where,

VC = Mean paw edema volume of control group

VT = Mean paw edema volume of treated group

 

Turpentine oil induced rat paw edema: Grouping of animals and drug treatments was same as above. 30min after administration of the vehicle/drug, edema was induced by administration of 0.05ml turpentine oil into the subplantar region of right hind paw of animal [8]. Paw volume of each rat from all groups was measured on 0, 1, 3 and 7th day after phlogistic agent administration. From the mean edema volume, the percentage inhibition of edema was calculated.

 

Method:

Research on inflammation has become the focus of global scientific study because of its implication in virtually all human and animal diseases. Plant based drugs used in the practice of traditional treatment of diseases including inflammation have become the focus of current research because they are cheap and have great therapeutic potential without much of the side effects associated with synthetic drugs[9]. Measuring anti inflammatory agent by a substance to reduce local edema induced in the rat paw by a phlogistic agent. In this study Egg albumin, Turpentine oil and Formaldehyde were used as irritants. Egg albumin induced paw edema is similar to carrageenan induced acute inflammation and is a biphasic system. The early phage is due to release of histamine, serotonin and increase prostaglandins (PG) synthesis in the damaged tissue surroundings. The second phage is occurred from 3 to 5 hours after administration by PG release and mediated by bradykinin, leukotriene’s, polymorphonuclear cells [10]. The test formulation showed the better anti-inflammatory activity and this is may be due to the inhibition of release of histamine, serotonin, kinins and this also retarded the release of PG like substances.

 

Turpentine oil in the sub plantar region causes paw edema that was maintained throughout the observation period i.e., 7 days as it is subacute model due to the triphasic release of inflammatory mediators. The initial phase is due to the release of histamine and serotonin, intermediate phase is due to kinin like substances and the late phase is due to cyclooxygenase and lipoxygenase products.  Kinin is considered to be the main mediator of granuloma as it causes vasodilatation and increases vascular permeability in the early stages of inflammation[11]. BA exhibited maximum inhibition of paw edema during the late phase of inflammation which suggest a prominent cyclooxygenase/ lipoxygenase inhibitory activity. Formaldehyde induced paw edema is one of the most suitable test procedure to evaluate the anti-inflammatory and anti-arthritic agents as it closely resembles human arthritis. Formaldehyde is reported to produce inflammation through proliferation and migration of fibroblasts, which are mainly concerned with the formation of connective tissue [12]. The paw swelling due to 2%v/v formaldehyde was maintained throughout the observation period i.e.10 days due to the release of histamine, serotonin, and PG. Histamine and PG are the key mediators in inflammatory hyperalgesia which are mediated through the activation of local pain receptors and nerve terminals producing hypersensitivity in the area of injury [13]. Inhibition of paw edema may be due to the ability of the BA to inhibit histamine, serotonin and PG. In all the experimental procedures, the results showed a significant inhibition of paw edema by BA and standard drug when compared with the control. Further studies are required on isolation of potent chemical constituents of the plant and to investigate the mechanism of antiinflammatory activity.

 

Egg albumin Induced Rat Paw Edema:

Five groups of adult rats (n=6) were used in this study. Animals were fasted over night with free access to water before the experiment. On the day of experiment, base line paw volume was recorded by using a digital plethysmometer (UGO Basile, 7140 Italy). Thereafter group-I (normal rats) received the vehicle (Distilled water 5ml/kg). Group-II (control rats) received the inducing agent and vehicle. Group-III (standard rats) received diclofenac sodium (10mg/kg) along with inducing agent. Group-IV and V received extract at doses of 250mg/kg and 500mg/kg respectively along with inducing agent. 1hr after administration of vehicle/drugs, edema was induced by administration of 0.1ml of fresh undiluted egg albumin solution into the subplantar region of right hind paw. Paw volume of each rat from all groups was measured at 0, 1, 3, 6, 12 and 24hr after phlogistic agent administration. From the mean edema volume, the percent inhibition of edema was calculated by using following formula:

 

 % Inhibition of edema = 100 (VC-VT/VC)

Where,

VC = Mean paw edema volume of control group

VT = Mean paw edema volume of treated group

Turpentine oil Induced Rat Paw Edema:

Grouping of animals and drug treatments was same as above. 30min after administration of the vehicle/drug, edema was induced by administration of 0.05ml turpentine oil into the subplantar region of right hind paw of animal. Paw volume of each rat from all groups was measured on 0, 1, 3 and 7th day after phlogistic agent administration. From the mean edema volume, the percent inhibition of edema was calculated.

 

Formaldehyde Induced Rat Paw Edema:

Grouping of animals and drug treatments was same as above. Drugs/vehicles were administered for a duration of 10days. 30min after administration of the drug/vehicle, edema was induced by administration of 0.1ml of 2%v/v Formaldehyde into the subplantar region of right hind paw of all animals on days 1 and 3[8]. Increase in paw edema volume was measured on 0, 1, 3, 7 and 10th day, 30min after administration of the respective vehicle/drug. From the mean edema volume, the percent inhibition of edema was calculated.

 

Statistical Analysis

The statistical significance was measured by using one way analysis of variance (ANOVA) and followed by Dunnett’s comparison test. All the data are presented as mean ± SEM and p < 0.001 was considered as significant.

 

RESULTS:

The phlogistic agents induced inflammation was significantly inhibited by the treatment given when compared with the standard drug. BA exhibited significant anti-inflammatory activity in a dose dependent manner.

 

Egg albumin induced paw edema:

The effect of BA on egg albumin induced paw edema was depicted in the table 1. The BA at a dose of 500mg/kg showed significantly greater inhibitory activity (46.25%) against standard diclofenac sodium (47.02%).

 

Table 1: Anti-inflammatory activity of  BA on Egg albumin induced paw edema Percentage inhibition of paw edema

 

Graph1: Concentration Vs Paw volume

All values are expressed as mean ± SEM, n=6, one way Analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test; ***p<0.001 as compared to control group; ns=non-significant.

 

 

Turpentine oil induced paw edema:

The inhibitory activity on turpentine oil induced paw edema are shown in table 2. The BA at a dose of 500mg/kg showed inhibitory activity of 44.30% against standard (46.50%).

 

 

Table 2: Anti-inflammatory activity of BA on Turpentine oil induced paw edema Percentage inhibition of paw edema

 

Graph2: Concentration Vs Paw volume

All values are expressed as mean ± SEM, n=6, one way Analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test; ***p<0.001 as compared to control group; ns=non-significant

 

.

Formaldehyde induced paw edema:

As shown in table 3 the BA at a dose of 500mg/kg showed greater inhibitory activity (46.40%) against standard (48.57%).

 

 

Table 3: Anti-inflammatory activity of BA on Formaldehyde induced paw edema Percentage inhibition of paw edema

 

Graph3: Concentration Vs Paw volume

All values are expressed as mean ± SEM, n=6, one way Analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test; ***p<0.001 as compared to control group; ns=non-significant.

 

 

DISCUSSION

The phlogistic agents induced inflammation was significantly inhibited by the treatment given when compared with the standard drug. BA exhibited significant anti-inflammatory activity in a dose dependent manner. Egg albumin induced paw edema: The effect of BA on egg albumin induced paw edema was depicted in the table 1. The BA at a dose of 500mg/kg showed significantly greater inhibitory activity (46.25%) against standard diclofenac sodium (47.02%). Turpentine oil induced paw edema: The inhibitory activity on turpentine oil induced paw edema are shown in table 2. The BA at a dose of 500mg/kg showed inhibitory activity of 68.75% against standard (65.28%). Formaldehyde induced paw edema: As shown in table 3 the BA at a dose of 400mg/kg showed greater inhibitory activity (44.30%) against standard (46.50%).

 

CONCLUSION:

Interestingly, the test compound showed potential anti inflammatory activity like standard Diclofenac sodium and justified the traditional use of Basella alba in the treatment of various types of pain and inflammation in all experimental models. Based on the above results, we conclude that the Ethanolic extract of Basella alba has significant anti-inflammatory activity and might prove efficacious for further design and development of agents with significant biological activities. Further, studies are required to elucidate the detail mechanism of action of these agents at molecular level to explore the therapeutic benefits.

 

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Received on 08.04.2017       Accepted on 17.05.2017     

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