Evaluation of In-vitro Anti-Inflammatory activity of Petroleum Ether Extract of Butea monosperma Flowers

 

Vishwanadham Yerragunta1*, Ayesha Saba2, Ayesha Sadia2, Amreen Begam2, Syeda Kausar Fatima2, Hajera Nausheen3, E. Sathish Reddy2

 

1Vishnu Institute of Pharmaceutical Education and Research, Narsapur, Medak, Telangana, India.

2PNR College of Pharmacy, Shamshabad, Rangareddy, Telangana, India.

3Azad College of Pharmacy, Moinabad, Rangareddy, Telangana, India.

*Corresponding Author E-mail: vishwanadham.y@gmail.com

 

ABSTRACT:

Butea monosperma is a species of Butea native to tropical and sub-tropical parts of the Indian Subcontinent and Southeast Asia, ranging across India. Common names include flame-of-the-forest and bastard teak. Butea monosperma is investigated for antibacterial activity, anti - inflammatory activity. From the study of all the activities already done, the authors found out that anti - rash or anti- allergic activity is not investigated. Dried flowers of Butea monosperma were extracted using Petroleum ether using continuous soxhlet extraction apparatus. The extract was evaporated to dryness using rota evaporator followed by drying in the desiccator. This dry extract was treated as whole drug and tested for in- vitro anti- inflammatory activity using Carrageenan induced rat paw edema method with Diclofenac sodium as standard drug. Successful in-vitro results were confirmed by animal study where anti-inflammatory activity was proved on Male Wistar albino rats.

 

KEYWORDS: Butea monosperma, In-vitro anti-inflammatory activity, Petroleum ether, Diclofenac sodium.

 

 

INTRODUCTION:

Butea monosperma is a species of Butea native to tropical and sub-tropical parts of the Indian Subcontinent and Southeast Asia, ranging across India. Common names include flame-of-the-forest and bastard teak.

 

Butea monosperma belongs to family fabeacae. It is a medium sized tree [1]. Butea monosperma is known for several medicinal properties these are widely used to internally in treatment of hepatic disorders, viral hepatitis, microbial and diarrhea [2,3,4]. The flowers are good source of flavanoids tannins [5-6].

 

The contents of flowers are butrin, butein, isobutrin, plastron, coreipsin and isocoreipsin [7]. The present work is a study about in -vitro Anti inflammatory activity of Petroleum ether extract of flowers of Butea monosperma. Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants.

 

Plant Description:

 

Kingdom:                Plantae

Order:                      Fabales

Family:                    Fabaceae

Genus:                     Butea

Species:                   B. monosperma

Binomial name :                Butea monosperma     (Lam.)

Synonyms:          Butea frondosa Roxb. ex Willd.
                              Erythrina monosperma Lam.[8]
                              Plaso monosperma

 

Inflammation:

Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants .Inflammation was recognized as a simple allergic reaction for many decades [9]. There are four cardinal signs of inflammatory conditions and they are redness, heat, swelling, and pain.

 

Inflammation is a protective response that involves immune cells, blood vessels, and molecular mediators. The purpose of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and to initiate tissue repair [10]. The present work was done to screen anti inflammatory activity of flowers of Butea monosperma.

 

MATERIALS AND METHODS:

Collection of plant materials [11]:

Butea monosperma flowers were collected from telangana forestry, hyderabad and was identified and authenticated by Department of Botany, Osmania University, Hyderabad, Telangana. The voucher specimen of the plant has been deposited.

 

Preparation of flower extracts [11-12]:

The flowers of Butea monosperma were shade‐dried, powdered and passed through a 60‐mesh sieve; this powder was subjected to continuous hot extraction in Soxhlet apparatus with petroleum ether, until color less solvent appeared in soxhlet apparatus. The extract was evaporated under reduced pressure using rotary evaporator until all the solvent had been removed to give an extract sample with a yield of 9.5% w/w.

 

Formulation of topical preparation [13]:

Flowers gel was prepared using Sodium CMC as a gelling agent in 1% w/w concentration with deionized water as a vehicle under mechanical stirrer. Flower extract of Butea monosperma (5% w/w) was added to the 1% Sodium Carboxy methyl cellulose, stirred and stored at cool place.

 

In-Vivo anti-inflammatory activity [12]:

Animals: Male wistar albino rats of either sex, weighing 150–250 g were subjected to carrageenan induced rat paw edema inflammation tests (n=5, in each group). All the animals were housed in animal house, standard environmental conditions and fed with rodent diet with water ad libitum. Three groups (control, Standard and Test) of five animals in each group were used for experiment. The animals had free access to food and water. The institutional animal ethical committee approved the protocol and CPCSEA guidelines.

 

Carrageenan induced rat paw edema

Male albino wistar rats were fasted for 24 hrs before the experiment. The rats were divided into six groups containing five rats in each group.

 

Acute inflammation was induced by o.1 ml of 1.0% of carrageenan in normal saline solution. This solution was injected to the left hind paw of sub planter region of rats. Rats of the control groups containing vehicle and 0.2 g 1% diclofenac sodium applied in the same manner was used as a standard. The 5% flowers gel containing extract was applied to the paws of the test group of five rats, 1 hr before carrageenan injection. Paw volume was measured immediately after carrageenan injection and at 1, 2, 3 and 4 hrs intervals by using a Digital plethysmometer.

 

Different groups treated were as follows:

Group 1: Carrageenan (1.0% 1ml) + vehicle

Group 2: Carrageenan (1.0% 1ml) + Diclofenac Sodium (1%)

Group 3: Carrageenan (1.0% 1ml) + Petroleum ether extract (5%)

 

The paw volume was measured at half, 1, 2, 3, 4 hrs after carrageenan injection using plethysmometer. The anti-inflammatory activity was evaluated based on the ratio of the changes in paw diameter in treated and untreated groups as per the formula given below:

 

Anti inflammatory activity (%) = [1- (Vt / Vc )] x 100

 

Vt   and  Vc are edema volumes in drug treated and control group respectively

 

In Vitro- Anti-inflammatory screening [14]:

The Butea monosperma flower extract of for anti- inflammatory activity using inhibition of albumin denaturation technique [15]. The standard drug and test compounds were dissolved in minimum amount of DMF and diluted with phosphate buffer saline (pH 7.4) in such a way that concentration of DMF in all solutions was less than 2.5%. Test solution (1 ml, 100 μg/ml) was mixed with 1 ml of 1% albumin solution in phosphate buffer saline and incubated at 27 ± 1° C in an incubator for 15 min.

 

Denaturation was induced by keeping the reaction mixture at 60 ± 1° C in a water bath for 10 min [16]. After cooling, the turbidity was measured at 660 nm with UV visible spectrophotometer. % of inhibition of denaturation was calculated from control where no drug was added. Each experiment was done in triplicate and average was calculated. Diclofenac sodium was used as standard drug.

 

The percentage of inhibition was calculated using the following formula.

 

% Inhibition of denaturation= [(Vt / Vc ) - 1 ] × 100

 

Where,

Vt = mean absorbance of test compound,

Vc = mean absorbance of control.

 

RESULTS:

From the Table no.1 that Butea monosperma flower extract (200 mg/kg), has shown significant in-vitro anti‐inflammatory activity determined using albumin denaturation method. The diclofenac sodium was used as standard drug. The percentage of inhibition was calculated using the above formula.

 

The results of anti inflammatory activity after topical application of petroleum ether (pet. ether) extract are reported in Table no.2. Statistical analysis showed that the inhibition of edema preparations containing extract were significantly different from control group at all the concentrations tested.

 

Statistical analysis:

Data obtained from pharmacological experiments were expressed as mean±SEM. The data were statistically analyzed by one way ANOVA followed by Dunnett’s test.

 

 

 

 

Table 1: Anti-inflammatory activity of petroleum ether extract of Butea monosperma flowers in carrageenan induced paw edema method:

 

Dose (mg/kg)

0min

15min

30min

60min

2hr

4hr

Control

-

0.039±  0.0001

0.041± 0.0003

0.043±

0.0001

0.047±

0.0001

0.050± 0.0004

0.052± 0.0007

Standard

(Diclofenac)

100

0.033±

0.0001*

0.023±

0.0002**

0.030±

0.0001**

0.023±

0.0001**

0.021±

0.0008**

0.017± 0.0003**

pet.ether

100

0.036±  0.0001*

0.033±

0.0004*

0.030±   0.0004*

0.029 ± 0.0001*

0.027±

0.0009**

0.025±

0.0005**

pet.ether

200

0.035±

0.0001*

0.030±

0.0001**

0.025±

0.0001**

0.024±

0.0001**

0.021±

0.0001**

0.019±

0.0004**

Statistical analysis was done by ANOVA followed by Dunnet’s test.  All the values are expressed as mean ± SEM.*P < 0.05, **P < 0.01.

 

 

Table 2:  Anti-inflammatory activity of Butea monosperma flowers (% inhibition of paw volume)

 

Dose (mg/kg)

0min

15min

30min

60min

2hr

4hr

Standard

100

15%

43%

30%

51%

58%

66%

pet.ether

100

7.6%

19%

30%

38%

46%

50%

pet.ether

200

10%

19%

41%

48%

58%

62%

Statistical analysis was done by ANOVA followed by Dunnet’s test.  All the values are expressed as mean ± SEM.*P < 0.05, **P < 0.01.

 

 

Anti-inflammatory activity by using carrageenan-induced rat paw edema method  results indicated that the Petroleum ether extract of flowers of Butea monosperma doses of 100 and 200 mg/kg significant  showed  significant activity (p<0.01) reduction in paw volume  and  petroleum ether  extract  of Butea monosperma flowers at doses of 100 and 200 mg/kg  exhibited a marked (P<0.05), inhibition of paw volume, which was comparable to diclofenac (100 mg).

 

DISCUSSION:

The anti-inflammatory activities after topical administration of Butea monosperma flowers extract of gel formulations were studied, The results of in-vitro anti-inflammatory activity by albumin denaturation method of petroleum ether extract are reported in Table no.2 where as results of in-vivo anti-inflammatory activity after topical application of extract gel are reported in Table 1.

 

Statistical analysis was done by ANOVA followed by Dunnet’s test showed that the edema inhibition by preparation containing extract is significantly more from control group. The results showed that the anti-inflammatory effect of the formulation containing 5% of the flower extract gel (1% Na CMC) was equivalent to the effect of standard Diclofenac gel formulation.

 

From these overall results, we can conclude that topical flower gel preparation containing at least 5 % of extract possesses anti-inflammatory effect which can be useful for the treatment of local inflammation. The data presented in this study demonstrated that the petroleum ether flower extract of Butea monosperma possess significant anti-inflammatory properties.

 

CONCLUSIONS:

In the present study, results indicate that the petroleum ether extracts of Butea monosperma possess anti-inflammatory properties. These activities may be due to the strong occurrence of polyphenolic compounds such as tannins, flavonoids. The extract fractions serve as free radical inhibitors or scavenger or acting possibly as primary oxidants and inhibited the heat induced albumin denaturation. This study gives on idea that the compound of the flowers extracts of Butea monosperma can be used as lead compound for designing a potent anti-inflammatory drug.

 

REFERENCES:

1.       Kushagra N, Mukesh KS, Dhansay D, Verma VK, Tripathi DK., Anti- inflammatory activity and chemo profile of plants used in traditional medicine: A review. J. Chem. Pharm, 2010, 5vol, 2nd ed. p.122-130

2.       Indian Materia Medica. Bombay: Popular Prakashan 2002, vol 1 p.223

3.       Kasture VS, Chopade CT, Deshmukh YK. Anticonvulsive activity of Albizria lebbeck, Hibiscus rosa and Butea monosperma in experimental animals. Journal of Ethanopharmacology. 2000 p.71:65‐75.

4.       Mishra M, Shukla Y, Sushilkumar., Euphane triterpenoid and lipid constituents from Butea monosperma. Phytochemistry, 2000, 54 vol 8thedi p.835‐38.

5.       Wagner H, Geyer B, Fiebig M, Kiso Y, Hakino H. Isoputrien and butrien, the antihepatotoxic principles of Butea monosperma flowers. Planta Med, 1986, 52 vol 2ndedi p.77‐79.

6.       Gupta SR, Ravindranathan B, Sheshadrai TR. The glucosides of Butea monosperma. Phytochemistry, 1970, (9), p.2231‐35.

7.       Agrawal AK, Tripathi DM, Sahai R, Gupta N. Management of giardiasis by herbal drug Pipali Rasayana; a clinical study. Journal of Ethanopharmacology, 1997, p.56: 223‐36.

8. "Butea monosperma (Lam.) Taub.". Germplasm Resources Information Network. United States Department of Agriculture. 2006-05-18. Retrieved 2009-10-24.

9.       Ferrero-Miliani L, Nielsen OH, Andersen PS, Girardin SE; Nielsen; Andersen; Girardin. "Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation". Clin. Exp. Immunol. (2007). 147 (2): 06

10.    Kulkarni S. K., Hand book of Experimental Pharmacology Vallabh Prakashan, Delhi, p.53-54.

11.    Phanse M, Kulkarni P, Kewatkar S, Lande M, Bhujbal S, Chaudhari P. Evaluation of anti-inflammatory activity of herbal gel formulation J. Nat. Prod. Plant Resour., 2011, 1 vol 2ndedi p.25-28

12.    Sosa S., Balick M., Arvigo R., Esposito R., Pizza C., Altinier G., Tubaro A., J Ethnopharmacology., 2002, 81 vol p.211-15.

13.    Winter CA., Risley EA., Nuss GW., Carragenan induced edema in hind paw of rats as an assay for anti inflammatory drug, Proceeding of society for Experimental Biology and Medicine; 1962, (11) p.544-47.

14.    Gelias and M.N. A.Rao,Inhibition of Albumin Denaturation and Antiinflammatory Activity of Dehydrozingerone and Its Analogs, Indian. J. Exp. Biol., 1998 p.26.

15.    Sharma Gajraj, Jayachandran E, Sreenivasa GM., Synthesis of Bioactive Molecule Fluoro Benzothiazole Comprising Quinazolylinyl Oxadiazole Derivative For Biological and Pharmacological Screening ,Ultra Chemistry, 2008, 4 vol 1stedi p.103-14.

16.    Williams L, Connar A., Latore L., The in vitro Antidenaturation Effects Induced by Natural Products and Nonsteroidal Compounds in Heat Treated (Immunogenic) Bovine Serum Albumin is Proposed as a Screening Assay for the Detection of Anti‐inflammatory Compounds, without the use of Animals, in the early Stages of the Drug Discovery Process, West Indian Med J., 2008, 57 vol 4thedi p.327.

 

 

 

 

 

Received on 28.04.2016          Modified on 08.05.2016

Accepted on 23.05.2016        © RJPT All right reserved

Research J. Pharm. and Tech. 2016; 9(6):755-758

DOI:10.5958/0974-360X.2016.00143.8