Effect of Ethanolic Extract of Borassus flabellifer L. Male Flowers (Inflorescences) on Chemically Induced Inflammation in Wistar Rats.

 

Sachin R Patil*1, MB Patil2, Ravi Kumar1 and Mahesh S. Paschapur*3

1 Department of Pharmaceutics, 2 Department of Pharmacognosy, 3 Department of Pharmacology, K.L.E.S’s College of Pharmacy, Ankola-581314, Karnataka, India

 

ABSTRACT

Objective: The present study is designed to investigate anti-inflammatory activities of ethanolic extract of male flowers (inflorescences) of Borassus flabellifer L. (Arecaceae).

 

Methods: Acute inflammation models like histamine-induced and egg-albumin-induced rat paw edema model and xylene induced ear oedema in mice were employed to investigate the anti-inflammatory activity. The biochemical parameters like serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), lipid per oxidation and alkaline phosphatase (ALP) were also estimated as supportive study.

 

Results: The extract at doses 150mg/kg b.w. and 300mg/kg b.w. and diclofenac sodium (standard) showed significant anti-inflammatory in all the models, as compared to control (p< 0.0001). The extract and standard drug also showed significant (p<0.0001) results for biochemical parameters.

 

Conclusion: The results of the present further confirm the use of Borassus flabellifer L. traditionally for the treatment of painful inflammatory conditions.

 

KEY WORDS Borassus flabellifer L., inflorescences, male flowers, anti-inflammatory, histamine, egg-albumin, SGOT, SGPT, Lipid per oxidation, ALP.           

 

INTRODUCTION

Inflammation is defined as a local response of living mammalian tissues to injury due to any agent. It is a body defense reaction in order to eliminate or limit the spread of injurious agent as well as to remove the consequent necrosed cells and tissues1.

 

An initial inflammatory stimulus triggers the release of chemical mediators from plasma or connective tissue cells. Such soluble mediators, acting together in sequence, amplify the initial inflammatory response and influence its evolution by regulating the subsequent vascular and cellular responses. The inflammatory response is terminated when injurious stimulus is removed and the inflammatory mediators have been dissipated, catabolized or inhibited2.

 

A large number of Indian medicinal plants are attributed with various pharmacological activities because they contain a diversified class of phytochemicals. It is believed that current analgesia-inducing drugs such as opioids and non-steroidal anti-inflammatory drugs are not useful in all cases, because of their side-effects and potency3. Traditional and folklore medicines play an important role in health services around the globe. About three quarters of the world population relies on plants and plant extracts for healthcare. India has an extensive forest cover, enriched with plant diversity. Several plants have been used in folklore medicine4.

 

 

The rational design of novel drugs from traditional medicine offers new prospects in modern healthcare. Ayurveda the traditional medicinal system in India describes certain plants which strengthen the host immune system.

 

Borassus flabellifer L. (Arecaceae) is a tall palm attaining a height of about 30m, with a black stem with a crown of leaves at the top; leaves are 0.9-1.5m in diameter, palmately fan shaped, petiole edges with hard horny spinescent serratures; flowers unisexual, male spadix branched, female spadix simple; fruits large, subglobose drupes, on the greatly enlarged perianth. The plant has been used traditionally as a stimulant, anti-laprotic, diuretic, antiphlogistic. The fruit is stomachic, sedative, laxative and aphrodisiac in nature. The roots and juice of the plant are useful in inflammatory reactions5-7.

 

Lack of scientific data with respect to the pharmacological properties of the flowers of Borassus flabellifer encouraged for the evaluation of its anti-inflammatory potential.

 

MATERIAL AND METHODS                                                                                                        

Plant material

The male flowers (inflorescences) of Borassus flabellifer L. (Arecaceae) were collected from various parts of Uttar Kannada district, Karnataka during November to December and were authenticated from Mr. Shivanand Bhat, Department of Botany, Government Arts and Science College, Karwar, Karnataka, India. The selected parts of these plants were then dried in shade at temperatures between 21-30°C for 15 to 30 days, after which these parts of plants were chopped and ground. Finally extraction was carried out by the following procedure.

 

Preparation of the extract

Around 1kg of this powder was subjected to continuous hot extraction with 95% ethanol in a soxhlet extractor for 48h. The total ethanolic extract was filtered and concentrated to dryness in Rotovapor rotary evaporator unit (Buchi Labortechnik, Flawil, Switzerland) and then stored.

 

Experimental animals

Swiss albino mice (18–20 g) and Wistar rats (100-150 g) of either sex were procured from Venkateshwara Enterprises, Bangalore, Karnataka. The experimental protocol was initially approved from the Institutes animal ethics committee and then experimental studies were undergone according to their rules and regulations. The animals were housed under standard environmental conditions and had free access to standard pellet diet (Goldmohar brand, Lipton India Ltd.) and water ad libitum.

 

Chemicals

All the drugs used in this study were of pharmaceutical grade. Histamine and egg-albumin were purchased from Sigma Chemicals Company, St. Louis, USA. Xylene was supplied by SISCO research laboratories, Mumbai, India. SGOT, SGPT and ALP standard kits were procured from Span Diagnostics, Surat, India. Pure Diclofenac Sodium was gifted by Dr. Reddy’s Laboratories, Hyderabad, India. 

 

 

Acute toxicity studies

The acute toxicity of ethanolic extract of male flowers of Borassus flabellifer was determined by using female albino mice (18-22g). The animals were fasted 3h prior to the experiment according to OECD guideline no. 425, up and down procedure8. Animals were administered with single dose of extract and observed for 48h. No adverse effect or mortality was detected to 4g/kg, p.o. Based on the results obtained from this study, and the dose for anti-inflammatory activity was fixed to be 150mg/kg b.w. and 300mg/kg for dose dependent study.

 

Anti-inflammatory activity

The animals were divided into four groups (n=6). Group I served as Control received the vehicle only (1% Carboxymethylcellulose, CMC, 10ml/kg p.o.). Group II served as Standard, received Diclofenac Sodium at dose of 100mg/kg b.w.  Group III and IV served as test, received ethanolic extract at doses of 150mg/kg and 300mg/kg b.w. p.o. respectively.  

 

1.      Histamine-induced hind paw oedema

This experiment was conducted on healthy rats9. Right hind paw oedema was induced by the sub plantar injection of 0.1ml of histamine (1mg/ml in 1% CMC). Extract and diclofenac sodium were administered 1h prior to the inflammatory insult. The paw volume compared to that of the control animals was recorded at 0, 1, 2, 3, 6, 12 and 24h and considered as anti-inflammatory response.

 

2.      Egg albumin-induced paw oedema

This test was performed by inducing egg-albumin to healthy rats10. All 4 groups were pretreated with vehicle or diclofenac sodium or the extract at both doses respectively. After 30 min, each group was injected with 0.5ml raw egg albumin sub-plantar to the left hind-paw. The paw volume compared to that of the control animals was recorded at 0, 1, 2, 3, 6, 12 and 24h and considered as anti-inflammatory response.

 

3.          Xylene induced ear oedema in mice

The xylene induced ear oedema was tested on healthy mice11-12.One hour after administration of the extract at both the doses and diclofenac sodium (100mg/kg b.w.), 0.03ml of xylene was applied to the anterior and posterior surfaces of the right ear. The left ear was considered as control. 30min. after xylene application, mice were killed and both ears were removed. Circular sections were taken, using a cork borer with a diameter of 7mm and weighed. The increase in weight caused by the irritant was measured by subtracting the weight of the untreated left ear section with that of the treated right ear sections.

 

Biochemical Estimation

In earlier experiments, especially carrageenan and histamine induced paw oedema; the biochemical changes observed were maximum at 6h as compared to 12h and 24h. Hence, biochemical changes in histamine and egg-albumin induced paw oedema and xylene induced ear oedema in mice were estimated at 6h only.

 

The rats were anaesthetized under light ether anaesthesia and blood samples were collected by retro-orbital plexus route for biochemical estimation. Serum

 

Table 1: Effect of ethanololic extract of Borassus flabellifer male flowers on histamine induced rat paw oedema

Groups

Dose (mg/kg)

Paw Volume (ml)

 

0h

1h

3h

6h

12h

24h

 

Control

1% CMC

1.292±

0.01352

1.817±

0.01406

2.348±

0.01887

1.973±

0.02512

1.800±

0.01633

1.678±

0.02007

 

Standard

100

1.292±

0.01014

1.740±

0.01653**

1.382±

0.01922***

1.222±

0.01939***

1.145±

0.02187***

1.083±

0.01333***

 

Alc 150

150

1.258±

0.01778

1.790±

0.01483

1.620±

0.01653***

1.533±

0.01909***

1.458±

0.01493***

1.348±

0.01662***

 

Alc 300

300

1.284±

0.01503

1.772±

0.01302

1.545±

0.01688***

1.415±

0.01384***

1.292±

0.01014***

1.180±

0.01211***

 

Standard: Diclofenac sodium (100mg/kg b.w.), Alc 150: Ethanolic extract at dose 150mg/kg b.w.,Alc 300: Ethanolic extract at dose 300mg/kg b.w. Each value is the Mean ± S.E.M. for 6 rats; *P < 0.05; **P < 0.01; *** P < 0.001 compared with control; One-way ANOVA followed by Dunnett’s test.

             

Table 2: Effect of ethanololic extract of Borassus flabellifer male flowers on histamine induced rat paw oedema

Groups

Percentage Inhibition (%)

0h

1h

3h

6h

12h

24h

Control

-

-

-

-

-

-

Standard

0.00

4.23

41.14

38.06

36.38

35.45

Alc 150

2.63

1.48

31.00

22.30

19.66

19.00

Alc 300

2.78

1.58

34.19

24.13

15.04

14.06

Standard: Diclofenac sodium (100mg/kg), Alc 150: Ethanolic extract at dose 150mg/kg b.w., Alc 300: Ethanolic extract at dose 300mg/kg b.w.

 

 

Table 3: Effect of ethanololic extract of Borassus flabellifer male flowers on egg-albumin induced rat paw oedema

Groups

Dose (mg/kg)

                                                                                                                          Paw Volume (ml)

0h

1h

3h

12h

24h

Control

1% CMC

1.272±0.01956

1.825±0.02320

2.417±0.01745

1.502±0.01662

1.420±0.01238

Standard

100

1.275±0.01432

1.748±0.01424**

1.393±0.01382***

1.197±0.008819***

1.150±0.01528***

Alc 150

150

1.250±0.01461

1.832±0.01078

1.647±0.01498***

1.485±0.01057

1.402±0.006540

Alc 300

300

1.265±0.01803

1.782±0.009098

1.505±0.01118***

1.300±0.008165***

1.248±0.009457***

Standard: Diclofenac sodium (100mg/kg), Alc 150: Ethanolic extract at dose 150mg/kg b.w., Alc 300: Ethanolic extract at dose 300mg/kg b.w. Each value is the Mean ± S.E.M. for 6 rats, *P < 0.05; **P < 0.01; *** P < 0.001 compared with control; One-way ANOVA followed by Dunnett’s test.

 

 

Table 4: Effect of ethanololic extract of Borassus flabellifer male flowers on egg-albumin induced rat paw oedema

Groups

Percentage Inhibition (%)

0h

1h

3h

6h

12h

24h

Control

-

-

-

-

-

-

Standard

0.23

4.21

42.36

38.38

20.30

19.01

Alc 150

1.96

-0.38

31.85

21.97

1.13

1.40

Alc 300

0.55

2.35

37.73

30.65

13.44

12.11

 

 

Table 5: Effect of ethanololic extract of Borassus flabellifer male flowers on xylene induced ear oedema in mice

Groups

Dose (mg/kg b.w.)

Ear oedema (in mg)

% inhibition

Control

1% CMC

8.133±0.1965

--

Standard

100

3.755±0.1397***

53.83

Alc 150

150

6.007±0.1451***

26.22

Alc 300

300

4.872±0.1239***

40.09

Standard: Diclofenac sodium (100mg/kg b.w.), Alc 150: Ethanolic extract at dose 150mg/kg b.w., Alc 300: Ethanolic extract at dose 300mg/kg b.w. Each value is the Mean ± S.E.M. for 6 rats; *P < 0.05; **P < 0.01; *** P < 0.001 compared with control; One-way ANOVA followed by Dunnett’s test.

 

 

Table 6: Effect of ethanololic extract of Borassus flabellifer male flowers on various biochemical changes in histamine induced rat paw oedema

Groups

Dose (mg/kg)

SGOT  (U/ml)

SGPT (U/ml)

Lipid peroxidation (%)

Alkaline Phosphate (U/ml)

Control

1% CMC

100.8±0.9458

75.17±1.014

100

82.83±1.014

Standard

100

61.00±1.291***

35.00±1.414***

50.83±0.9458***

65.00±1.065***

Alc 150

150

83.83±1.302***

63.50±0.7638***

74.00±1.317***

74.50±1.360***

Alc 300

300

73.67±1.453***

53.50±1.176***

63.17±1.138***

71.67±0.8028***

Standard: Diclofenac sodium (100mg/kg b.w.), Alc 150: Ethanolic extract at dose 150mg/kg b.w., Alc 300: Ethanolic extract at dose 300mg/kg b.w. Each value is the Mean ± S.E.M. for 6 rats; *P < 0.05; **P < 0.01; *** P < 0.001 compared with control; One-way ANOVA followed by Dunnett’s test.

 

 

Table 7: Effect of ethanololic extract of Borassus flabellifer male flowers on various biochemical changes in egg-albumin induced rat paw oedema

Groups

Dose (mg/kg)

SGOT (U/ml)

SGPT(U/ml)

Lipid peroxidation (%)

Alkaline Phosphate (U/ml)

Control

1% CMC

109.2±1.167

72.67±1.229

100.0

84.00±1.317

Standard

100

54.33±1.256***

27.83±1.078***

61.33±1.430***

48.33±1.202***

Alc 150

150

87.17±1.302***

43.50±1.118***

82.33±1.256***

73.67±1.022***

Alc 300

300

67.00±1.211***

35.17±1.195***

68.33±0.9888***

64.50±1.025***

Standard: Diclofenac sodium (100mg/kg b.w.), Alc 150: Ethanolic extract at dose 150mg/kg b.w., Alc 300: Ethanolic extract at dose 300mg/kg b.w. Each value is the Mean ± S.E.M. for 6 rats;  *P < 0.05; **P < 0.01; *** P < 0.001 compared with control; One-way ANOVA followed by Dunnett’s test.

 

Table 8: Effect of ethanololic extract of Borassus flabellifer male flowers on various biochemical changes in xylene induced rat paw oedema

Groups

Dose (mg/kg)

SGOT  (U/ml)

SGPT (U/ml)

Lipid peroxidation (%)

Alkaline Phosphate (U/ml)

Control

1% CMC

112.0±1.751

41.17±1.249

100.0

67.17±1.249

Standard

100

62.17±1.078***

20.00±0.7303***

74.67±1.498***

44.00±1.291***

Alc 150

150

101.5±1.384***

33.00±1.065***

86.17±1.078***

57.17±1.138***

Alc 300

300

74.17±1.537***

25.50±1.176***

80.17±0.7032***

49.00±0.6831***

Standard: Diclofenac sodium (100mg/kg b.w.), Alc 150: Ethanolic extract at dose 150mg/kg b.w.,  Alc 300: Ethanolic extract at dose 300mg/kg b.w. Each value is the Mean ± S.E.M. for 6 rats; *P   *P < 0.05; **P < 0.01; *** P < 0.001 compared with control; One-way ANOVA followed by Dunnett’s test.

 

 

 

was separated and SGOT, SGPT, ALP were determined by the colorimetric method13-14 using standard kits.

Liver was removed and subjected homogenization to measure liver per oxidation by the standard method15. The % inhibition of lipid per oxidation by the test or standard drug was calculated by using following formula;

[(A-B)/B] X 100

Where; A: Control group, B: Test or Standard group

 

Statistical analysis

All data were expressed as Mean ± S.E.M. and analyzed statistically by using One-way Analysis of Variance (ANOVA) followed by Dunnett’s test. A difference was considered significant at P value less than 0.0001.

 

RESULTS

The effect of ethanolic extract of Borassus flabellifer male inflorescences showed significant and dose dependant anti-inflammatory activity in all the tested models. Its effect on histamine induced hind paw oedema in rats is shown in Table 1. The extract at both the dose levels (150mg/kg b.w. and 300mg/kg b.w.) was found to have significant anti-inflammatory activity in rats. The extract showed the inhibition of paw oedema by 31.00% and 34.19% at the dose 150mg/kg b.w. and 300mg/kg b.w. respectively at 3h as compared to the control group. Diclofenac sodium at 100mg/kg b.w. inhibited the oedema volume by 41.14% as shown in Table 2.

 

The effect of ethanolic extract of Borassus flabellifer male inflorescences on egg albumin-induced hind paw edema in rats is shown in Table 3. The result showed that the extract at dose 300mg/kg produced significant inhibition of egg-albumin-induced oedema from 3h to 24h. Whereas at 150mg/kg b.w. extract showed anti-inflammatory activity at 3h and 6h only. The maximum inhibition of oedema was observed at 3h for all the treated groups i.e. diclofenac sodium, 150mg/kg b.w. and 300mg/kg b.w. which was found to be 42.36%, 31.85% and 37.73% respectively as shown in Table 4.

 

The ethanolic extract of Borassus flabellifer male inflorescences at the doses 150 mg/kg b.w. and 300 mg/kg b.w. showed 26.22% and 40.09% inhibition in xylene induced ear oedema while the standard drug diclofenac sodium at 100mg/kg b.w. showed 53.83% inhibition when compared to control (Table 5).

 

The results of biochemical changes in histamine-induced and egg-albumin induced rat paw oedema and xylene induced ear oedema in mice are shown in Table 6, 7 and 8 respectively. There was significant (P<0.0001) decreased levels of SGPT, SGOT, ALP and Lipid peroxidation in all the models as compared to their respective control groups.

DISCUSSION

Inflammation has different phases; the first phase is caused by an increase in vascular permeability, second one by infiltration of leukocytes and the third one by granuloma formation. Hence, in this present study, we have examined the effect of ethanolic extract of Borassus flabellifer male inflorescences on these phases of inflammation.

 

Acute inflammation in rats was induced by sub-plantar injection of phlogistic agents like egg-albumin. Various mediators are released like histamine and serotonin (initial phase), kinins (middle phase) and prostaglandins (final phase after 3 to 5h) after egg-albumin injection, which play an important role in the development of inflammation16. (The ethanolic extract of Borassus flabellifer male inflorescences exhibited maximum anti-inflammatory activity at 3h, acting on final phase. Thus, we can assume that the anti-inflammatory activity of ethanolic extract is possibly mediated by inhibiting action of prostaglandins.

 

Xylene induced ear edema test provides a skin inflammation model suitable for evaluation of topical and systemic anti-inflammatory agents. The majority of its activities appear to involve or depend on arachidonic acid release and metabolism and interaction with protein kinase C. It has a good predictive value to screen anti-inflammatory agents. Inflammatory mediators such as kinin, serotonin, and PGs are released by Xylene and other irritants17. There is increasing evidence that lysosomal enzymes play an important role in the development of acute and chronic inflammation18-21. Most of the anti-inflammatory drugs exert their beneficial effects by inhibiting either release of these enzymes or by stabilizing lysosomal membrane, which is one of the major events responsible for the inflammatory process22. So, we can assume that our drug extract might be acting by either inhibiting the lysosomal enzymes or stabilizing the membrane.

 

CONCLUSION

Thus, it can be concluded that the ethanolic extract of male flowers (inflorescences) of Borassus flabellifer possess potent anti-inflammatory activities. The inhibitory activity of the extract justified the use of the plant as a non-specific anti-inflammatory activity in folk medicine. Further detailed investigations needs to be underway to determine the exact phytoconstituents, which are responsible for the anti-inflammatory activity.

 

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Received on 10.04.2009

Accepted on 30.05.2009     

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 1(1): July.-Aug. 2009, 59-63