INTRODUCTION:

Inflammation is a pervasive form of body’s defense,^[1] a complex physiological response of vascular tissues to harmful stimuli or injury associated with pain and increases vascular permeability, protein denaturation, and membrane alteration^[2] and initiates the healing process.^{[3, 4]}

Inflammatory mediators such as histamine, serotonin, slow reacting substances of anaphylaxis (SRS-A), prostaglandins etc^[5] induces characteristic inflammatory changes including vasodilatation, increases capillary permeability, destruction and healing of tissues.^{[6, 7]}

Anti-inflammatory drugs stabilize lysosomal membrane^[8] and inhibit the release of inflammatory mediators and there by inhibits the process of inflammation.^[9] Human red blood cell membrane resemblances lysosomal membrane,^[10] hence the erythrocyte membrane stabilization may correlate with lysosomal membrane stabilizing effect.^{[11, 12]} The in vitro human red blood cell membrane lysis was taken as measure for screening anti-inflammatory activity.^[13-15] In the present work, erythrocyte stabilization^[10] has been used as a method in estimating anti-inflammatory property.^[16]

MATERIALS AND METHODS:

Collection of plant sample:

The fresh fruits of Ananas cosmosus were procured from the local market, Visakhapatnam, washed under tap water and cleaned. The fruit peel was removed and shade dried for about two to three weeks at room temperature. The dried fruit peel was coarse powdered in a mixer grinder, sieved and stored in air tight container for study.

Preparation of extract:

The powdered fruit peel was weighed, packed in the Soxhlet column and extracted with hydro alcohol (30:70) at elevated temperatures by Soxhlet extraction method.^[17] Then the extracted liquid was concentrated by using distillation apparatus for a period of two to three hours and then air dried over night at room temperature. The extract was labeled, stored in stock vials and kept in refrigerator for further studies.

HRBC membrane stabilization assay:

The invitro anti-inflammatory activity of hydroalcoholic extract of Ananas cosmosus fruit peel was determined by HRBC membrane method.^[18] The blood was collected from healthy volunteer who was not taken any NSAID's for two weeks prior to the experiment. The collected blood was mixed with the equal volume of sterilized Alsever solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid and 0.042% sodium chloride in distilled water) and centrifuged at 3000 RPM. The packed cells were washed with isosaline solution (0.85%, pH 7.2) and a 10% v/v suspension was made with isosaline. This HRBC suspension was used for the estimation of invitro anti-inflammatory activity. The assay mixture contains 1mL phosphate buffer (0.15 M, pH 7.4), 2 mL of hyposaline (0.36%), 0.5 mL of 10% v/v HRBC suspension and different concentrations (50 to 250 µg/mL) of hydroalcoholic extract of Ananas cosmosus fruit peel prepared in isosaline solution.

All the assay mixtures were incubated at 37ºc for 30 minutes and centrifuged. The supernatant solution was estimated for hemoglobin content using spectrophotometry at 560nm. The control was prepared without any plant extracts which served as negative control and diclofenac was used as the reference standard.

The percentage haemolysis was estimated by assuming the haemolysis protection in the control as 100%. The percentage of HRBC membrane protection was calculated by using the formula.^[19]

Optical density of test sample

% Protection = 100 ---------------------------------------------- x 100

Optical density of control

Statistical analysis:

Percentage protection of HRBC membrane lysis results were expressed as Mean±Standard Deviation, (n=3) and were compared using ANOVA followed by Bonferroni post tests. All the results were statistically significant with p<0.001. The graphical representation and statistical analysis of the data were performed using GraphPad Prism version 5.02 by GraphPad software, Inc.

RESULTS:

Invitro anti-inflammatory activity of hydroalcoholic extract of Ananas cosmosus fruit peel was tested at different concentrations ranging from 50 - 250µg/ml using HRBC membrane stabilization method and compared with standard drug Diclofenac sodium at the same concentrations [Table 1]. The hydroalcoholic extract at concentration 250µg/ml showed significant stabilization towards HRBC membrane and was a dose dependent increase in percent protection of HRBC membrane lysis [Figure 1].

Table 1: Percentage protection of HRBC membrane by Diclofenac sodium (standard drug) and hydroalcoholic extract of Ananas cosmosus fruit peel. % Protection of HRBC membrane

Conc. (µg/mL)	Std. drug (Diclofenac sodium)	Hydroalcoholic extract of Ananas cosmosus
50	69.81±1.44	58.26±0.71 ^***
100	72.15±1.06	61.44±0.71^***
150	74.17±0.8	64.36±0.94^***
200	79.45±0.97	67.51±1.25^***
250	83.57±0.53	72.86±1.05^***

*Percentage protection of HRBC membrane values represented as Mean±SD, n=3). SD=Standard deviation, statistically significant with^***p<0.001 compared with standard drug.

Figure 1: Effect of Diclofenac sodium and hydroalcoholic extract of Ananas cosmosus fruit peel on HRBC membrane stabilization.

DISCUSSION:

Inflammation is a common response by the living tissue towards injury characterized by redness, swelling, heat, pain and loss of function. Diclofenac sodium in doses of 50 – 100 mg is the most widely prescribed non steroidal anti-inflammatory agent worldwidethat inhibits the release of lysosomal enzyme or stabilizes the lysosomal membrane.^[20] Frequent administration of Diclofenac sodium is essential to maintain its therapeutic concentration due to its short biological half life^[21] and fast mean elimination half life which could increases the risk for serious dose-dependent adverse effects such as gastrointestinal side effects,^[22] cardiovascular effects^[23] and renal effects.^[24]

The current investigation is based on the need for newer anti-inflammatory agents from natural origin with significant activity and fewer side effects as substitutes for chemical therapeutics. Many fruits such as black berry, raspberry, pomegranate, camu camu, etc reported to have anti-inflammatory property and possess proven medical activities.^[25] In the present study, hydroalcoholic extract of the Ananas cosmosus fruit peel is assessed for anti-inflammatory activity by using HRBC membrane stabilization. Since HRBC membrane is similar to lysosomal membrane components, the HRBC membrane lysis prevention is taken as a measure of anti-inflammatory activity. The present result indicates the efficacy of Ananas cosmosus as an effective therapeutic agent in the treatment of acute inflammation as it showed percent protection of the HRBC membrane lysis of 72.86% for 250µg/ml tested dose. Further in vivo and detailed studies are required to confirm and isolate the active chemical constituent in Ananas cosmosus fruit peel that is responsible for this property and for identification of the possible mechanism of its anti-inflammatory activity.

CONCLUSION:

The present investigation concluded that the hydroalcoholic extract of Ananas cosmosus fruit peel has significant membrane stabilization property which could be a result of inhibition of hypotonicity induced hemolysis. These results may contribute to the literature for the isolation and identification of the active chemical constituent in Ananas cosmosus.

CONFLICT OF INTEREST:

We have no conflict of interest in the subject matter or materials discussed in the manuscript.

REFERENCES:

1. Naoh T. Ashley, Zachary M. Weil and Randy J. Nelson. Inflammation: Mechanisms, Costs and Natural Variation. Annual Review of Ecology, Evolution and Systematics. 2012;43:385-406.

2. Leelaprakash G and Mohan Dass S. In vitro anti-inflammatory activity of methanol extract of Enicostemma axillare. International Journal of Drug Development and Research. 2011; 3(3):189-196.

3. Ferrero-Miliani L, Nielson OH, Andersen PS and Girardin SE. Chronic inflammation: Importance of NOD2 an NALP3 in interleukin-1β generation. Clinical and Experimental Immunology. 2007; 147(2):227-235.

4. Soehnlein O and Lindbon L. Phagocyte partnership during the onset and resolution of inflammation. Nature Reviews Immunology. 2010; 10: 427-439.

5. Perianayagam JB, Sharma SK and Pillai SK. Anti-inflammatory activity of Trichodesma indicum root extract in experimental animals. Journal of Ethanopharmacology. 2006; 104: 410-414.

6. Okoli CO, Akah PA, Nwafor SV, Anasiobi AI, Ibegbunan IN, Arojikwe O, et al. Anti-inflammatory activity of hexane leaf extract of Aspilia africanum C.D. Adams. Journal of Ethanopharmacology. 2007; 109(2):219-225.

7. Eming SA, Krieg J and Davidson JM. Inflammation in wound repair: Molecular and cellular mechanisms. Journal of Investigative Dermatology. 2007; 127(3):514-525.

8. Amujoyegbe OO, Agbedahunsi JM, Akinpelu BA and Oedapo OO. In vitro evaluation of membrane stabilizing activities of leaf and root extracts of Calliandra portoricensis (JACQ) benth on sickle and normal human erythrocytes. International Research Journal of Pharmacy. 2012; 2:198-203.

9. Omale J and Okafor PN. Comparative antioxidant capacity, membrane stabilization, polyphenol composition and cytotoxicity of the leaf and stem of Cissus multistriata. African Journal of Biotechnology. 2008; 7:3129-3133.

10. Ejebe DE, Siminialayi IM, Emudainowho JOT, Ofesi U and Morka L. Analgesic and anti-inflammatory activities of the ethanol extract of the leaves of Helianthus annus in wistar rats. Asian Pacific Journal of Tropical Medicine. 2010;3(5):341-347.

11. Azeem AK, Dilip C, Prasanth SS, Junice V and Hanan Shahima. Anti-inflammatory activity of the glandular extracts of Thunnus alalunga. Asian pacific Journal of Tropical Medicine. 2010;3(10):794-796.

12. Chaitanya, Sandhya S, David Banji, Vinod KR, Murali S. HRBC membrane stabilizing property of root, stem and leaf of Veletinum. International Journal of Research in Pharmaceutical and Biomedical Research. 2011;2(1):2229-3701.

13. Chioma A Anosike, Onyechi Obidoa, Lawrence US Ezeanyika. Membrane stabilization as a mechanism of the anti-inflammatory activity of methanol extract of garden egg (Solanum aethiopicum). Daru Journal of Pharmaceutical Sciences. 2012; 20:76.

14. Boniface PK, Verma S, Shukla A, Khan F, Srivastava SK, Pal A. Membrane stabilization: a possible anti-inflammatory mechanism for the extracts and compounds from Spathodea campanulata. Natural Product Research. 2014;28(23):2203-2207.

15. Pankaj Debnath, Abhijit Das, Amirul Islam, Sultan Md. Gias Uddin. Membrane stabilization – a possible mechanism of action for the anti-inflammatory activity of a Bangladeshi medicinal plant: Erioglossum rubiginosum (Bara Harina). Pharmacognosy Journal. 2013; 5(3); 104-107.

16. Yerramsetty Nagaharika, Valluri Kalyani, Shaik Rasheed, Ramadosskarthikeyan. Anti-inflammatory activity of leaves of Jatropha gossypifolia L. by HRBC membrane stabilization method. Journal of Acute Disease. 2013; 2(2):156-158.

17. William B. Jensen. The Origin of the Soxhlet Extractor. Journal of Chemical Education. 2007;84(12):1913.

18. Yerramsetty Nagaharika, Valluri Kalyani, Shaik Rasheed, Ramadosskarthikeyan. Anti-inflammatory activity of leaves of Jatropha gossypifolia L. by hrbc membrane stabilization method. Journal of Acute Disease.2013; 2(2):156-158.

19. Banani Das, Manabendra Duttachoudhury, Amitabha Dey, Lokesh Deb. Antioxidant and anti-inflammatory activity of aqueous and methanolic extracts of rhizome part of drynaria quercifolia (L.) J. Smith. International Journal of Pharmacy and Pharmaceutical Sciences. 2014;6(6):43-49.

20. Rajendran Vadivu, K. S. Lakshmi. In vitro and In vivo anti-inflammatory activity of leaves of Symplocos cochinchnesis (Lour) Moore ssp laurina. Bangladesh Journal of Pharmacology. 2008;3(2):121-124.

21. Willis JV, Kendall MJ, Flinn RM, Thornhill DP, Welling PG. The pharmacokinetics of diclofenac sodium following intravenous and oral administration. European Journal of Clinical Pharmacology. 1979; 16(6):405-410.

22. Geis GS, Stead H, Wallemark CB, Nicholson PA. Prevalence of mucosal lesions in the stomach and duodenum due to chronic use of NSAID in patients with reeumatoid arthritis or osteoarthritis, and interim report on prevention by mirsoprostol of diclofenac associated lesions. The Journal of Rheumatology. 1991; 28:11-14.

23. Trelle S, Reichenbach S, Wandel S, Hildebrand P, Tschannen B, Villliger PM, et al. Cardiovascular safety of non-steroidal anti-inflammatory drugs: network meta-analysis. BMJ. 2011; 342:c7086.

24. Brater DC. Anti-inflammatory agents and renal function. Seminars in Arthritis and Rheumatism. 2002; 32(3):33-42.

25. Tariq Ismail, Piero Sestili, Saeed Akhtar. Pomegranate peel and fruit extracts: A review of potential anti-inflammatory and anti-infective effects. Journal of Ethnopharmacology. 2012; 143(2):397-405.

Received on 20.01.2018 Accepted on 18.02.2018

Asian J. Pharm. Res. 2018; 8(1):33-35.

DOI: 10.5958/2231-5691.2018.00006.0