The present study was aimed to explore the anti–inflammatory potential of selected Pteridophytes from Western Ghats of South India using heat induced haemolytic activity. Stabilization of RBCs membrane was studied to further establish the mechanism of anti–inflammatory action of Cyclosorus interruptus (Willd.) H. Ito., Pityrogramma calomelanos (L.) Linkvar. calomelanos, Pteris vittata L., Pteris confusa T. G. Walker., Pteris biaurita L., Pteris multiaurita Ag., Adiantum incisum Forssk., Adiantum latifolium Lam. , Hemionitis arifolia (Burm.) T. Moore. and Ceratopteris thalictroides (L.) Brong. aqueous extracts with varied inhibition.Except A. latifolium and P. confusa all other tested extracts effectively inhibited the heat induced haemolysis. The studied extracts percentage of inhibition were as follows A. incisum > Pityrogramma calomelanos var. calomelanos = Cyclosorus interruptus > P. multiaurita > P. biaurita = H. arifolia = C. thalictroides > P. vittata > P. confusa > A. latifolium. The result of the present study suggested that further studies on the isolation of active principles from the aqueous extracts of Cyclosorus interruptus, Pityrogramma calomelanos var. calomelanos, P. biaurita, P. multiaurita, A. incisum, H. arifolia, and C. thalictroides may bring out an alternative drug for the inflammation.

Keywords: anti–inflammatory, pteridophytes, haemolysis, antibiotics, neutrophils

Synthetic drugs are employed for the pain and inflammatory administration possess various side effects, toxic to animals.¹ and they are very expensive.² In addition, the increase in dominance of multiple drug resistance leads the researcher to develop new anti–inflammatory drugs. Previous studies on the phytochemical potentials proved the versatile utility of secondary metabolites.^3–7 Extraction and isolation of various bioactive compounds from plants paved a pathway to discover new active principles with therapeutic application.^8–10 The ancient history reported the utilization of Pteridophytes in Homeopathic, Ayurvedic and Unani medicines as insecticides, antibiotics, food and ornamentation.¹¹ Lamichhane et al. ¹² revealed the anti–inflammatory potentials of Cheilanthes albomarginata. Anti–inflammatory activity of Cyathea nilgirensis was reported by Shaya Mary and Mahesh ¹³. Ismiarni Komala et al.¹⁴ determined the anti–inflammatory potentials of Nephrolepis falcata and Pyrrosia lanceolate. With this knowledge the present study was aimed to explore the anti–inflammatory potential of selected Pteridophytes from Western Ghats of South India using heat induced haemolytic activity.

The healthy and disease free young sporophytes of selected pteridophytes viz., Cyclosorus interruptus (Willd.] H. Ito., Pityrogramma calomelanos (L..] Linkvar. calomelanos, Pteris vittata L., Pteris confusa T. G. Walker., Pteris biaurita L., Pteris multiaurita Ag., Adiantum incisum Forssk., Adiantum latifolium Lam. , Hemionitis arifolia (Burm.] T. Moore. and Ceratopteris thalictroides (L.] Brong. were collected from their natural habitats, Tamil Nadu, India. The collected sporophytes were washed in the running tap water to remove the debris and unwanted materials. The materials were dried and the excess water was removed using blotting paper. 10gm of young sporophytes were cut into small pieces and boiled with 100 ml of distilled water for 30 min. After 30 min, the aqueous extracts were filtered using Whatman No. 1 filter paper. The filtered extracts were centrifuged at 3000rpm for 10 min. The supernatants were collected and used for anti–inflammatory studies.

Membrane stabilization test

Preparation of red blood cells (RBCs] suspension: Fresh whole human blood (10 ml) was collected and transferred to the centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10 min and were washed three times with equal volume of normal saline. The volume of blood was measured and reconstituted as 10% v/v suspension with normal saline.^15,16

Heat induced haemolytic assa:The reaction mixture (2ml] consisted of 1ml of Cyclosorus interruptus, Pityrogramma calomelanos var. calomelanos, P. vittata, P. confusa, P. biaurita, P. multiaurita, A. incisum, A. latifolium, H. arifolia, and C. thalictroides aqueous extracts and 1 ml of 10% RBCs suspension, instead of test sample only saline was added to the control test tube. Aspirin was taken as a standard drug. All the centrifuge tubes containing reaction mixture were incubated in water bath at 56 °C for 30 min. At the end of the incubation the tubes were cooled under running tap water. The reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants was taken at 560 nm. The experiment was performed in triplicates for all the test samples. Percent membrane stabilization activity was calculated by the following formula.^16,17

% of inhibition = Control ABS – Sample ABS / Control ABS * 100

Stabilization of RBCs membrane was studied to further establish the mechanism of anti–inflammatory action of Cyclosorus interruptus, Pityrogramma calomelanos var. calomelanos, P. vittata, P. confusa, P. biaurita, P. multiaurita, A. incisum, A. latifolium, H. arifolia, and C. thalictroides aqueous extract with varied inhibition (Figure 1).Except A. latifolium and P. confusa all other tested extracts effectively inhibited the heat induced hemolysis (Figure 1). The results provided evidences for membrane stabilization effect of the selected plant drug as an additional mechanism for their anti–inflammatory effect. Due to the resemblance of RBC membrane with lysosomal membrane, this effect may possibly inhibit the release of lysosomal content of neutrophils at the site of inflammation. The studied extracts percentage of inhibition were as follows A. incisum > Pityrogramma calomelanos var. calomelanos = Cyclosorus interruptus > P. multiaurita > P. biaurita = H. arifolia = C. thalictroides > P. vittata > P. confusa > A. latifolium.

Figure 1 Antinflammatory Activity of Selected Pteridophytes.

The aqueous extracts of Cyclosorus interruptus, Pityrogramma calomelanos var. calomelanos, P. vittata, P. biaurita, P. multiaurita, A. incisum, H. arifolia, and C. thalictroides inhibited the heat induced haemolysis of RBCs. These results provide evidence for membrane stabilization as an additional mechanism of their anti inflammatory effect. This effect may possibly inhibit the release of lysosomal content of neutrophils at the site of inflammation. In recent years, the search for plant derived drug with anti inflammatory properties has been focussed by many researchers due to their potential use in the therapy of various chronic and infectious diseases.¹⁸ The present study also examined anti inflammatory potential of selected plants viz., Cyclosorus interruptus, Pityrogramma calomelanos var. calomelanos, P. vittata, P. biaurita, P. multiaurita, A. incisum, A. latifolium, P. confusa, H. arifolia, and C. thalictroides using heat induced haemolytic assay. Plant derived phenolic, flavonoids, terpenoids, tannins and saponins possess potent anti inflammatory activity.^19–27

The results of the present study showed varied percentage of inhibition, this may be due to the occurrence of phenolic, flavonoids, terpenoids, tannins and saponins in the studied plant extracts. Williams et al.²⁸ suggested that extracts which can inhibit the denaturation greater than 20% over the range concentration were considered as having anti–inflammatory property.²⁸ In the present study the aqueous extracts of Cyclosorus interruptus, Pityrogramma calomelanos var. calomelanos, P. biaurita, P. multiaurita, A. incisum, H. arifolia, and C. thalictroides showed more than 20% of inhibition. The result of the present study suggest that further studies on the isolation of active principles from the aqueous extracts of Cyclosorus interruptus, Pityrogramma calomelanos var. calomelanos, P. biaurita, P. multiaurita, A. incisum, H. arifolia, and C. thalictroide may bring out an alternative drug for the inflammation.

None.

Author declares that there are no conflicts of interest.

1. Thomas MC. Diuretics, ACE inhibitors and NSAIDs – the triple whammy. Med J Aust. 2000;172(4):184-185.
2. Ahmad F, Khan RA, Rasheed S. Study of analgesic and anti–inflammatory activity from plant extracts of Lactuca scariola and Artemisia absinthium. Journal of Islamia Academy Sciences. 1992;5(2):111-114.
3. Fabricant DS, Fansworth NR. The value of plants used in traditional medicine for drug discovery. Environ Health Perspect. 2001;109(Suppl 1):69-75.
4. Prachayasittikul S, Buraparuangsang P, Worachartcheewan A, et al. Antimicrobial and antioxidant activity of bioreactive constituents from Hydnophytum formicarum Jack. Molecules. 2008;13(4):904-921.
5. Chen IN, Chang CC, Wang CY, et al. Antioxidant and antimicrobial activity of Zingiberaceae plants in Taiwan. Plant Foods Hum Nutr. 2008;63(1):15-20.
6. Pesewu GA, Cutler RR, Humber DP. Antibacterial activity of plants in traditional medicine of Ghana, with particular reference to MRSA. J Ethnopharm. 2008;116(1):102-111.
7. Turker AU, Usta C. Biological screening of some Turkish medicinal plants for antimicrobial and toxicity studies. Nat Prod. 2008;22(2):136-146.
8. Kumar RA, Sridevi K, Kumar NV, et al. Anticancer and immunostimulatory compounds from Andrographis paniculata. J Ethnopharm. 2004;92(2–3):291-295.
9. Sheeja K, Kuttan G. Activation of cytotoxic T lymphocyte responses and attenuation of tumour growth in vivo by Andrographis paniculata extract and andrographolide. Immunopharmacol Immunotoxicol. 2007;29(1):81-93.
10. Mukherjee PK, Kumar V, Houghton PJ. Screening of Indian medicinal plants for acetyl cholinesterase inhibitory activity. Phyto Res. 2007;21(12):1142-1145.
11. Hynniewta SR, Kumar Y. Herbal remedies among the Khasi traditional healers and village folks in Meghalaya. Indian J Tradit Knowl. 2008;7(4):581-586.
12. Lamichhane R, Se–Gun Kim, Amrit Poudel, et al. Evaluation of in vitro and in vivo Biological activities of Cheilanthes albomarginata. BMC Complement Altern Med. 2014;14:342.
13. Sahaya Mary, Mahesh MK. Anti–inflammatory activity of Cyathea nilgirensis Holttum, against Carrageenan induced Paw edema. International Journal of Recent Scientific Research. 2015;6(8):5807-5809.
14. Ismiarni Komala, Azrifitria, Yardi, et al. Antioxidant and anti–inflammatory activity of the Indonesian ferns, Nephrolepis falcata and Pyrrosia lanceolate. Int J Pharm Pharm Sci. 2015;7(12):162-165.
15. Sadique J, Al–Rqobahs WA, Bughaith EI–Gindi Ar. The bioactivity of certain medicinal plants on the stabilization of RBS membrane system. Fitoterapia. 1989;60:525-532.
16. Sakat S, Juvekar AR, Gambhire MN. In vitro antioxidant and anti–inflammatory activity of methanol extract of Oxalis corniculata Linn. I. J Pharm Pharm Sci. 2010;2(1):146-155.
17. Shinde UA, Phadke AS, Nari AM, et al. Membrane stabilization activity– a possible mechanism of action for the anti–inflammatory activity of Cedrus deodara wood oil. Fitoterapia. 1999;70(3):251-257.
18. Halliwell B. Antioxidants in human health and disease. Annu Rev Nutr. 1996;6:33-50.
19. Roy SP, Niranjan CM, Jyothi TM, et al. Antiulcer and anti–inflammatory activity of aerial parts Enicostemma littorale Blume. J Young Pharm. 2010;2(4):369-373.
20. Garg VKR, Jain M, Sharma PKR, et al. Anti inflammatory activity of Spinacia oleracea. Intern J Pharma Prof Res. 2010;1(1):1-4.
21. Lai HY, Yau YY, Kim KH. Blechnum orientale Linn – a fern with potential as antioxidant, anticancer and antibacterial agent. BMC Complem Altern Med. 2010;10:15.
22. Lopez–Lazaro M. Distribution and biological activities of the flavonoid luteolin. Mini Rev Med Chem. 2009;9(1):31–59.
23. Yoshida T, Konishi M, Horinaka M, et al. Kaempferol sensitizes colon cancer cells to TRAIL–induced apoptosis. Biochem. Biophys Res Comm. 2008;375(1):129-133.
24. Amaral S, Mira L, Nogueira JM, et al. Plant extracts with anti–inflammatory properties––a new approach for characterization of their bioactive compounds and establishment of structure–antioxidant activity relationships. Bioorg Med Chem. 2009;17(5):1876-1883.
25. Neukirch H, D'Ambrosio M, Sosa S, et al. Improved anti–inflammatory activity of three new terpenoids derived, by systematic chemical modifications, from the abundant triterpenes of the flowery plant Calendula officinalis. Chem Biodiv. 2005;2(5):657-671.
26. Fawole OA, Amoo SO, Ndhlala AR, et al. Anti–inflammatory, anticholinesterase, antioxidant and phytochemical properties of medicinal plants used for pain–related ailments in South Africa. J Ethnopharmacol. 2010;127(2):235-241.
27. Gepdireman A, Mshvildadze V, Suleyman H, et al. Acute anti–inflammatory activity of four saponins isolated from ivy: alphahederin, hederasaponin–C, hederacolchiside–E and hederacolchiside–F in carrageenan–induced rat paw edema. Phytomedicine. 2005;12(6–7):440-444.
28. Williams LAD, Connar AO, Latore L, et al. The in vitro anti–denaturation effects induced by natural products and non–steroidal 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(4):327-331.