1 INTRODUCTION:

The species of Phyllanthus has long been used in folk medicine for liver protection, intestinal infections, hepatitis, diabetes, and jaundice.[1,2]Phyllanthus fraternus Webster Linn [PF] (family, Euphorbiaceae) is a is an annual herb, whose stem is non-erect and 30 cm long, the leafy shoots are 5–10 cm long, oblong and joined to the brachlets of the stem, six sepals in the flower and well distributed in India. It is used as a folklore remedy for the treatment of various diseases of liver by traditional healers and tribals. [1-2]

The hydroalcoholic extract (HE) of the Phyllanthus fraternus, when given intraperitoneally, produced significant inhibition of acetic acid-induced abdominal constrictions, with mean ID50 values of 7.4 mg/kg and also produced graded inhibition against both phases of formalin-induced licking, being more active in relation of the late phase.

The HE of the Phyllanthus elicited significant inhibition of the capsaicin-induced neurogenic pain, with mean ID50 value of >30 mg/kg. [3] Gopi and Setty have investigated the protective effect of an aqueous extract of Phyllanthus fraternus (AEPF) against bromobenzene induced mitochondrial dysfunction in rat liver mitochondria. [4] It has been reported earlier that mitochondrial dysfunction caused by the administration of alcohol or thioacetamide or carbon tetrachloride could be prevented by prior administration of an aqueous extract of Phyllanthus fraternus and the protective effect was attributed mainly to the antioxidant property of the extract, while the effect of administration of allyl alcohol on the oxidative stress and the protective effect due to administration of an aqueous extract of Phyllanthus fraternus against allyl alcohol-induced damage in liver mitochondria were studied previously. [5] The scientific data indicates that the therapeutic intervention of Phyllanthus, which is a potent antihepatotoxin and antiviral agent, can be used to prevent or reduce the deterioration caused by free radicals thereby preventing subsequent pathological and biochemical changes which occur during cerebral ischemia.[6]

Ramakrishna et al have suggested that the Phyllanthus fraternus extract is an efficient armament against nephrotoxicity induced by bromobenzene and may constitute a lead for the discovery of a novel drug for the treatment of drug-induced nephrotoxicity. [7] Kumari & Setty suggests that administration of aqueous extract of P. fraternus can enhance the therapeutic potential of anticancer drugs by reducing drug related toxicity. [8]

Studies have established analgesic activity of Phyllanthus fraternus Webster. [9] While genus phyllanthus has reported to posses’ good anti-inflammatory potential. [10]Therefore, with the aim to investigate the possible anti-inflammatory activity of PF, present study of was carried using in-vitro models.

2. MATERIALS AND METHODS:

2.1 Plant material:

P. fraternus Webster was obtained from different places in Karad western Maharashtra. The plant was identified and authenticated by Botanical survey of India, Pune.

2.2 Preparation of the extract:

The dried leaves, stems and roots of P. fraternus was minced and extracted with 70% ethanol-water in the proportion of 70:30, being stirred and macerated at room temperature (22-28°C) for 15 days. The ethanol was evaporated and the extract (yield 5-7%) was concentrated to the desired level and stored in a refrigerator. The extracts were dissolved in 0.9% NaCl solution or distilled water to the desired concentration just before use.

2.3 Drugs and chemicals:

Diclofenac sodium was procured from Indoco Remedies, Mumbai. All the other chemicals were of analytical grade obtained commercially. Double distilled water was used throughout the study.

2.4 Evaluation of anti-inflammatory effects by membrane stabilizing property, [11-12]:

Alsever solution was prepared by dissolving 2% dextrose, 0.8% Sodium citrate, 0.05% citric acid and 0.42% sodium chloride in distilled water, which was later sterilized. Blood was collected from median cubital vein of the first author [ARC 29 years old male]. The collected blood was mixed with equal volume of sterilized alsever solution. The blood was centrifuged at 1000-2000 rpm and packed cells were washed with isosaline and a suspension in 10% (V/V) Isosaline was made. Various concentrations of the phyllanthus extracts were prepared in a mixture of 1ml Phosphate buffer, 2ml Hyposaline and 0.5ml HRBC suspension. Diclofenac sodium was used as the reference drug. Instead of hyposaline, 2ml of distilled water was used in control. The assay mixtures were incubated at 37ºC for 30 minutes and centrifuged. The haemoglobin content in the supernatant solution was estimated using UV spectrophotometer (Shimadzu, UV 1800) at 560nm. The percentage haemolysis was calculated by assuming the haemolysis produced in the presence of distilled water as 100%.

The percentage of HRBC [Human Red Blood Cells] membrane stabilization or protection was calculated using this equation,

Percentage inhibition of Haemolysis =

100 X [OD1-OD2 /OD1]

Where, OD1= Optical Density of hypotonic buffered saline solution alone and OD2 = Optical Density of test sample (phyllanthus extracts and diclofenac) in hypotonic medium.

2.5 Evaluation of in vitro anti-inflammatory activity by Protein denaturation method [13]

The reaction mixture (10 mL) consisted of 0.4 mL of egg albumin (from fresh hen’s egg), 5.6 mL of phosphate buffered saline (PBS, pH 6.4) and 4 mL of varying concentrations of Phyllanthus fraternus HE extract so that final concentrations become 25, 50, 100, 200, 400 and 800 µg/mL. Similar volume of double-distilled water served as control. Then the mixtures were incubated at (37⁰c ±2) in a incubator for 15 min and then heated at 70⁰ c for 5 min. After cooling, their absorbance was measured at 660 nm (SHIMADZU, UV 1800) by using vehicle as blank and their viscosity was determined by using Ostwald viscometer. Diclofenac sodium at the final concentration of (25, 50, 100 & 200 µg/mL) was used as reference drug and treated similarly for determination of absorbance and viscosity. The percentage inhibition of protein denaturation was calculated by using the following formula,

% inhibition = 100 x ( Vt / Vc - 1)

Where, Vt = absorbance of test sample, Vc = absorbance of control.

The extract/drug concentration for 50% inhibition (IC50) was determined by plotting percentage inhibition with respect to control against treatment concentration.

3. RESULTS:

In the current study in-vitro results confirm the reported anti-inflammatory activity of P. fraternus Webster.

3.1 Membrane stabilizing activity:

Since HRBC membranes are similar to lysosomal membrane components, the prevention of hypotonicity-induced HRBC membrane lysis was taken as a measure of anti-inflammatory activity of drugs. The results obtained demonstrate that PF can significantly and dose-dependently inhibits RBC haemolysis (Table 1). In the study PF extract at concentration range of 25-800 μg/ml protects significantly the erythrocyte membrane against lysis induced hypotonic solution. Also Diclofenac sodium offered a significant protection of the RBC’s against the damaging effect. The extract at concentration range of 25-800 μg/ml protects the human erythrocyte membrane against lysis induced by hypotonic solution. At concentration of 200 μg/ml, the PF extract produced 28.121% inhibition of RBC haemolysis as compared with 52.48 % produced by diclofenac sodium.

Table 1. Effect of P. fraternus Webster on hypotonic solution-induced haemolysis of erythrocyte membrane

Treatment		Concentration (μg/ml)		% Inhibition of haemolysis
Control		0		0
P. fraternus Webster		25		3.515
	50		10.787
	100		16.00
	200		28.121
	400		36.848
	800		50.181
Diclofenac sodium		50		31.878
	100		40.848
	200		52.848

Table 2. Effect of PF on protein denaturation.

Treatment	Concentration (μg/ml)	% Inhibition of protein denaturation	Viscosity (cps)
Control	0	0	1.46
P. fraternus Webster	25	26.128	0.67
	50	34.679	0.72
	100	97.862	0.76
	200	142.99	0.85
	400	225.412	0.89
	800	334.679	0.98
Diclofenac sodium	25	28.741	0.57
	50	35.154	0.71
	100	111.11	0.84
	200	159.38	1.02

3.2 Inhibition of protein denaturation:

The inhibitory effects of different concentrations of PF on protein denaturation are summarized in Table 2. The present findings exhibited a concentration dependent inhibition of protein (albumin) denaturation by PF extracts throughout the concentration range of 25 to 800 µg/mL. Diclofenac sodium (at the concentration range of 78.125 to 2 500 µg/mL) was used as reference drug which also exhibited concentration dependent inhibition of protein denaturation. PF extract at concentration of 800 μg/ml and Diclofenac sodium at concentration of 200 μg/ml showed significant inhibition 334.67 % and 159.38 % respectively of protein denaturation when compared with control. This was further confirmed by comparing their IC50 values. PF possessed IC50 value 52 µg/mL whereas that of diclofenac sodium was found to be 44 µg/mL.

4. DISCUSSION:

4.1 Membrane stabilizing activity of P. fraternus Webster-

Some of the extracts of P. emblica were found effective in preventing human polymorphonuclear leukocytes and platelet functions. They were capable of inhibiting calcium ionophore A23187-induced leukotriene B4 release from human polymorphonuclear, thromboxane A2 production in platelets and also adrenaline-induced platelet aggregation. [14] It is well known that vitality of cells depends on the integrity of their membranes. Exposure of RBC to injurious substances such as hypotonic medium, methyl salicylate or phenyl hydrazine results in the lysis of membrane accompanied by haemolysis and oxidation of haemoglobin [15-16]. The haemolytic effect of hypotonic solution is related to excessive accumulation of fluid within the cell resulting in the rupturing of its membrane. Injury to RBC membrane will render the cell more susceptible to secondary damage through free radical-induced lipid peroxidation. This action is consistent with the observation that breakdown of bimolecules leads to the formation of free radicals which in turn enhance cellular damage. [15-16] The progression of bone destruction seen in rheumatoid arthritis is due to increased free radical activity.[16-17]

Also the Extract with membrane-stabilizing properties are well known for their interfering activity with the early phase of the inflammatory mediators release, namely the prevention of phospholipases release that trigger the formation of inflammatory mediators.[11 & 15-16]

A possible explanation for the stabilizing activity of P. fraternus Webster could be an increase in the surface area to volume ratio of the cells which could be brought about by an expansion of membrane or shrinkage of the cell, and an interaction with membrane proteins.[18] Moreover, it has also been shown that the deformability and cell volume of erythrocytes is closely related to the intracellular content of calcium. Hence, it may be speculated that the cytoprotective effect on erythrocyte membrane may be due to the ability of the test extract to alter the influx of calcium into the erythrocytes. The present investigation suggests that the membrane stabilizing activity of P. fraternus Webster may be playing a significant role in its anti-inflammatory activity.

4.2 Denaturation of proteins and activity of P. fraternus Webster-:

Denaturation of proteins is a well documented cause of inflammation and rheumatoid arthritis.[19] Production of auto antigens in certain arthritic diseases may be due to denaturation of proteins in vivo[19-21] Several anti-inflammatory drugs have shown dose dependent ability to inhibit thermally induced protein denaturation. It has been reported that one of the features of several non-steroidal anti-inflammatory drugs is their ability to stabilize (prevent denaturation) heat treated albumin at the physiological pH (pH, 6.2-6.5) [19-21]. This anti-denaturation effect was further supported by the change in viscosities. It has been reported that the viscosities of protein solutions increase on denaturation[13]. In the present study, the relatively high viscosity of control dispersion substantiated this fact. Ability of PF extract to bring down thermal denaturation of protein is possibly a contributing factor for its anti-inflammatory activity.

4.3 Phytoconstituents and anti-inflammatory activity of P. fraternus Webster-

Phytochemical studies from the Phyllanthus fraternus extract showed the presence of alkaloids, triterpenoids, glycosides, flavonoids, tannins. While previous studies also suggest presence of several bioactive molecules, such as lignans namely phyllanthin, hypophyllanthin, niranthin, nirtetralin and phyltetralin in the plant extract.[22] The anti-inflammatory activity of PF extract in the present study may be due to the presence of therapeutically active lignans and flavonoids.[23] The anti-inflammatory and analgesic activity of phyllanthus extracts rich in flavonoids, lignans and their rich fractions on inflammation have previously been reported.[14, 16 & 22]

CONCLUSION:

The data of our studies suggests that PF showed significant anti-inflammatory activity in both the models tested. Further studies involving the purification of the phyto constituents of the plant and their further investigations may result in the development of a potent anti-inflammatory agent.

ACKNOWLEDGEMENTS:

The authors are thankful to UGC [University Grants Commission] for providing fellowship under RGNFS [Grant reference no.- F. 16-1917(SC)/2010 (SA-III)] to ARC for his doctoral studies.

5. REFERENCES:

1. Binay Sen, S.D. Dubey, K. Tripathi Pharmacognostical study of Tamalaki ( Phyllanthus fraternus Webster), a herb used in Tamaka-svasa, AYU, 2011, 32, 398-401.

2. Sharma S.K,Sheela M.A.Pharmacognostic evaluation of leaves of certain Phyllanthus species used as a botanical source of Bhumyamalaki in Ayurveda, AYU, 2012, 32, 250-253.

3. Adair R.S. Santos a, Rafael O.P. De Campos a, Obdu´ lio G. Miguel, Valdir Cechinel Filho, Antonio Carlos Siani, Rosendo A. Yunes, Joao B. Calixto, Antinociceptive properties of extracts of new species of plants of the genus Phyllanthus (Euphorbiaceae) Journal of Ethnopharmacology, 2000, 72, 229–238.

4. Sriram Gopi, Oruganti H. Setty Protective effect of Phyllanthus fraternus against bromobenzene induced mitochondrial dysfunction in rat liver mitochondria, Food and Chemical Toxicology, 2010, 48, 2170–2175.

5. R. Sailaja, O.H. Setty, Protective effect of Phyllanthus fraternus against allyl alcohol-induced oxidative stress in liver mitochondria Journal of Ethnopharmacology, 2006, 105, 201–209

6. Kranthi Kumari Naga, A Padmini, Manas Panigrahi, Phanithi Prakash Babu, Protective efficacy of phyllanthus fraternus against cerebral ischemia reperfusion in rat, Annals of Neurosciences, 2008, 15, 36-42.

7. Vadde Ramakrishna, Sriram Gopi, Oruganti H. Setty, Protective effect of Phyllanthus fraternus against bromobenzene- induced mitochondrial dysfunction in rat kidney, Chinese Journal of Natural Medicines 2012, 10(5): 0328-0333

8. Kumari KK, Setty OH. Protective effect of Phyllanthus fraternus against mitochondrial dysfunction induced by co-administration of cisplatin and cyclophosphamide. J Bioenerg Biomembr. 2012, 44, 179-88.

9. Bagalkotkar G, Sagineedu SR, Saad MS, Stanslas J., Phytochemicals from Phyllanthus and their pharmacological properties, a review. J Pharm Pharmacol. 2006, 58, 1559-70.

10. Jay Ram Patel, Priyanka Tripathi, Vikas Sharma, Nagendra Singh Chauhan, Vinod Kumar Dixit, Phyllanthus amarus: Ethnomedicinal uses, phytochemistry and pharmacology: A review Journal of Ethnopharmacology, 2011, 138, 286– 313.

11. Gambhire M, Juvekar A and Wankhede S. Evaluation of anti-inflammatory activity of methanol extract of Barleria cristata leaves by in vivo and in vitro methods. The Internet Journal of Pharmacology, 2009, 7(1) 1-4.

12. Gambhire MN, Juvekar AR and Sakat SS. Evaluation of anti-inflammatory activity of methanol extract of Murraya koenigi leaves by in vivo and in vitro methods. Pharmacologyonline, 2009, 1(10) 1072-1094.

13. Sangita Chandra, Priyanka Chatterjee, Protapaditya Dey, Sanjib Bhattacharya, Evaluation of in vitro anti-inflammatory activity of coffee against the denaturation of protein, Asian Pacific Journal of Tropical Biomedicine, 2012, S178-S180.

14. Ihantola-Vormisto, A., Summanen, J., Kankaanranta, H., Vuorela, H., Asmawi, Z.M., Moilanen, E., Anti-inflammatory activity of extracts from leaves of Phyllanthus emblica. Planta Medica, 1997, 63, 518–524.

15. Varadarasou Mouttaya Mounnissamy, Subramanian Kavimani, Vaithilingam Balu, Sabarimuthu Darlin Quine, Evaluation of anti-inflammatory and membrane stabilizing properties of ethanol extract of Cansjera rheedii J.Gmelin (Opiliaceae), Iranian Journal of Pharmacology & Therapeutics, 2007, 6, 235-237.

16. Raja Chakraborty, Biplab De, Nayakanti Devann, Saikat Sen, Antiinflammatory, antinociceptive and antioxidant activities of Phyllanthus acidus L. extracts, Asian Pacific Journal of Tropical Biomedicine (2012)S953-S961

17. L.D.A.M. Arawwawala, L.S.R. Arambewela, W.D. Ratnasooriya, Alpinia calcarata Roscoe: A potent antiinflammatory agent, Journal of Ethnopharmacology, 2012, 139, 889– 892

18. Chopade, A. R. Somade, P M. & Sayyed F J., Membrane stabilizing activity & protein denaturation a possible mechanism of action for the anti-inflammatory activity of Phyllanthus amarus, JKIMSU, 2012, 1, 67-72.

19. Williams LAD, O’Connar A, Latore L, Dennis O, Ringer S, Whittaker JA, 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, 327-331.

20. Umapathy E, Ndebia EJ, Meeme A, Adam B, Menziwa P, Nkeh-Chungag BN, et al. An experimental evaluation of Albuca setosa aqueous extract on membrane stabilization, protein denaturation and white blood cell migration during acute inflammation. J Med Plants Res., 2010, 4, 789-795.

21. A Vijayalakshmi, V Ravichandiran, Malarkodi Velraj, S Hemalatha, G Sudharani, S Jayakumari, Anti-anaphylactic and anti-inflammatory activities of a bioactive alkaloid from the root bark of Plumeria acutifolia Poir, Asian Pacific Journal of Tropical Biomedicine, 2011, 401-405

22. Khatoon, S., Rai, V., Rawat, A.K.S., Mehrotra, S., Comparative pharmacognostic studies of three Phyllanthus species. J. Ethnopharmacol., 2006, 104, 79–86.

23. Havsteen BH. The biochemistry and medical significance of the flavonoids. Pharmacol. Ther. 2002, 96, 67-202.

Received on 18.01.2013 Modified on 02.02.2013

Research J. Pharm. and Tech. 6(3): March 2013; Page 251-254