In vitro antioxidant and anti-inflammatory potential of Polyalthia longifolia in rats.

Sir,

Polyalthia longifolia is a large genus of shrubs and trees, belonging to family Annonaceae, distributed in tropics and subtropics. It is used for the treatment of skin diseases, fever, diabetes, and hypertension. A number of biologically active compounds have been isolated from this nonvolatile fraction of the plant.[1] Its aqueous extract also helps lower blood pressure and respiration rate in experimental animals. The hypoglycemic and antihyperglycemic activities of various solvent extracts of P. longifolia var. pendula leaf extracts were evaluated in alloxan-induced experimental diabetic rats.[2] Hence an attempt is made to evaluate the antioxidant and anti-inflammatory potential of the ethanolic and aqueous extracts of leaves of P. longifolia.

The leaves of P. longifolia (sonn.) was procured and authenticated by Dr. Siddamallaya, Survey officer, Regional Research Institute (Ay.), Bengaluru. The coarse powder of the leaves was Soxhlet extracted with 90% ethanol. The aqueous extract was prepared by the processes of maceration. Experimental protocol was approved by the Institutional Animal Ethics committee of KLE College of Pharmacy, Bangalore (Ref. No. IAEC/KLECP/BNG/06/2009), and was taken for conducting anti-inflammatory activity.

The 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging capacity of P. longifolia (aqueous and ethanolic extracts) was measured. One ml of 0.1 mM ethanolic solution of DPPH was added to 3 ml of P. longifolia solution in water for aqueous extract and in ethanol for ethanolic extract of different concentrations (5, 25, 50, 75, 100, 250 μg/ml). After 30 min, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging effect.[3]

Nitric oxide generated from Sodium Nitroprusside (SNP) was measured using Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride in 2% H3PO4). About 100 mM of SNP was prepared in Phosphate Buffered Saline (PBS) (pH 7.4). The reaction mixture (3 ml) containing 100 mM SNP (0.2 ml, final concentration 10 mM) and PBS (1.8 ml) and extracts at various concentrations (1 ml) were incubated at 25°C for 180 min. At the end of 30 min, 1 ml of incubated sample was mixed with 1 ml of Griess reagent. The absorbance of the chromophore formed during the diazotization of nitrite with sulfanilamide and subsequent coupling with naphthyl ethylenediamine was read at 540 nm.[4] Superoxide anions were generated using Phenazine Methosulfate-Nicotinamide Adenine Dinucleotide (PMS-NADH) system. The superoxide anions are subsequently made to reduce nitroblue tetrazolium, which yields a chromogenic product, which is measured at 560 nm. The PMS-NADH system was used for generation of superoxide anion. It was assayed by the reduction of Nitroblue Tetrazolium (NBT).[5] About 1 ml of NBT (156 μM), 1 ml NADH (468 μM) in 100 mM phosphate buffer (pH 7.8) and 0.1 mL of sample solution of different concentrations were mixed. The reaction was started by adding 100 μl PMS (60 μM). The reaction mixture was incubated at 25°C for 5 minute and absorbance of the mixture was measured at 560 nm against blank samples. The percentage inhibition was determined by comparing the results of control and test samples.[6]

Total reduction capability of P. longifolia (aqueous and ethanolic extracts) was estimated by using the method of Oyaizu. Different concentrations of P. longifolia extracts (25, 50, 100, 150, 200, 250, 500 μg/ml) in 1 ml of distilled water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 mi, 1%). The mixture was incubated at 50°C for 20 min and then 2.5 ml of trichloro acetic acid (10%) was added to the mixture. After that it was centrifuged for 10 min at ×1000g. The upper layer of the solution (2.5 ml) was taken and mixed with distilled water (2.5 ml) and ferric chloride (0.5 ml, 0.1%) and the absorbance was measured at 700 nm. Higher absorbance of the reaction mixture indicates greater reducing power.[6-8]

Albino rats (150-200 gm) were divided into six groups, each containing six animals. Group I served as control, Group II-VI received aqueous and ethanolic extracts (200 mg/kg and 300 mg/kg body weight) of P. longifolia and indomethacin at the dose of 10 mg/kg body weight, respectively. After 1 h of drug administration, 0.1 ml of 1% Carrageenan (Sigma) in normal saline solution was injected into the sub-plantar region of the right hind paw of each rat. The paw volumes were measured at 0, 1, 2, 3, 4, 5, and 24 h intervals with the help of plethysmograph. The percentage decrease in paw volume was determined using the formula (Control reading – Test reading)/Control reading ×100).[9]

The percentage DPPH scavenging capacity of ethanolic and aqueous extracts from 25 to 250 μg/ml was 19.28 ± 2.18 to 72.78 ± 1.003 and 19.46 ± 0.507 to 62.90 ± 2.85, respectively. At 250 μg/ml ethanolic extract of P. longifolia produced DPPH scavenging capacity comparable (P<0.01) to that of ascorbic acid. When nitric oxide scavenging activity of ethanolic and aqueous extracts of P. longifolia was compared with standard ascorbic acid at 75 and 100 μg/ml ethanolic extract produced better activity (P<0.01 and P<0.05, respectively) and at 125, 150, and 250 μg/ml, it produced nitric oxide scavenging activity comparable (P<0.05) to that of ascorbic acid. Extracts of P. longifolia produced superoxide radical scavenging activity from 25 μg/ml and increased up to 200 μg/ml (53.56 ± 0.810 to 60.52 ± 0.510 and 85.54 ± 4.860 to 92.41 ± 2.230, respectively). Both extracts of P. longifolia at 25, 50, 100, 150, 200 μg/ml produced superoxide radical scavenging activity, which was better (P<0.01) than standard Butylated Hydroxyanisole (BHA) at all concentrations. The extract was found to possess reduction capability, which increased with increase in concentration (25 to 250 μg/ml). A 50 μg/ml aqueous extract of P. longifolia produced reducing capability similar to that of (P<0.05) standard ascorbic acid. Ethanolic extract of P. longifolia was found to have better antioxidant activity when compared to aqueous extracts of P. longifolia.

The ethanolic and aqueous extracts of P. longifolia were found to reduce the edema induced by Carrageenan. Both extracts of P. longifolia were found to exhibit significantly (P<0.05) anti-inflammatory activity than the standard indomethacin [Table 1].

Table 1

Effects of aqueous and ethanolic extracts of leaves of Polyalthia longifolia on Carrageenan-induced paw edema in albino Wistar rats

Extracts possess antioxidative and radical scavenging capacity. Both the ethanolic and aqueous extracts of P. longifolia produced anti-inflammatory activity due to the presence of flavonoids and phenolic compounds at various time intervals that was evaluated for by Carrageenan-induced paw edema model. This suggests that P. longifolia may produce the anti-inflammatory activity at all three phases of inflammation by inhibiting more than one mediator, and therefore producing the activity. Ethanolic extract was found to possess better anti-inflammatory activity than aqueous extracts.