IN VITRO ANTIOXIDANT AND IN VIVO ANTI-INFLAMMATORY ACTIVITY OF Curcuma albiflora THW

Curcuma albiflora Thw. is a poorly explored plant which is termed as Harankaha locally. Among other Harankaha plants, C. albiflora is threaten and restricted to Sabaragamuwa province only. Therefore, current study was conducted to quantify phytochemicals in terms of total phenolic content (TPC), total flavonoid content (TFC) and prove or disprove its antioxidant and anti-inflammatory activity using ferric reducing antioxidant power (FRAP), DPPH, ABTS+, and oxygen radical absorbance capacity (ORAC) assays, and carrageenaninduced paw oedema, formaldehyde-induced paw oedema, and cotton pellet induced granuloma tests in Wistar rats respectively. Whole plant extract from composite samples was prepared using 50% ethanol (in water) and 50% dichloromethane (in methanol) as solvents mixture by continuous extraction (6 h). Antioxidant values of TPC, TFC, and FRAP were 31.25 ± 1.48, 11.22 ± 0.13, and 17.90 ± 0.38 mg/g respectively. IC50 values (ppm) of DPPH and ABTS+ were 827.78 ± 6.06, and 188.84 ± 2.99 respectively. ORAC value was 128.10 ± 3.29 mg/g. The correlation of TFC was shown that it may link to flavonoid content. Since, C. albiflora inhibited (61% at 1h) 1st phase in the acute model, and at the late phase on sub-chronic model considering insignificant on cotton pellet granuloma test was shown that its anti-inflammatory activity (as 19.5% on 400 mg/kg) may not relate on prostaglandin. Further experiments are warrant to identify a mechanism of antiinflammatory activity of C. albiflora.


Introduction
Harankaha botanically known as C. albiflora Thw. is claimed to be used as an anti-inflammatory medicinal plant in traditional medicine (DOA, 2002).Under the same vernacular name, three plants (Curcuma albiflora, C. zedoaria Rosc., and Zingiber zerumbet Smith.) are being used (Dassanayaka, 1983).Amongst C. albiflora is an endemic, threaten and endangered plant (MOE, 2012).It is also an unexplored.The current study was conducted to study antioxidant activity of C. albiflora by using total phenolic content (TFC), total flavonoid content (TPC), ferric reducing antioxidant power (FRAP), DPPH, ABTS + , and oxygen radical absorbance capacity (ORAC), and also anti-inflammatory activity of C. albiflora by using formaldehyde-induced paw oedema, carrageenan-induced paw oedema, and cotton pellet induced granuloma tests in Wistar rats.

Ethical approval`
The study protocol and procedures were reviewed and approved by FGS, Colombo ethics committee (2015/MPhil-PhD/013). Since, C. albiflora is an endemic threaten specie, approval was taken from Forest department, Sampathpaya, Battaramulla (My ref.R&E/RES/NFSRC/12) to collect samples.

Total flavonoid content (TFC)
DCM/methanol extract of C. albiflora was determined according to Siddhuraju et al. (2003) with slight modification.Briefly, 100 μL of extract, 70 μL of AlCl3 (2%) were mixed and incubated at room temperature for 10 min.Absorbance was measured at 415 nm using micro-plate reader (Spectra Max +384).Quercetin was used as standard, and water was used as blank.Replicated (5) results expressed as quercetin acid equivalents (mg/g).

Ferric reducing antioxidant power (FRAP) assay
The ferric reducing antioxidant power of C. albiflora DCM/methanol extract was determined according to Benzine et al. (1999)  and extract (20 μL) were incubated at room temperature for 8 min and absorbance was measured at 600 nm using micro-plate reader (SpectraMax+384).Trolox was used as standard and buffer was used as blank.Replicated (5) results were expressed as Trolox equivalents (mg/g).
A reaction volume of 200 μL, containing DPPH radical (65 μL), Methanol (85 μL) and extract (50 μL) were incubated at room temperature for 10 min and absorbance was measured at 517 nm using micro-plate reader (Spectra Max +384).Trolox was used as standard and methanol was used as control.DPPH radical scavenging activity percentage was measured using the formula; Percentage radical scavenging activity = (1 -A/B) × 100 where A: absorbance of extract at concentration and B: absorbance of control.
IC50 at each concentration of the extract/standard were calculated.

ABTS + radical scavenging assay
The ABTS + radical scavenging in plant material was determined according to Re et al. (1999) with slight modification.Briefly, 50 mM PBS (phosphate buffer saline, pH 7.4) was prepared.Fresh ABTS + (2, 2-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical solution was prepared.A reaction volume of 200 µL, containing PBS (110 µL), extract (50 μL), and ABTS + solution (40 µl) were incubated at room temperature for 10 min.Absorbance was measured at 734 nm using micro-plate reader (SpectraMax+384).Trolox was used as standard, and water was used as control.PBS and water were used as the blank.ABTS + radical scavenging activity percentage and IC50 was calculated as per section 2.4.4.
Decay of florescence was scanned for 35 min at 1 min interval at 37 o C (Ex 494 nm, Em 535 nm) using micro-plate reader (Spectra Max Gemini EM).The area was recorded under the curve for extracts and Trolox.ORAC value was calculated using the formula; where net AUC extract = AUC extract -AUC blank, net AUC Trolox = AUC Trolox -AUC blank, and AUC = area under the curve.

Anti-inflammatory experiments
Required concentrations of C. albiflora (200 mg/kg, 400 mg/kg, and 600 mg/kg) drug groups were prepared using water as solvent.The animal dose was calculated based on human equivalent dose (HED) using the formula HED (mg/kg) = animal dose (mg/kg) x animal Km factor (6)/ Human Km factor (37) (Reagan-Shaw et al., 2007).

Carrageenan induced paw oedema in Wistar rats
Inflammations were induced by 0.05 ml of 1% carrageenan powder was dissolved in 1% methyl cellulose (analytical grade, from Sigma-Aldrich) into the plantar surface of the left hind paw under mild anesthetic ether anesthesia.Treatments were performed prior 1h of carrageenan injection.The paw volumes of pretreatment of the paws and 1h, 2h, 3h, and 4h after the treatment were measured.The percentage inhibition was determined using the formula; Inhibition percentage = (controltreated)/control x 100

Formaldehyde induced paw oedema in Wistar rats
Inflammations were induced by 0.1 ml of 2% formaldehyde in distilled water into the plantar surface of the left hind paw under mild anesthetic ether anesthesia on days 1 and 3. Treatments were performed for 7 consecutive days.The paw volumes of these rats were measured prior to the injection of formaldehyde, at 4 h after the injection on day 1 and at 1 h of oral treatment from days 2-7.

Cotton pellet induced granuloma in Wistar rats
Granulomatous lesions were induced by surgically implanting two cotton pellets subcutaneously in the dorsal region of the rats near the axila.C. albiflora extract was administered orally before 1 h of the surgery.Rats were anaesthetized using ketamine (0.6 ml kg-1) and autoclaved sterile pellets of cotton (8 ± 0.5 mg each) were implanted.The rats of the control group were administered with water and standard group by Indomethacin (5 mg/kg).Drugs and water were administered for consecutive 7 days.Rats were anaesthetized on the eighth day using ketamine and the pellets were dissected out carefully and dried at 60 o C (3 d).Mean weight of the granuloma tissue formed around each dried pellets were recorded.

Statistical analysis
Calculation and statistical analysis was performed using Minitab 17 (version 17.1.0.0).Results were expressed as mean ± standard deviation.DPPH, TPC, and TFC values were further analyzed by Pearson correlation coefficient.Anti-inflammatory activity results were analyzed by ANOVA comparing control group values using Turkey test.

Antioxidant activity
TPC, TFC, FRAP, and ORAC values were reported in Table 1.IC50 values of DPPH, and ABTS + were reported in Table 2.The standard curves were included in Plate 1.  3. DPPH, TPC, and TFC values were further analyzed by Pearson correlation coefficient and reported in Table 4.

Carrageenan induced paw-oedema in Wistar rats
The effect of oral treatment of C. albiflora whole plant extract of rats was reported in Table 5.

Formaldehyde induced paw-oedema in Wistar rats
The effect of C. albiflora extract on the formaldehyde-induced paw oedema was reported in Table 6.

Cotton pellet induced granuloma in Wistar rats
Wet cotton pellets which were dissected out on the 8 th day and mean dry weight of cotton pellets were shown in the Plate 2. The effect of C. albiflora on cotton pellet granuloma test in Wistar rats was reported in Table 7.

Antioxidant activity
All in vitro antioxidant experiments were performed on dichloromethane/ methanol (1:1) extract of C. albiflora in the concentration of the 200 mg/kg.Polar and non-polar dichloromethane/ methanol solvent system was used to extract polar and non-polar compounds present in C. albiflora.The standard and the plant extracts were shown their maximum %inhibitory activity against concentration; standard was shown 70.56 ± 0.30 at 12.50 ppm, and C. albiflora DCM/methanol (1:1) extract was shown 65.76 ± 0.72 % at 1250 ppm assay concentration on DPPH bioassay (Table 3).Moreover, C. albiflora DCM/methanol (1:1) extract was shown 99.19 ± 0.42 % at 500 ppm assay concentration on ABTS + bio assay.In vitro antioxidant studies were shown that the radical scavenging effect (DPPH, ABTS+) was found to increase with increasing concentrations (Table 3).The correlations of TFC against the antioxidant activity based on the DPPH, ABTS + , FRAP, and ORAC assay were significant (Table 4).Further, the negative correlation between TPC and antioxidant activity were suggested that it could be related to other antioxidant compounds contained in the plants (Kolar et al., 2011).Although TPC, and TFC values of C. albiflora were reported as 31.25 ± 1.48 (mg GAE/g of extract), and 11.22 ± 0.13(mg QE/g of extract), in terms of C. longa were reported as 260± 0.25 (mg GAE/ g of extract) and 79.36 ± 0.01(mg QE/g of extract) respectively (Rajeswari et al., 2013).Whereas the concentration of 1250 μg/mL of C. albiflora.DCM/methanol extract was shown that 65.76 ± 0.72 %, the concentration of 100 μg/mL of water extract of C. zedoaria reported to have 98.95% inhibition (Himaja et al., 2010).Although, IC50 values of C. albiflora DCM/methanol extract on DPPH and ABTS + assays were 827.78 ± 6.06 ppm, and 188.84 ± 2.99 ppm respectively, Water extract of C. aromatica was reported to have 427.75 ± 1.43 ppm and 11.674±1.98ppm respectively (Ammayappan et al., 2012).Low IC50 values were shown higher antioxidant activity.Although, ORAC value of C. albiflora was reported as 128.10 ± 3.29 mg TE/g of extract in C. longa.It was reported 1592.77μM TE/g of extract (Reşat et al., 2013).According to the results obtained from the current study, C. albiflora was shown a low antioxidant active species, comparatively to C. longa, C. aromatica and C. zedoaria.

Carrageenan induced paw-oedema in Wistar rats
The 200 mg/kg was significantly impaired the paw oedema, at 1h (by 61%).In contrast, the 400 and 600 mg/kg tested were significantly inhibited the paw oedema measured; 1h (by 45-58%), 2h (by 24-46%), 3h (by 21-27%).Therefore it was shown that the antiinflammatory effect of C. albiflora was inversely dose dependent (See Table 1).Indomethacin induced significantly impairment of oedema at all-time points measured (58-89%).Initial phase lasting primarily mediated via production of cox-1, histamine, serotonin, bradykinins etc (Ratnasooriya et al., 2015).Since, C. albiflora inhibited 1 st phase, the antiinflammatory effect may relate with above mentioned pathways.C. zedoaria shows 54-56% inhibition in the initial phase and 56-59% in late phase by the concentration 200 mg/kg of petroleum ether on carrageenan induced paw oedema test.However it was shown that the 58% inhibition at 2h by the concentration of 200 mg/kg of chloroform (Kaushik et al.2011), Therefore, it was shown that the antiinflammatory activity of C. zedoaria on both the initial and late phases.In contrast, C. albiflora was shown its antiinflammatory activity in initial phase.

Formaldehyde induced paw-oedema in Wistar rats
The drug group (400 mg/kg and 600 mg/kg) significantly (P<0.05)reduced the paw oedema from the day 5 to 7 by 400 mg/kg when compared with the control (see Table 5).This effect related prostaglandin synthesis can be proved by cotton pellet induced granuloma test.From the present study, it can be concluded that 400 mg/kg treated group was shown higher inhibition percentage than 200 and 600 mg/kg treated groups.Since, C. albiflora was shown low (as 19.5% on 400 mg/kg) anti-inflammatory activity on Cotton pellet granuloma test, it can be concluded that anti-inflammatory activity of C. albiflora is not linked with prostaglandin synthesis.

Cotton pellet induced granuloma in Wistar rats
Inhibition percentages were found as 51.3 % (Standard), 8.1 % (200 mg/kg treated group), 19.5 % (400 mg/kg treated group), and 13.4 % (600 mg/kg treated group).From the present study, it can be concluded that 400 mg/kg treated group was shown higher inhibition percentage than 200 and 600 mg kg-1 treated groups.

Conclusions
It can be concluded from the current study that C. albiflora was shown correlation with flavonoid content which is responsible for its antioxidant property.C. albiflora inhibited 1 st phase in the carrageenan-induced paw oedema test, but with low concentrations.By formaldehyde induced paw oedema and cotton pellet granuloma, tests were shown anti-inflammatory activity at the late phase (from 5 to 7 day) and its activity does not relate to prostaglandin synthesis, on other autacoids.Further experiments have to be conducted to identify a mechanism of antiinflammatory activity of C. albiflora.

Table 3 :
DPPH and ABTS + free radical scavenging activity of the extract C. albiflora Table 4: Correlations between the IC50 values of DPPH assay, ABTS + assay, FRAP, and ORAC assays with phenolic and flavonoids content of C. albiflora Table 5: Effect of oral treatment of C. albiflora whole plant extract on the carrageenan induced paw oedema

Table 6 :
Effect of C. albiflora extract (water) on the formaldehyde-induced paw oedema in Wistar rats Table 7: Effect of C. albiflora on cotton pellet granuloma test in Wistar rats Plate 2: Cotton pellet experiment: A: Picture of wet cotton pellets after surgical removal according to group, B: mean of dry weight of cotton pellets of each treatment groups