In-vitro antioxidant and in-vivo anti-inflammatory activities of aerial parts of Cassia species

2.1 Plant material

Leaves, bark, seeds and flowers of C. siamea were collected from the University campus of Maharaja Krishnakumarsinhji Bhavnagar University Bhavnagar, Gujarat, India. Leaves, bark, flowers and seeds of C. javanica were collected from Gandhinagar City, Gujarat, India. Voucher specimens of C. siamea (BU/PG/CHHERBS/01/11) and C. javanica (BU/PG/CH/HERBS/02/11) were deposited. Both species were authenticated at Botany Department, Sir P. P. Institute of Science, Bhavnagar, India.

2.2 Preparations of extracts

The aerial parts of C. siamea, and C. javanica were cleaned with deionized water, oven dried at 50 °C for 48 h and powdered in a grinder. The plant material (100 g) was extracted with different solvents (1500 mL) using Soxhlet apparatus for 24 h at a temperature not exceeding the boiling point of the respective solvents. The obtained extracts were filtered using Whatmann filter paper No. 1 and concentrated under vacuum at 40 °C using a rotary vacuum evaporator (Büchi Laboratoriums, Switzerland) to dryness. The extractive values of the extracts were calculated.

2.3 Chemicals and reagents

2,2′-Diphenyl-1-picrylhydrazyl hydrate (DPPH), β-carotene, linoleic acid, butylatedhydroxytoluene (BHT), ferrous chloride and Folin–Ciocalteu reagent were purchased from Hi-Media Lab. Pvt. Ltd., Mumbai, India. Sodium dihydrogen orthophosphate, di-sodium hydrogen orthophosphate dehydrate and sodium acetate was purchased from E-Merck India Ltd. Other chemicals and solvents were procured from SD Fine-Chem Ltd., Mumbai, India and were of analytical grade, while Hydrogen peroxide (H₂O₂) was purchased from RFCL Limited (Rankem), Mumbai, India. λ-Carrageenan was purchased from Sigma–Aldrich Chemicals, India. Reference standard diclofenac sodium was obtained as gift sample from Unique Pharmaceuticals, Ankleshwer, India.

2.4 Instrumentation

GC–MS analysis was performed on Shimadzu GC–MS-QP2010 Plus system equipped with RTX-5 m.s. capillary column (0.25 mm × 30 m × 0.25 μm). The interface temperature and the MS scan range of the instrument were set at 230 °C and 35–450 atomic mass units (AMU), respectively. Helium was used as the carrier gas at a flow rate of 10 mL/min. Column temperature was started at 60 °C, held for 2 min, ramped up to 170 °C at a rate of 2 °C/min, held for 3 min, ramped to 250 °C at a rate of 3 °C/min and finally held for 120 min. The samples (1 μL) were injected in split mode at 220 °C and compounds identified using NIST98 MS data library. A total of 15 compounds were identified based on GC–MS results in which few were known and two to three compounds were not previously reported in both species.

2.5 Animals

An inbred colony of female Swiss albino mice (25–60 g) were used for acute toxicity study, while adult female Wistar albino rats (170–300 g) were used for anti-inflammatory study. They were kept in polypropylene cages at 25 ± 2 °C, with relative humidity 45–55% under 12 h light and dark cycles. All the animals were acclimatized to the laboratory conditions for a week before use. They were fed with standard animal feed (Government Medical College, Bhavnagar) and water ad libitum. The test extracts and the standard drugs were administered in the form of a suspension in water using 1% carboxymethyl cellulose as suspending agent. All pharmacological activities were carried out as per CPCSEA (Committee for the purpose of control and supervision of experiments on animals) norms. All the pharmacological experimental protocols were approved by the Institutional animal ethics committee (IAEC No. 24/2011, pharmacology No. 24/2011, date: 13/09/2011), Government Medical College, Bhavnagar.

2.6 Determination of total phenols and flavonoids

Total phenolic content was determined according to Folin–ciocalteu method (Sabir and Rocha, 2008). The results are expressed as grams of gallic acid equivalents per 100 g of dry extract. The concentration of phenolic compounds was calculated according to the following Eq. (1) obtained from the standard Gallic acid (5–50 μg) curve.

2.7 DPPH radical scavenging assay

The radical-scavenging activity was evaluated using an improved DPPH assay (Huang et al., 2011). For 0.2 mM DPPH, 2.7 mL was added to 0.3 mL of the extract solution at various concentrations. The mixture was shaken vigorously and incubated at room temperature for 1 h before the absorbance was measured at 517 nm. The radical scavenging activity (RSA) was calculated as a percentage of DPPH discoloration using the Eq. (3).

2.8 β-Carotene bleaching inhibition assay

Antioxidant activity was also assessed using the β-carotene linoleate model system (Shon et al., 2003) Briefly, 6 mg of β-carotene was dissolved in 20 mL of chloroform, and 4 mL of this solution was pipetted into a round-bottomed 500 mL flask containing 80 mg linoleic acid and 800 mg Tween-80. After removing chloroform using a rotary evaporator, 200 mL of distilled water was added slowly to the mixture with vigorous agitation to form a stable emulsion. Then, 3 mL aliquots of the emulsion were transferred into different test tubes containing 0.2 mL of the samples and were incubated in a water bath at 50 °C for 2 h. Ascorbic acid was used as standards for comparison. As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm. The antioxidant activity (AA) was measured in terms of successful bleaching of β-carotene using Eq. (4).

2.9 Assay of H₂O₂ scavenging activity

The ability of all seed extracts to scavenge hydrogen peroxide was determined according to the method described previously (Ruch et al., 1989). A solution of hydrogen peroxide (2 mM) was prepared in phosphate buffer (pH 7.4). Hydrogen peroxide concentration was determined spectrophotometrically from absorption at 230 nm. Extracts samples (10–50 μg/mL) in distilled water were added to a hydrogen peroxide solution (0.6 mL). Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentages of scavenging of hydrogen peroxide of extracts of aerial parts and standard compounds are calculated by using the following Eq. (5).

2.10 Acute toxicity test studies

Acute oral toxicity study was performed as per OECD-423 guidelines (acute toxic class method; Ecobichon, 1997). Female Swiss albino mice (n = 6) selected by random sampling technique and were used for acute toxicity study. The animals were kept fasting overnight providing only water. Subsequent to administration of the extracts, the animals were observed closely for the first 3 h for any toxic manifestations such as increased locomotors activity, salivation, clonic convulsion, coma and death. Subsequent observations were made at regular intervals for 24 h. The animals were observed for a further week. The ethanolic extracts were safe up to a dose of 2000 mg/kg body weight, so 200 mg/kg (p.o.) and 400 mg/kg (p.o.) were used as moderate dose for the evaluation of anti-inflammatory activities (Ghose, 2005).

2.11 Carrageenan-induced rat paw edema of ethanolic extract

Paw edema was induced by injecting 0.1 ml of 1% W/V $\lambda$ -carrageenan in physiological saline into the subplantar tissues of the left hind paw of each rat (Winter et al., 1962). The extracts of aerial parts of C. siamea and C. javanica (200 mg/kg and 400 mg/kg p.o.) were administered orally 30 min prior to $\lambda$ -carrageenan administration. The paw volume was measured at 60, 120, 180 and 240 min by the mercury displacement method using a plethysmograph. The percentage inhibition of paw volume in drug treated group was compared with the control group. Diclofenac sodium (5 mg/kg p.o.) was used as standard reference (Nsonde Ntandou et al., 2010).

2.12 Statistical analysis

All results were obtained with means ± standard deviation of three parallel measurements. The data were analyzed by a one-way analysis of variance (ANOVA) using Graph pad INST-3 to determine the effects of extracts. Significant differences between extract means were determined by two-way ANOVA tests.

3.1 Total phenolic content

Natural polyphenols have chain-breaking antioxidant activities and are believed to present many degenerative diseases including cancer and atherosclerosis (Jayaprakasha et al., 2001). Phenolics are aromatic secondary plant metabolites and are called high-level antioxidants because of their ability to scavenge free radicals and active oxygen species such as singlets, superoxide free radicals and hydroxyl radicals (Roginsky, 2003). Many studies have reported the antioxidant activities of phenolics from plant sources in different systems (Hagerman et al., 1998; Sekretar et al., 2004; Tachakittirungrod et al., 2007; Kobus et al., 2009).The contents of total phenolics in crude extracts of aerial parts of C. siamea and C. javanica are depicted in Fig. 1. The crude extracts obtained from aerial parts of C. siamea and C. javanica were characterized by their contents of total phenolics expressed as gallic acid equivalent (GAE) for C. siamea leaf (CSL), C. siamea bark (CSB), C. siamea flower (CSF), C. javanica leaf (CJL) and C. javanica seeds (CJS) in 95% ethanol, methanol and water. Lowest total phenolics were obtained from the water extract of CSF: 69.37 mg/g (GAE) and highest total phenolics were obtained from the 95% ethanolic extract of CJL: 910.85 mg/g (GAE). This is due to fact that solubility of phenolics is less in water. The calculations of total phenolics were carried out by using standard gallic acid curve with least square regression (y = 0.016x + 0.029; R² = 0.9968; standard deviation of residuals from line = 0.01855). Similarly, total flavonoid content equivalent to quercetin (QU. E) for C. siamea and C. javanica was calculated from the standard curve of QU with least square regression (y = 0.0001x + 0.011; R² = 0.9941; standard deviation of residuals from line = 0.001514) and is depicted in Fig. 2.

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Figure 1

Total phenolic contents of 95% ethanol, methanol and water extracts of C. siamea leaf, C. siamea bark, C. siamea flowers, C. javanica leaf and C. javanica seeds. Results of total phenolic contents were calculated as average ± S.D for each group.

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Figure 2

Total flavonoid contents of 95% ethanol, methanol and water extracts of C. siamea leaf, C. siamea bark, C. siamea flowers, C. javanica leaf and C. javanica seeds. Results of total flavonoid contents were calculated as average ± S.D for each group.

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Aerial parts of both plants and different solvents used for extraction were responsible for the variation in total phenolic contents. The order of phenolic content obtained from three solvents was 95% ethanol > methanol > water for aerial parts. Variations in phenolic contents of various extracts are attributed to polarities of different compounds present in various aerial parts, and such differences have been reported elsewhere (Duh et al., 1990). Similarly, the order of flavonoid content obtained from three solvents was 95% ethanol > methanol > water for both plants. The results of the extracted material showed that leaves of C. javanica had highest phenolic and flavonoid contents.

3.2 DPPH radical scavenging assay

DPPH is a stable free radical in aqueous or methanol and ethanol solution and accept an electron or hydrogen radical to become a stable diamagnetic molecule. It is used as a substrate to evaluate the antioxidative activity of antioxidants (Baumann et al., 2002). The reduction capability of DPPH radicals was determined by a decrease in its absorbance at 517 nm. The decrease in absorbance of DPPH radical was caused by antioxidant, and was due to the scavenging of hydrogen donation. DPPH assay showed that extracts of CSB in water had the highest scavenging with 39.47% (R² = 0.9624; standard deviation of residuals from line = 3.869) scavenging at 50 μg/mL concentration. Results of DPPH assay also showed that extracts of CSL, CSB, CSF, CJL and CJS were in the order of methanol > 95% ethanol > water. The scavenging effect of extracts on the DPPH radical increased with an increasing concentration of each extract and is shown in Fig. 3. The degree of discoloration indicates the scavenging capacity of the extract. The effect of antioxidants on the DPPH radical scavenging was due to the result from their hydrogen donating ability (Hallivel and Guttreridge, 1989). IC₅₀ value for water extract of CJS was moderate (83.5 μg/mL ± 7.07; p < 0.001; R² = 0.8233) in comparison to the standard BHT (Table 1). Similarly, many researchers reported that some plant extracts showed a lower activity against the DPPH, if compared with BHT (Ramarathnam et al., 1995; Halliwell, 1997). In addition, there was a statistically significant correlation between the amount of phenolic compounds and DPPH scavenging activity in all the extracts.

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Figure 3

Percentage scavenging of DPPH in different dosages of aerial parts of C. siamea and C. javanica in ethanolic extract. (i) –■– CSL (ii) –∗– CSB (iii) –▴– CSF (iv) –▾– CJL (v) –♦– CJS.

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3.3 H₂O₂ scavenging activity

The results showed excellent H₂O₂ scavenging activities for all aerial parts at a concentration range from 10 to 50 μg/mL and is depicted in representative Fig. 4.

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Figure 4

Percentage scavenging of H₂O₂ in different dosages of aerial parts of C. siamea and C. javanica in ethanolic extract. (i) –■– CSL (ii) –●– CSB (iii) –▴– CSF (iv) –▾– CJL (v) –♦– CJS.

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IC₅₀ for standard ascorbic acid was 49.47 ± 1.22 μg/mL (R² = 0.9891), while IC₅₀ for methanolic extract of CJL was 49.74 ± 0.29 μg/mL (R² = 0.9553; standard deviation of residuals from line = 1.848). Similarly, water extract of CJL had IC₅₀ value 52.07 ± 0.41 μg/mL with linear regression (R²) = 0.9792, while 95% ethanolic extract of CJL had IC₅₀ value 53.75 ± 0.84 μg/mL with linear regression (R²) = 0.9941(Table 1). These results suggest that bioactive compound(s) present in the different extracts of CJL have very good impact on the scavenging activity of H₂O₂ system. Hydrogen peroxide itself is not very reactive but it can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cell (Gordon, 1990). Thus, the removing of H₂O₂ is very important for antioxidant defense in cell or food systems. Scavenging of H₂O₂ by extracts is attributed to their phenolics, which could donate electrons to H₂O₂. The extracts might be used to provide a good H₂O₂ scavenger for humans and foods especially, CJL extracts in all three solvents showed excellent ability to scavenge H₂O₂.

3.4 β-Carotene bleaching effect

The bleaching effect was measured by the peroxidation of β-carotene. Antioxidants can reduce the extent of β-carotene destruction by neutralizing the linoleate-free radical and other free radicals formed in the system (Vongadow et al., 1997). Accordingly, the absorbance decreased rapidly in reaction mixtures without extracts, whereas in the presence of extracts, the reaction mixtures retained their color and thus, absorbance for a longer time (Fig. 5).

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Figure 5

Percentage bleaching of β-carotene in different dosages of aerial parts of C. siamea and C. javanica in ethanolic extract. (i) –■– CSL (ii) –●– CSB (iii) –▴– CSF (iv) –▾– CJL (v) –♦– CJS.

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The IC₅₀ of different extracts in inhibiting the bleaching of β-carotene suggest that bleaching is moderately inhibited by extracts of aerial parts of both plants in three different solvents with linear regression (R²) ranging from 0.8471 to 0.9991 with p < 0.003, which is extremely significant if compared with standard propyl galate solution (Table 1). This is because the presence of different antioxidant molecules in extracts might be responsible for the inhibition of β-carotene destruction by neutralizing the effect of linoleate-free radical and other free radicals formed in the system (Cheung et al., 2003).

3.5 In vivo anti-inflammatory assay

The acute toxicity results showed that etahnolic extracts of C. siamea and C. javanica was safe up to a dose of 2000 mg/kg body weight. Based on acute toxicity data, two different dosages 200 and 400 mg/kg (p.o.) were selected for in vivo anti-inflammatory study and results are depicted in Table 2. The results of aerial parts were compared with positive control of diclofenac sodium (NSAID) (5 mg/kg p.o.) and were authenticated by statistical analysis.

The ethanolic extracts of aerial parts of C. siamea and C. javanica (200 and 400 mg/kg p.o.) reduced the swelling induced in the rats paw by carrageenan on each of the five occasions. It was measured and it was observed to be dose dependent. Bark extracts of C. javanica in ethanol showed a significant impact on the reduction of edema rate at a lower dosage that is 200 mg/kg (p.o.) on each of the four occasions with p < 0.001, which is considered as extremely significant. The anti-inflammatory activity is demonstrated here for the first time by using aerial parts. However, the Cassia genus presents in other anti-inflammatory species such as Cassia italica (Jain et al., 1997). It was observed from results of percentage inhibition of paw edema that leaf extracts of C. siamea and C. javanica in ethanol have a very significant impact on the reduction of paw edema if compared with standard positive control and it is shown in Fig. 6. This study confirmed the anti-inflammatory properties of ethanolic extracts of aerial parts of C. siamea and C. javanaica. The traditional methods of extraction are promising since the yield extracts are devoid of cytotoxicity or acute toxicity on rats and therefore, in the present study we have given focus to traditional methods for the extraction of active ingredients from aerial parts of both plants.

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Figure 6

Reduction of paw edema of rats with percentage inhibition in two dosages of aerial parts of C. siamea and C. javanica in ethanolic extract.

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