Recently, focus on plant research has increased throughout the world to show immense potential of medicinal plants used in various traditional systems and already more than 13,000 plants have been studied during the last 5 year period (Bensky et al., 2004). Probably, the history of plants being used for medicinal purpose is as old as the history of mankind (Riaz et al., 2013). Our traditional  system  of  medicine  and  folklore usually uses the whole medicinal plant or a part of it for the treatment of all types of diseases successfully (Imran et al., 2014). The medicinal plant Stephania japonica Linn. (Family - Menispermiaceae) is a slender wiry climber or twining shrub (Senthamarai et al., 2012). The family Menispermaceae is a family of about 65 genera and 350 species, distributed in warmer parts of the world. The members of this family are commonly herbs or shrubs but rarely trees. The plants of the genus Stephania have recognized medicinal values and traditionally have been used for the treatment of asthma, tuberculosis, dysentery, hyperglycemia, cancer, fever, intestinal complaints, sleep disturbances and inflammation (Chopra et al., 1958; Kirtikar et al., 1987). The leaves and roots are bitter and astringent and used in the treatment of fevers, diarrhea, dyspepsia and urinary disease (Ghani, 2003). The present study focuses on screening of phytochemicals, antioxidant activities and analgesic effect of ethanolic extract of S. japonica Linn.

Phytochemicals are well known to show a variety of pharmacological actions in human body (Akinmoladun et al., 2007). Antioxidants are molecules which are capable of preventing or inhibiting oxidation by counteracting reactive oxygen species (ROS) (Zia-Ul-Haq et al., 2013a). ROS which are produced during cellular metabolism mediate many acute and chronic diseases. So the balance between antioxidation and oxidation is essential for proper maintaining of a healthy biological system (Amin et al., 2013). By acting in the central ner-vous system (CNS) or on the peripheral pain mechanism analgesics relieve pain as a symptom without affecting its cause (Riaz et al., 2014).

Plant

The fresh leaves of S. japonica Linn. for the proposed study were collected from Karamjal, Sundarban, Bangladesh on January, 2010. The plant was identified and authenticated by expert botanist of Bangladesh National Herbarium, Mirpur, Dhaka, where the voucher specimen has been deposited for future reference. Its DACB Accession No. is 34527.

Preparation of plant extract

The plant material was shade dried with occasional shifting, powdered mechanically with a mechanical grinder and stored in a tight container. About 500 g of powered material was taken in a clean, sterilized flat-bottomed glass container and soaked in 1500 ml of 80% ethanol (Merck KGaA, Darmstadt, Germany). The con-tainer with its contents was sealed and kept for a period of 14 days accompanying occasional shaking and stirring.  The  whole  mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then it was filtered through whatman filter paper (Bibby RE200, Sterilin Ltd., UK). The resultant filtrate was then evaporated in water bath maintained 40°C to dryness. It rendered a gummy concentrate of reddish black color. The gummy concentrate was designated as crude extract of Ethanol (Zia-Ul-Haq et al., 2013b).

Test animals and drug

For the screening of analgesic potential of crude ethanolic extract of S. japonica leaves, young Swiss-albino mice aged 4 to 5 weeks (either sex), average weight 20 to 25 g were used. They were collected from the animal Research Branch of the International Center for Diarrheal Disease and Research, Bangladesh (ICDDRB). Animals were kept in favorable condition for one week for adaptation and fed rodent food and water ad libitum formulated by ICDDRB. They were maintained carefully under standard environ-mental conditions (temperature: 24.0 ± 1.0°C, relative humidity: 55 to 65% and 12 h light/dark cycle) and had free access to feed and water ad libitum. All protocols for animal experiment were approved by the animal ethical committee of Noakhali Science and Tech-nology University (NSTU) research cell. In this anlgesic experiment, as standard, diclofenac sodium (donated by Opsonin Pharma Ltd., Bangladesh) was used, Tween 80 and acetic acid used were of analytical grade (Merck KGaA, Darmstadt, Germany).

Chemicals for antioxidant assay

1,1-Diphenyl-2-picryl hydrazyl (DPPH), trichloro acetic acid (TCA), L-ascorbic acid, butylated hydroxy anisole (BHA), gallic acid, folin-ciocalteu phenol reagent, phosphate buffer (pH 6.6), potassium ferricyanide [K₃Fe(CN)₆] (1%), distilled water, EDTA, ferrozine, FeCl₂ and FeCl₃ (0.1%) were of analytical grade and purchased from Merck KGaA, Darmstadt, Germany.

Pharmacological evaluation

Phytochemical screening

The freshly prepared crude ethanolic extract was qualitatively tested for the presence of chemical constituents. Phytochemical screening of the extract was performed using the following reagents and chemicals: Alkaloids with Dragendorff’s reagent and Mayer’s reagent, flavonoids with the use of Mg and HCl; tannins with ferric chloride and potassium dichromate solutions and saponins with ability to produce stable foam and steroids with Libermann-Burchard reagent, reducing sugars with Benedict’s reagent and Fehling’s reagent. These were carefully identified by characteristic color changes using standard procedures (Ghani, 2003; Amin et al., 2013; Evans, 1989).

Antioxidant activity

Here four complementary test methods namely DPPH free radical scavenging, reducing power assay, ferrous ion chelating ability and total phenolic content were used to determine the antioxidant properties of plant extract.

Determination of DPPH-free radical scavenging activity

The stable DPPH free radical-scavenging activity was measured using a slightly modified method described by Chang et al. (2001). Stock solution (1 mg/ml) of the ethanol extract of the leaves of S. japonica was prepared in ethanol from which serial dilutions were carried out to obtain the concentrations of 5, 10, 20, 40, 60, 80, 100 µg/ml. In this assay, 2 ml of 0.1 mM ethanolic DPPH solution was mixed to 2 ml of extract solution at different concentrations and the contents were stirred vigorously for 15 s. Then, the solutions were allowed to stand at dark place at room temperature for almost 30 min for reaction to occur. Thereafter absorbance was measured against a blank at 517 nm with a double beam UV spectropho-tometer (UV-1800, UV-Vis spectrophotometer, Shimadzu, Japan). The percentage of DPPH free radical-scavenging activity of each plant extract was calculated as:

DPPH radical-scavenging activity (I %) = [(A₀ – A) / A₀ ] × 100

Where A₀ is the absorbance of the control solution containing all reagents except plant extracts, A is the absorbance of the DPPH solution containing plant extract. Finally, the concentration of sample required to scavenge 50% DPPH free radical (IC₅₀) was calculated from the plot of inhibition (%) against the concentration of the extract. Ascorbic acid and BHA were used as positive control standard for this experiment.

Reducing power assay

The reducing power assay of S. japonica was determined according to the method reported by Oyaizu (1986), with slight modifications. Here 1 ml of extract solution of different concentrations (5, 10, 20, 40, 60, 80, 100 µg/ml) was mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide [K₃Fe(CN)₆] (1% w/v). The mixture was incubated at 50°C for almost 30 min. Thereafter, the reaction was terminated by adding 2.5 ml of trichloroacetic acid (10%, w/v), then the mixture was centrifuged at 3000 rpm for 10 min. The supernatant solution (2.5 ml) was mixed carefully with distilled water (2.5 ml) and ferric chloride (0.5 ml, 0.1% w/v) solution. Then the absorbance was recorded at 700 nm against a blank using UV spectrophotometer. Here increased absorbance value of the reaction mixture indicates increased reducing power. Three replicates were made for each test sample and average data was recorded. Ascorbic acid and BHA were used as positive control standard too.

Ferrous ion chelating ability

The ferrous ions chelating activity of ethanolic extract of the leaves of S. japonica and standards was investigated according to the method of Dinis et al. (1994). Briefly, ethanol extracts (5 ml) was mixed to 0.1 ml solution of 2 mM FeCl₂ and ethanol. The reaction was initiated by the addition of 0.2 ml of 5 mM ferrozine and mixture was shaken vigorously and left standing at room temperature for 10 min. After the mixture had reached equilibrium, the absorbance of the solution was then recorded spectrophotometrically at 562 nm in UV spectrophotometer, wherein the Fe⁺² chelating ability of extracts was monitored by measuring the ferrous ion-ferrozine complex. The percentage of inhibition of ferrozine-Fe²⁺ complex formation was calculated as:

Ferrous ions chelating ability (%) = [(A₀ - A) / A₀] × 100

Here, A₀ is the absorbance of the control solution (containing all reagents except plant extract), A is the absorbance in the presence of the sample  of  plant  extracts.  Three replicates  were  made  for each test sample and average data was noted where EDTA was used as positive control standard.

Determination of total phenolic content

The total phenolic contents of the extract were determined by the modified Folin-Ciocaltu method (Wolfe et al., 2003). 0.5 ml of each extract (1 mg/ml) was added with 5 ml Folin-Ciocaltu reagent (1:10 v/v distilled water) and 4 ml (75 g/L) of sodium carbonate. The mixture was vortexed for 15 s and allowed to stand for almost 30 min at 40°C for color development. The absorbance was measured at 765 nm with a spectrophotometer (UV-1800, Shimadzu, Japan). Finally total phenolic content was determined as mg of gallic acid equivalent per gram using the equation obtained from a standard gallic acid calibration curve (y = 6.9103x - 0.0937, R² = 0.0936).

Analgesic potential

Analgesic potential of the ethanolic extract of S. japonica leaves was tested using the model of acetic acid induced writhing in mice (Ahmed et al., 2004; Whittle, 1964). Briefly, experimental animals (n = 5) were randomly selected and divided into four groups denoted as group-I, group-II, group-III, group-IV. Each mouse was weighed carefully and the doses of the test samples and control materials were adjusted accordingly. Here each group received a particular treatment, that is, control, positive control (standard Diclofenac Na) and two doses (250 and 500 mg/kg body weight) of the extract solution. Positive control group was administered at the dose of 25 mg/kg body weight and control group was treated properly with 1% Tween 80 in water at the dose of 15 ml/kg body weight. Test samples, standard drug and vehicle were administered orally almost 30 min before intraperitoneal administration of 0.7% acetic acid. After an interval of 5 to 10 min, the mice were observed for specific contraction of the body referred to as ‘writhing’ (constriction of abdomen, turning of trunk and extension of hind legs) for the next 10 min.

Statistical analysis

For antioxidant determination, data were presented as mean ± standard deviation (SD). Statistical analysis for animal experiment was carried out using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons using SPSS 16.0 for Windows^®. The results obtained were compared with the control group. P-values < 0.05 were considered to be statistically significant.

Phytochemical screening

Phytochemical analysis of the crude extract revealed the presence of alkaloid, tannin, gum, flavonoid and saponin (Table 1).

Antioxidant activity

DPPH free radical scavenging activity

The DPPH radical scavenging activity of ethanolic extract of the leaves of S. japonica  was  found  to  increase  with the increasing concentration. Maximum inhibition 84.11 ± 0.016 was observed at 100 µg/ml concentration. The IC50 value was determined as 18.57 ± 0.079. Ascorbic acid (AA) and butylated hydroxyl anisole (BHA) which were used as standard compounds showed maximum inhibition 95.58 ± 0.017 and 93.09 ± 0.019, respectively at 100 µg/ml and IC50 value was determined as 1.93 ± 0.027 and 4.10 ± 0.035, respectively (Table 2).

Reducing power assay

Reducing power of ethanol extract of S. japonica was found   to    increase  with  the  increasing  concentration. Maximum absorbance 2.013 ± 0.024 was observed at 100 µg/ml concentration. On the other hand, ascorbic acid and BHA which were used as positive control showed maximum absorbance 2.811 ± 0.013 and 2.031 ± 0.019, respectively at the same concentration (Table 3).

Fe²⁺ ion chelating ability

Ferrous ion chelating ability of the extract was found toincrease with the increasing concentration. Maximum chelating (%) ability 86.11 ± 0.022 was observed at 100 µg/ml concentration and the IC₅₀ value was determined as 18.68 ±  0.029.  EDTA  which  was  used  as  standard compound showed maximum chelating (%) ability of 99.75 ± 0.011 at 100 µg/ml, and 50% inhibition was found at 8.87 ± 0.035 (Table 4).

Determination of total phenolic content

Based on the absorbance values of the extract solutions, the colorimetric analysis of the total phenolics of extracts were determined and compared with that of standardsolution of gallic acid equivalents. The amount of total phenolic content was determined in the ethanolic crude extract of S. japonica as 279.05 ± 0.73 mg/g of gallic acid equivalent (Table 5).

Analgesic potential

The results of the test showed that the 500 mg/kg ethanolic extract leaves of S. japonica leaves exhibit highly significant (P < 0.001) inhibition of writhing reflex by 41.47% while the standard drug diclofenac sodium inhibition was found to be 45.02% at a dose of 25 mg/kg body weight (Table 6).

Medicinal plants have a long history of serving people in many regions of the world and  about  80%  of  the  world population still uses plants for various medical purposes (Schulz et al., 2001; Kong et al., 2003), because medicinal plants contain various types of phytochemicals and these phytochemicals are well known to show a variety of pharmacological actions in human body (Akinmoladun et al., 2007). In our study, preliminary phytochemical screening showed the presence of various phytochemicals.

Polyphenolic compounds like flavonoids, tannins and phenolic acids, commonly found in plants which contain multiple biological effects, including antioxidant activity (Brown et al., 1998; Vinson et al., 1995; Gil et al., 1999; Kahkonen et al., 1999). In this investigation, the extract showed moderate antioxidant activities with an IC₅₀ which were compared with the values of standard drugs used. Antioxidant effect of this study could be attributed to the presence of tannin found with the plant extract. Antioxidant activities of ethanolic extract of the leaves of S. japonica was found to increase with the increasing concentration. The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability (Shirwaikar et al., 2006). DPPH^? is a stable free radical which accepts an electron or hydrogen radical and thus become a stable diamagnetic molecule (Nakayama et al., 1993) and is generally used as a substrate for evaluating the antioxidant activity of a compound (Chang et al., 2002).

Based on the data obtained from this study, DPPH radical scavenging activity of extract of S. japonica was moderate. The reducing power assay of S. japonica extract was also determined. The reducing properties are normally associated with the presence of reductones, which are responsible to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom (Duh, 1994). Ferrous ion chelating ability of extract was also evaluated to determine the antioxidant activity and this chelating ability was found to increase with the increasing concentration. Phytochemical compoonds, mainly phenolic compounds (such as flavonoids, phyenyl propanoids, phenolic acids, tannins etc.) are very important components for the free radical scavenging and antioxidant activities of plants. Phenolic compounds react as hydrogen donors and thus neutralize the free radicals (Kulisic et al., 2004; Tanaka et al., 1988). In the present study, the total amount of phenolic compounds was calculated as moderate in the ethanol extract of S. japonica leaves. Phenols are important components of plants which may contribute directly to antioxidant effect of the system (Duh, 1994).

Analgesic potential of the ethanolic extract of S. japonica leaves was tested using the model of acetic acid induced writhing in mice. This acetic acid induced writhing response is a sensitive procedure to evaluate peripherally acting analgesics and represents pain sensation by triggering localized inflammatory response (Ahmed et al., 2006). It is known that non-steroidal anti-inflammatory and analgesic drugs reduce the inflame-matory pain by inhibiting the formation of pain mediators at the peripheral target sites, where prostaglandins and bradykinin are proposed to play a significant role in the pain process (Hirose et al., 1984). The oral administration of doses of S. japonica extract significantly (p < 0.001) inhibited writhing response induced by acetic acid in a dose dependent manner. It is likely that the plant extract might have exerted its peripheral antinociceptive action by interfering with the local reaction caused by the irritant or by inhibiting the synthesis, release and/or antagonizing the action of pain mediators at the target sites. Interes-tingly, compounds like flavonoids (Kim et al., 2004) and steroids, triterpenes in part, have been shown to possess anti-inflammatory and analgesic activity (Pritam et al., 2001). Besides, tannins are also found to have a nice contribution in antinociceptive activity (Ramprasath et al., 2006).

In the context of the discussion, it can be concluded that the ethanolic extract of S. japonica possesses moderate antioxidant and potent analgesic activity. These activities increase with the increasing of concentrations. The potency of the extract may be due to the presence of phytochemicals like tannins, flavonoids, phenolics etc. However, extensive researches are necessary to find out the active principles responsible for these activities.

The  authors  are   grateful   to   Opsonin   Pharma    Ltd., Bangladesh for their generous donation of medicine and BNH to identify the plant. The authors are also thankful to the authority and Senior Scientific Officer Hemayet Hossain of BCSIR (Bangladesh Council of Scientific and Industrial Research) Laboratories, Dhaka to use their lab

All authors have none to declare