Abstract: In the present study crude methanolic extract of Stephania japonica leaf was investigated for possible antioxidant, analgesic and toxic activity. The extract showed antioxidant activity in DPPH radical scavenging activity, nitric oxide scavenging activity and reducing power assays. In both DPPH radical and NO scavenging assay, the extract exhibited moderate antioxidant activity and the IC50 values in DPPH radical scavenging and NO scavenging assays were found to be 105.55±1.06 and 129.12±0.15 μg mL-1, respectively while the IC50 values of ascorbic acid were 12.30±0.11 and 18.64±0.22 μg mL-1, respectively. Reducing power activity of the extract increased in a dose dependent manner. Analgesic activity of the crude extract was evaluated using acetic acid-induced writhing model of pain in mice. The crude extract at 200 and 400 mg kg-1 b.wt. doses displayed significant (p<0.001) reduction in acetic acid induced writhing in mice with a maximum effect of 75.89% reduction at 400 mg kg-1 b.wt. which is comparable to the standard, diclofenac sodium (86.52%). The extract was also investigated for toxic potentiality using Brine Shrimp lethality bioassay. In this bioassay the extract showed significant toxicity to Brine Shrimp nauplii with the LC50 value of 25.19±0.98 μg mL-1. The study clearly indicates that the extract possesses good analgesic and cytotoxic activity along with moderate antioxidant potential.
INTRODUCTION
Free radicals cause depletion of immune system antioxidants, change in gene expression and induce abnormal proteins and contribute to more than one hundred disorders in humans including atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, cancer and AIDS (Hela and Abdullah, 2010; Kumpulainen and Salonen, 1999). Although organisms have endogenous antioxidant defenses produced during normal cell aerobic respiration against ROS, other antioxidants are taken both from natural and synthetic origin. Synthetic antioxidants are widely used but their use is being restricted now-a-days because of their toxic and carcinogenic effects. Thus, interest in finding natural antioxidants, without any undesirable effect, has increased greatly (Rechner et al., 2002). Some natural antioxidant (e.g., rosemary and sage) are already exploited commercially either as antioxidant additives or a nutritional supplements but generally there is still a demand to find more information concerning the antioxidant potential of plant species. It has been mentioned the antioxidant activity of plants might be due to their phenolic, flavonoid, tannin and proanthocyanidin compounds (Hela and Abdullah, 2010; Frankle and Meyer, 2000). Pain is a sensorial modality and primarily protective in nature but often causes discomfort. It is the most important symptom that brings the patient to physician. Analgesics relieve pain as a symptom without affecting its cause (Tripathi, 1999). Currently available analgesic drugs such as opiates and NSAIDs are not useful in all cases due to their adverse effects. In this respect new compounds with improved pain management capacity and fewer side effects are being sought with urgency.
Stephania japonica (Thunb.) Miers, (Family-Menispermaceae) is a slender twining shrub with greenish yellow flowers and large tubers. It is native to eastern and southern Asia and Australasia. In Bangladesh, it is grown in many areas. The leaves and roots are bitter and astringent and used in fevers, diarrhea, dyspepsia and urinary disease (Ghani, 2003).
As a part of presant ongoing investigations on local medicinal plants of Bangladesh (Mazumder et al., 2009) in this study, we reported antioxidant, analgesic and toxic potentiality of the leaves of Stephania japonica.
MATERIALS AND METHODS
Plant materials: The plant, Stephania japonica (Thunb.) Miers was collected from the village Uthali under Jibannagar thana of Chuadanga district, Bangladesh during the month of August 2009. The plants were mounted on paper and the sample was identified by Mrs. Mahmuda Begum, senior scientific officer, Bangladesh National Herbarium, Dhaka, where the voucher specimen has been deposited. Its DACB Accession No. is 34,393.
Chemicals: Sodium nitroprusside were purchased from E. Merck (Germany). 1,1-diphenyl-2-picryl-hydrazyl (DPPH), sodium nitroprusside, ascorbic acid, quercetin and potassium ferric cyanide were purchased from Sigma Chemical Co. Ltd, (St. Louis, MO, USA). Diclofenac-Na was collected from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals and reagents were of analytical grade.
Preparation of plant extract: The plant material was shade-dried with occasional shifting and then powdered with a mechanical grinder, passing through sieve #40 and stored in a tight container. The dried powder material (1.5 kg) was refluxed with MeOH for three hours. The total filtrate was concentrated to dryness, in vacuo at 40°C to render the MeOH extract for investigation.
Animal: For the experiment, Twenty swiss albino mice of either sex, 3-4 weeks of age, weighing between 20-25 g, were collected from the animal research branch of the International Center for Diarrheal Disease and Research, Bangladesh (ICDDRB). Animals were maintained under standard environmental conditions (temperature: (24.0±1.0°C), relative humidity: 55-65% and 12 h light/ dark cycle) and had free access to feed and water ad libitum. The animals were acclimatized to laboratory condition for one week prior to experiments. All protocols for animal experiment were approved by the institutional animal ethical committee.
Phytochemical screening: The freshly prepared crude 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, 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. These were identified by characteristic color changes using standard procedures (Ghani, 2003).
In vivo analgesic screening
Acetic acid-induced writhing test: The analgesic activity of the
samples was also studied using acetic acid-induced writhing model in mice. Test
samples and vehicle were administered orally 30 min before intraperitoneal administration
of 0.7% acetic acid but Diclofenac-Na was administered intraperitonially 15
min before injection of acetic acid. After an interval of 5 min, the mice were
observed for specific contraction of body referred to as ‘writhing’
for the next 10 min (Ahmed et al., 2004).
In vitro tests for antioxidant activity
Free radical scavenging activity measured by 1,1-diphenyl-2-picryl-hydrazyl
(DPPH): The free radical scavenging activity of MeOH extract based on the
scavenging activity of the stable 1,1-diphenyl-2- picrylhydrazyl (DPPH) free
radical was determined by the method described by Braca
et al. (2001). Plant extract (0.1 mL) was added to 3 mL of a 0.004%
MeOH solution of DPPH. Absorbance at 517 nm was determined after 30 min and
the percentage inhibition activity was calculated from [(A0–A1)/A0]
x 100, where A0 is the absorbance of the control and A1 is
the absorbance of the extract/ standard. IC50 value was calculated
from the equation of line obtained by plotting a graph of concentration (μg
mL-1) versus % inhibition.
Nitric oxide radical scavenging assay: The procedure is based on the method, where sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (10 mM) in phosphate buffered solution (pH 7.4) was mixed with different concentrations of plant extract of S. japonica dissolved in 10% DMSO and incubated at room temperature for 150 min. The same reaction mixture without the extract but the equivalent amount of the solvent used served as the control. After incubation, 0.5 mL of Griess reagent (1% sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride was added. The absorbance was measured at 546 nm and the percentage inhibition activity was calculated from [(A0–A1)/A0] x 100, where, A0 is the absorbance of the control and A1 is the absorbance of the extract/ standard (Sreejayan, 1997). IC50 value was calculated from the equation of line obtained by plotting a graph of concentration (μg mL-1) versus % inhibition.
Reducing power activity: The reducing power of S. japonica was determined by according to the method previously described by Oyaizu (1986). Extracts at different concentrations in 1 mL of 10% DMSO were mixed with 2.5 mL of phosphatebuffer (0.2M, pH 6.6) and 2.5 mL potassium ferricyanide [K3Fe (CN) 6] (1%) and then the mixture was incubated at 50°C for 30 min. Afterwards, 2.5 mL of trichloroacetic acid (10%) was added to the mixture which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 mL of upper layer solution was mixed with 2.5 mL distilled water and 0.5 mL FeCl3 (0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.
Brine shrimp lethality bioassay: The cytotoxic activity of the plant was evaluated using Brine Shrimp lethality bioassay method (Meyer et al., 1982) where 6 graded doses (viz,. 5, 10, 20, 50, 100 and 200 μg mL-1) were used. Brine shrimps (Artemia salina Leach) nauplii Ocean 90, USA were used as test organisms. For hatching, eggs were kept in brine with a constant oxygen supply for 48 h. The nature nauplii were then used in the experiment. DMSO was used as a solvent and also as a negative control. The median lethal concentration LC50 of the test sample after 24 h was obtained by a plot of percentage of the dead shrimps against the logarithm of the sample concentration. Vincristine sulfate was used as a reference standard in this case.
Statistical analysis: All the in vitro experimental results were Mean±SEM of three parallel measurements. Results of in vivo study were given as Mean±SEM. and data were evaluated by using student’s ttest. pvalues<0.001 were regarded as significant.
RESULTS
Phytochemical screening: Phytochemical analyses of the crude extract revealed the presence of steroid, alkaloid, tannin and glycoside (Table 1).
In vivo Analgesic screening
Acetic acid-induced writhing test: Table 2 shows
the effects of the extract of on acetic acid-induced writhing in mice.
| Table 1: | Result of chemical group tests of the methanol extract of Stephania japonica leaf |
| ME: Methanolic extract, +: Present, -: Absent; +++: Reaction intensity is high, ++: Reaction intensity is medium, +: Reaction intensity is normal | |
| Table 2: | Effects of the methanolic extract of Stephania japonica leaf on acetic acid-induced writhing in mice |
| Values are Mean±SEM, (n =5); **p<0.001, Student’s t test as compared to vehicle control. Group-I aminals received vehicle (1% Tween 80 in water), Group-II received Diclofenac-Na 10 mg kg-1 body weight, Group-III and Group-IV were treated with 200 and 400 mg kg-1 body weight (p.o.) of the crude extract of S. japonica leaf | |
| Table 3: | Antioxidant activities of the S. japonica leaf extract on DPPH and NO |
| aDPPH is the free radical scavenging activity (IC50: μg mL-1), bNO is the inhibition of nitric oxide production (IC50: μg mL-1), *p<0.001 by student’s test for values between the sample and the control | |
The oral administration of both doses of S. japonica extract significantly (p<0.001) inhibited writhing response induced by acetic acid in a dose dependent manner.
In vitro antioxidant activity
DPPH radical scavenging activity: The percentage (%) scavenging of
DPPH radical was found to be concentration dependent i.e., concentration of
the extract between 10-200 μg mL-1 greatly increasing the inhibition
activity. The IC50 value of the extract was 105.55±1.06 μg
mL-1, as opposed to that of ascorbic acid (IC50 12.30±0.11
μg mL-1) which is a well known antioxidant (Table
3).
Nitric Oxide (NO) scavenging activity: Table 3 represents the result of nitric oxide scavenging activity (IC50 value) of the methanolic extract of S. japonica compared with various reference compounds such as ascorbic acid and quercetin. IC50 value of the extract of was 129.12±0.15 μg mL-1, whereas, standard ascorbic acid and quercetin showed 18.64±0.22 and 27.69±0.57 μg mL-1, respectively.
Reducing power ability: For the measurement of the reductive ability, presat study investigated the Fe3+ to Fe2+ transformation in the presence of methanolic extract.
| Table 4: | Brine shrimp lethality bioassay of crude methanol extract of stephania japonica leaf |
| Fig. 1: | Reducing power of Methanolic extract of S. japonica, ascorbic acid, galic acid and quercetin by spectrophotometric detection of Fe3+ to Fe2+ transformation. Results are Mean±SEM of three parallel measurements |
Like the antioxidant activity, the reducing power of S. japonica increased with increasing concentration of the sample. Figure 1 shows the reductive capabilities of the S. japonica compared with ascorbic acid, galic acid and quercetin.
Brine Shrimp lethality bioassay: The result of Brine Shrimp lethality bioassay is given in Table 4. S. japonica leaf extract displayed strong toxic potentiality. LC50 value for the extract was found to be 25.19±0.98 μg mL-1.
DISCUSSION
The oral administration of both doses of S. japonica extract significantly (p<0.001) inhibited writhing response induced by acetic acid in a dose dependent manner. Acetic acid-induced writhing model represents pain sensation by triggering localized inflammatory response. Such pain stimulus leads to the release of free arachidonic acid from tissue phospholipid (Ahmed et al., 2006). The acetic acid induced writhing response is a sensitive procedure to evaluate peripherally acting analgesics. The response is thought to be mediated by peritoneal mast cells (Ribeiro et al., 2000), acid sensing ion channels (Vogel and Vogel, 1997) and the prostaglandin pathways (Voilley, 2004).
Preliminary phytochemical screening showed the presence of tanin, alkaloid and glycoside in the plant extract. However, tetrandrine and fangchinoline alkaloids were isolated from S. japonica and showed anti-inflammatory effect through decrease leukotriene and prostaglandin generation. Furthermore tetrandrine has been shown to inhibit the production of TNF-alpha and IL-6 (Teh et al., 1990; Xue et al., 2008). Li et al. (2000) has been demonstrated that aconitum and S. tetrandra combinedly showed remarkable analgesic activity in rabbits and mice model. So, the observed analgesic activity may be attributed to these compounds. Moreover, recent studies suggest that the inflammatory tissue damage is due to the liberation of reactive oxygen species form phagocytes invading the inflammation sites (Parke and Sapota, 1996). Again the plant extracts demonstrated antioxidant action in the tested models. So it can be assumed that Cyclooxygenase (COX) inhibitory activity together with antioxidant activity may reduce the production of free arachidonic acid from phospholipid or may inhibit the enzyme system responsible for the synthesis of prostaglandins and ultimately relive pain-sensation.
Polyphenolic compounds, like flavonoids, tannins and phenolic acids, commonly found in plants have been reported to have multiple biological effects, including antioxidant activity (Brown and Rice-Evans, 1998; Vinson et al., 1995; Gil et al., 1999; Kahkonen et al., 1999). Fangchinoline and cepharanthine isolated from Stephania rotunda showed antioxidant activity in different in-vitro model (Gulcin et al., 2010). Tannic acid present in the plant extract, as evident from phytochemical screening, may be responsible for the antioxidant action. NO scavenging capacity of the extract may help to arrest the chain of reactions initiated by excess generation of NO that are detrimental to the human health. Nitric oxide is also implicated for inflammation, cancer and other pathological conditions (Moncada et al., 1991). A direct correlation between antioxidant capacity and reducing power of certain plant extracts has been reported (Tanaka et al., 1988). The reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom (Duh et al., 1999).
The extract also showed significant cytotoxicity on Brine Shrimp nauplii. This may be due to the fact that S. japonica contains isotrilobine and trilobine, bisbenzylisoquinoline alkaloids which was previously reported to possess multidrug-resistance-reversing activity in human breast cancer cell line (Andrea and Chang, 1997).
CONCLUSION
The study clearly indicates that the extract possesses antioxidant and cytotoxic substances. At the same time its ability to suppress abdominal writhes confirms the analgesic property of the extract. These findings justify the traditional uses of this plant in the treatment of diabetes, wounds, inflammatory conditions, worms, infestations and malarial fever. Further research is necessary for elucidating the active principles.