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| ORIGINAL ARTICLE |
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| Year : 2015 | Volume
: 35
| Issue : 2 | Page : 70-78 |
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Therapeutic potential of Polyalthia cerasoides stem bark extracts against oxidative stress and nociception
BC Goudarshivananavar1, V Vigneshwaran2, Madhusudana Somegowda2, Kattepura K Dharmappa3, Siddanakoppalu N Pramod2
1 Department of Studies in Chemistry, Sahyadri Science College (Autonomous), Kuvempu University, Shimoga, Karnataka, India
2 Department of Studies and Research in Biochemistry, Laboratory of Immunomodulation and Inflammation Biology, Sahyadri Science College (Autonomous), Kuvempu University, Shimoga, Karnataka, India
3 Department of Studies in Biochemistry, PG Centre of Mangalore University, Madikeri, Karnataka, India
| Date of Web Publication | 14-Dec-2015 |
Correspondence Address:
Siddanakoppalu N Pramod
Laboratory of Immunomodulation and Inflammation Biology, Department of Studies and Research in Biochemistry, Sahyadri Science College (Autonomous), Kuvempu University, Shimoga - 577 203, Karnataka
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0257-7941.171667
Background: Polyalthia cerasoides is a medicinal plant known for its ethnopharmacological importance. Despite this, investigation related to its therapeutic benefit is still unexplored.
Aim: To evaluate the stem bark extracts of Polyalthia cerasoides for pharmacological activities relating to inflammation, nociception and oxidative stress using in vivo and in vitro models.
Materials and Methods: Pet ether, ethyl acetate and chloroform fractions of the stem bark were evaluated for anti-inflammatory activity by carrageenan-induced hind paw edema in rats. Anti-nociceptive activity in mice was assessed using thermally and chemically induced analgesic models. The free radical quenching potential of the extracts was initially analyzed using the in vitro DPPH photometric assay, Hydroxyl radical scavenging and Lipid Peroxidation assays. Then modulatory effect of the extracts on in vivo antioxidant system was evaluated by carbon tetrachloride induced hepatotoxicity and subsequent measurements of antioxidant enzymes such as Superoxide dismutase, Catalase and Peroxidase from the liver homogenate.
Results: Among the tested fractions, ethyl acetate extract had substantially inhibited the inflammation by 68.5% that was induced by subcutaneous carrageenan injection whereas pet ether and chloroform extract showed only minimal inhibitory effect. Investigation of the anti-nociceptive activity revealed that the ethyl acetate fractions had significantly repressed the algesia in both the analgesic experimental models. In vitro and in vivo individual antioxidant assays demonstrated that the ethyl acetate fraction has strong free radical quenching potential which also restores the endogenous hepatic enzymes.
Conclusion: The ethyl acetate fraction enriched with flavinoids and steroids from Polyalthia cerasoides stem bark has potent bioactivity to combat inflammation, ROS and pain. This needs further characterization for potential therapeutic applications. Keywords: Analgesia, anti-inflamatory, carageenan, catalase, lipid peroxidation, nociception, reactive oxygen species
How to cite this article:
Goudarshivananavar B C, Vigneshwaran V, Somegowda M, Dharmappa KK, Pramod SN. Therapeutic potential of Polyalthia cerasoides stem bark extracts against oxidative stress and nociception. Ancient Sci Life 2015;35:70-8 |
How to cite this URL:
Goudarshivananavar B C, Vigneshwaran V, Somegowda M, Dharmappa KK, Pramod SN. Therapeutic potential of Polyalthia cerasoides stem bark extracts against oxidative stress and nociception. Ancient Sci Life [serial online] 2015 [cited 2023 Mar 31];35:70-8. Available from: https://www.ancientscienceoflife.org/text.asp?2015/35/2/70/171667 |
| Introduction | |  |
Inflammation is a tissue response to stimuli such as: Pathogens, damaged cell and irritants. It is the protective attempt by the organism to remove the injurious stimuli as well to initiate healing process of the tissue.[1] Acute inflammation is characterized by edema, erythema, pain, heat and above all, loss of function. These important signs of inflammation are triggered by the infiltration mediators which include histamine, tryptase, prostaglandins, leukotrienes and white blood corpuscles (leucocytes).[2],[3]
Reactive oxygen species (ROS) are natural by products of body metabolism and are charged molecules that attack cells, tear through cellular membranes, react and create havoc with nucleic acids, proteins and enzymes present in the body. Various reports have demonstrated the role of ROS in inflammation and it is also evident from the anti-inflammatory effects of the antioxidants. As inflammatory conditions induce pain and oxidative stress, drug products with combinational treatment plans are most preferred for inflammation therapy.[3],[4] Drugs currently used for management of pain and inflammatory conditions are either narcotics such as opioids or non-narcotics. Both of them have toxic side effects on chronic administration.[5] On the other hand many medicines of plant origin have been used since a long time without any adverse effects. Plants represent a large untapped source of structurally novel compounds that may serve as leads for the development of novel drugs.[6]
Plants belonging to the family Annonaceae have long been used as a major source of medicines for the prevention and treatment of a variety of diseases in India and many Asian countries. To date, ethnopharmacological claims for Annonaceae include the use of its bark to control blood pressure, diabetes and its use as a febrifuge.[7],[8],[9]Polyalthia cerasoides (Roxb.) Bedd.(Annonaceae) is a medium sized tree distributed in almost all forests of Deccan India up to 3000 ft. In southern India, the plant is almost exclusively used for its edible fruits and seeds.[10],[11] The stem bark of this plant is used as tonic to combat stress and pain by local medicinal practitioners and experimental studies have demonstrated its normalizing activity on brain neurotransmitters, moderate cytotoxicity and antioxidant activity in vitro.[11],[12] Various studies have reported the antiproliferative, apoptotic and antimutagenic activity of the seed extract.[13] Pharmacognostic reports relating to the extracts of stem bark of P. cerasoides is limited. The present study has been attempted to understand the pharmacological activities, particularly anti-inflammatory, analgesic and in vitro and in vivo antioxidant potential of the stem bark extracts of P. cerasoides to identify new potential phytoconstituents which may further provide information to develop novel drugs to manage pain, stress and inflammations.
| Materials and Methods | | |
Chemicals and reagents
Carrageenan suspension, formalin, acetic acid, ethyl acetate, chloroform, petroleum ether, ascorbic acid were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India. The reagent 1,1-diphenyl-1- picrylhydrazyl (DPPH) was procured from Sigma-Aldrich (St. Louis, MO, USA). Pentazocine and Diclofenac sodium injections were purchased from Novartis and Ranbaxy, India, respectively. All other solvents and chemicals used in this experiment were of analytical grade.
Animals
Swiss albino mice, 6-8 weeks old (20-25 g) and Wistar rats (150-200 g) of either sex were used in the study. The animals had free access to food and water, and were housed at room temperature, 24 ± 2°C in a natural (12 h each) light-dark cycle. The animals were acclimatized for at least 5 days to the laboratory conditions before conducting experiments. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) and the care of the laboratory animals was taken as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) regulations.
Plant collection and solvent extraction
The bark of P. cerasoides was collected from the forests of Kuvempu University campus area. The plant was authenticated with the taxonomist from Department of Botany, Kuvempu University, India. Fresh plant material was collected and sprayed with alcohol in order to arrest any enzymatic degradation. The collected materials were washed with running Water and subsequently tapped dry and chopped into pieces. The material was then shade dried and coarsely powdered.
The weighed amount of material was successively extracted using soxhlet apparatus with solvents of varying polarity starting from pet-ether, chloroform and ethyl acetate. Each extraction was carried out for 18 h (approximately 45 cycles) at room temperature. The extracts were concentrated after evaporating the solvent using flash evaporator, under reduced pressure and controlled temperature. The extracts so obtained were air dried, weighed, packed and stored at 4 deg. C.[14]
Phytochemical analysis of the extracts and acute toxicity studies
Qualitative analysis for the phytochemical constituents in the extracts of stem bark of P. cerasoides was performed based on the specific qualitative tests as reported previously.[15] All the three extracts, i.e. pet ether (PEPCF), chloroform (CLPCF) and ethyl acetate (EAPCF) fractions were evaluated for the presence of medicinally active phytochemicals. The tests include detection of alkaloids, saponins, tannins, glycosides, steroids or terpinoids, carbohydrates and flavonoids in the fractions and are carried out following the standard protocols as described. The presence of the phytochemicals were assessed and graded as -, +, ++ based on the absence and abundance in the fractions.
Acute toxicity study was carried out using Swiss albino mice (25-30 g) by up and down staircase method as per CPCSEA guidelines as described previously.[3] Suspension of pet ether, chloroform and ethyl acetate fractions of P. cerasoides was orally administered to different groups of mice at doses of 50, 300, 1000 and 2000 mg/kg body weight following standard intraoral protocol. Animals were observed for 48 h to study their general behavior, signs of discomfort and nervous manifestations. The death and behaviour of the animals in each group were recorded and were used for the assessment of approximate Lethal Dose (LD50) and acute toxicity level and also to fix the dose for the further pharmacological studies.
Analgesic activity
Analgesic activity for the fractions was assayed in mice by inducing pain chemically and thermally using acetic acid induced writhing model and hot plate assay respectively.
Acetic acid induced writhing assay
Acetic acid writhing assay was performed in accordance with the methods described earlier.[3] Briefly, five groups of mice (n = 6) were randomly formed. The groups were treated as (i) control (distilled water, p.o.) (ii) standard (Diclofenac, 5 mg/kg i.p.) while the three test groups received suspensions of pet ether, chloroform and ethyl acetate extract fractions of stem bark of P. cerasoides (100 and 200 mg/kg p.o.) respectively. Acetic acid solution 0.6% v/v (10 ml/kg) was injected by intraperitoneal route one hour after treatment and number of writhes (i.e., index of pain reaction against chemical stimuli characterized by abdominal muscle contraction together with turning of trunk and extension of hind limbs) was counted over a period of 20 min. Analgesic activity was also expressed as a percentage of inhibition of writhes with respect to the control group.
Eddy's hot plate assay
Eddy's Hot plate assay was performed by the method as described previously.[16] Briefly, a hot plate was maintained at 55 ± 1°C. Albino mice were divided in six groups (n = 6). The animals were placed on the hot plate and the basal reaction time taken to cause a discomfort (licking of paw or jumping response whichever appeared first) was recorded at 0 min. A cut-off period 15 sec. was established to prevent damage to the paws. The treatment and groupings of mice was done in the same manner as mentioned in the acetic acid induced writhing model except that in this case, the standard group received pentazocine (5 mg/kg i.p.). The reaction time in seconds was reinvestigated at 30, 60, and 120 min after the treatment. Changes in mean reaction time were noted.
Anti-inflammatory activity
Acute anti-inflammatory activity of suspensions of pet ether, chloroform and ethyl acetate extracts of stem bark of P. cerasoides (100 and 200 mg/kg p.o.) respectively, was evaluated using carragenan induced paw edema in rats as described previously with slight modifications.[17] Briefly, six groups of albino rats (n = 4) were randomly distributed as control, standard and test groups. The initial paw volumes of each animal were measured by means of a mercury plethysmometer. The standard group was treated with Diclofenac injection (5 mg/kg, i.p.) while suspensions of pet ether, chloroform and ethyl acetate extracts of stem bark of P. cerasoides (100 and 200 mg/kg p.o.) was administered to test groups and distilled water (10 ml/kg, i.p.) was given to the control group, 0.1 ml of 1% carrageenan solution was injected in the plantar region of the left hind paw of rats thirty minutes after treatment. Paw volumes were again measured 3 h after carrageenan injection. The difference in edema volume was calculated in each control, test and standard group and compared with the control group for determination of the percentage of inhibition of the paw edema.
In-vitro antioxidant activity of EAPCF from stem bark of P. cerasoides
Reaction with DPPH radical
The DPPH radical scavenging potential of P. cerasoides extracts was evaluated by previously described methods with slight modifications.[18] Briefly, 2.5 ml of 200 mM DPPH in methanol was mixed with 0.5 ml of different concentrations of ethyl acetate P. cerasoides fraction (1-100 mg/ml) in methanol and kept in dark for 30 min. The absorbance at 517 nm was measured. Ascorbic acid (ASC) was used as a standard for comparison. Plotting the percentage DPPH scavenging against ASC and EAPCF concentration gave the standard cuve from which the IC50 value is determined.
Reaction with hydroxyl radical
Hydroxyl radical scavenging activity was measured by the ability of the ethyl acetate extract of P. cerasoides
(6-500 mg/ml) to scavenge the hydroxyl radicals generated by the Fe 3+-ascorbate-EDTA-H2O2 system (Fenton reaction).[19] The reaction mixture containing deoxy-D-ribose (3 mM), ferric chloride (0.1 mM), EDTA (0.1 mM), hydrogen peroxide (2 mM) in phosphate buffer (20 mM, pH = 7.4), with different concentrations of the EAPCF in a volume of 0.3 ml were added, to give a final volume of 3.0 ml. After incubation for 30 min at ambient temperature, 1.0 ml of TCA-TBA reagent (equal volumes of TCA-2.8% and TBA-0.5% in 4mM NaOH) was added, followed by heating the tubes in a water bath for 30 min. The tubes were then cooled and the absorbance was measured at 532 nm. Mannitol was used as standard for comparison. Different concentrations (0.5-4.5 mg/ml) of mannitol were mixed as explained above. Plotting the percentage inhibition of hydroxyl radical scavenging against that of mannitol and EAPCF concentration gave the standard curve from which IC50 value was calculated.[19]
Lipid peroxidation (LPO) assay
Egg phospatidylcholine (20 mg) in chloroform (2 ml) was dried and further dispersed in normal saline (5 ml). The mixture was sonicated to get a homogeneous suspension of liposomes. Lipid peroxidation was initiated by adding 0.05 mM trolox to a mixture containing liposome (0.1 ml), 150 mM potassium chloride, 0.2 mM ferric chloride, EAPCF (0.10-300 mg/ml) in a total volume of 0.4 ml. The reaction mixture was incubated for 40 min at 37°C. After incubation, the reaction was terminated by adding 1 ml of ice cold 0.25 M hydrochloric acid containing 20% w/v of trichloroacetic acid, 0.4% w/v of thiobarbituric acid and 0.05% w/v of butylated hydroxytoluene. After heating at 80°C for 20 min, the samples were cooled. The pink chromogen was extracted with a constant amount of n-butanol, and the absorbance of the upper organic layer was measured at 532 nm. Trolox was used as standard for comparison. Plotting the percentage inhibition of LPO scavenging against trolox concentration gave the standard curve and IC50 value is caluculated for the samples.[20]
In-vivo antioxidant activity for EAPCF in mice
The in vivo antioxidant activity of ethyl acetate fraction of P. cerasoides was carried out using Swiss albino mice (6-8 weeks old) as described previously.[3] Briefly, animals were divided into groups (n = 6). Group I: Served as control (administered PBS, 5 ml/kg, p.o.). Group II: Served as negative control (CCl4/olive oil (1:1), 1 ml/kg, i.p on 3rd and 4th day). Group III: Treated with Silymarin 100 mg/kg, p.o for successive five days. Test groups IV and V: Suspensions of ethyl acetate fraction at the dose of 250 and 500 mg/kg, p.o. respectively for five days. All the animals except control group received CCl4 (1 ml/kg, i.p.) on 3rd and 4th day. On the fifth day 2 hr after the administration of the last dose, livers were isolated to measure the levels of antioxidant enzymes.
Five percent liver homogenate was prepared with 0.15 M KCl and centrifuged at 1000 rpm for 10 min. The cell free supernatant was used for the estimation of Super oxide dismutase (SOD), Catalase and Peroxidase by the methods described previously.[21],[22],[23]
SOD assay
0.5 ml of liver homogenate was taken, and 1 ml of 50 mM sodium carbonate, 0.4 ml of 24 µM NBT, and 0.2 ml of 0.1 mM EDTA were added. The reaction was initiated by adding 0.4 ml of 1 mM hydroxylamine hydrochloride. Zero time absorbance was taken at 560 nm followed by recording the absorbance after 5 min at 25° C. The control was simultaneously run without liver homogenate. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of NBT by 50%. The specific activity was expressed in terms of units per mg of proteins.
Catalase assay
1 ml of liver homogenate was taken with 1.9 ml of phosphate buffer in test tubes (50 mM, pH 7.4). The reaction was initiated by the addition of 1ml of H2O2 (30 mM). Control without liver homogenate was prepared with 2.9 ml of phosphate buffer and 1 ml of H2O2. The decrease in optical density due to decomposition of H2O2 was measured at the end of 1 min against the blank at 240 nm. Units of catalase were expressed as the amount of enzyme that decomposes 1 μM H2O2 per min at 25° C. The specific activity was expressed in terms of units per mg of proteins.
Peroxidase assay
0.5 ml of liver homogenate was taken, and to this were added 1 ml of 10 mM KI solution and 1 ml of 40 mM sodium acetate. The absorbance of potassium iodide was read at 353 nm, which indicates the amount of peroxidase. Then 20 μl of H2O2 (15 mM) was added, and the change in the absorbance in 5 min was recorded. Units of peroxidase activity were expressed as the amount of enzyme required to change the optical density by 1 unit per min. The specific activity expressed in terms of units per mg of proteins.
Statistical analysis
Data were expressed as Mean ± Standard Deviation (SD). The values were then subjected to one-way ANOVA followed by Turkey's multiple comparison tests for significant difference. The level of significance was considered at P ≤ 0.05 and P ≤ 0.01.
| Results | | |
Stem bark fractions and phytochemical constituents of P. cerasoides
Three different fractions were obtained after solvent based sequential extraction. The yields are shown in [Table 1]. The pet ether fraction (PEPCF) was semisolid (3.5%) with light brown color and was moderately positive for the content of saponins, terpenoids and sterols, the chloroform fraction (CFPCF) was light green, solid (2%) with a moderate content of terpenoids, sterols and alkaloids. The ethyl acetate (EAPCF) fraction was black semisolid with a maximum yield (5.5%) and was positive moderately for contents, tannins or phenols, saponins, quinones and it showed abundance for sterols and flavonoids. The results of phytochemical constituents are shown in [Table 2]. | Table 1: Percentage yield of crude fractions from the stem bark of P. cerasoides with the solvent extraction of petroleum ether, chloroform and ethyl acetate
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 | Table 2: Qualitative phytochemical analysis of crude fractions obtained from the extraction of stem bark of P. cerasoides
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Toxicity and anti-nociceptive activity of the P. cerasoids stem bark
Fractions were checked for toxicity in mice using the staircase method after administration at different doses ranging from 50-2000 mg/kg (p.o.). No toxicity was observed for all the fractions at 50, 100, 200, 500 and 1000 mg/kg with no change in behavior or movements among the mice. However, at 2000 mg/kg dose five mice showed movement reduction and suffered shock initially for 5-6 h. and recovered completely to normalcy after 24 h. It appears that the fractions obtained from P. cerasoides were not toxic to mice even at 2000 mg/kg.
In the acetic acid induced writhing method, Pet ether, and ethyl acetate fractions showed a significant analgesic activity against chemically induced pain. Both the fractions have shown a reduction in the number of writhes as compared to standard [Figure 1]a. PEPCF at 100 and 200 mg/kg doses are effective in reducing pain by 28.82%, 48.48% respectively and EAPCF at the same dose levels shows reduction in pain by 61.73%, 63.68%, whereas CFPCF fraction showed less than 10% [Figure 1]b. The EAPCF fraction is more significant (P < 0.001) compared to standard drug diclofenac (63.7%). On hot-plate test, PEPCF (6.6 sec) and EAPCF (4.5 sec) showed significant elevation in pain threshold when compared to standard drug pentozocin (7.6 sec) and control (3.4 sec) as represented in [Figure 2]. The thermal sensitivity which was reduced by EAPCF fraction at 200 mg/kg dose was very significant (P < 0.01) and comparable with the standard drug pentazocine. | Figure 1: Anti-nociceptive activity of P. cerasoides stem bark extracts in acetic acid induced writhing in Swiss albino mice. (a) P. cerasoides stem bark fractions (100 and 200 mg/kg, p.o.). Control distilled water (10 ml/kg, p.o.), standard drug diclofenac sodium (5 mg/kg. i.p.). (b) Percentage reduction of writhing in groups treated with the extracts and standard drug. One way ANOVA followed by multiple Tukey's comparison test. Values are presented as the mean ± SEM (standard error); n = 6 for all groups (Statistically significant values are *P < 0.05; **P < 0.01)
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 | Figure 2: Anti-nociceptive activity of P. cerasoides stem bark determined on Eddy's hot plate test in Swiss albino mice. P. cerasoides stem bark fractions (PEPCF, CFPCF, EAPCF, 100 and 200 mg/kg, p.o.). Control distilled water (10 ml/kg, p.o.), standard drug pentazocine (5 mg/kg, i.p.). One way ANOVA followed by multiple Tukey's comparison test. Values are the mean ± SEM, n = 6 in each group, (statistically significant values are *P < 0.05; **P < 0.01)
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Anti-inflammatory potential of P. cerasoides stem bark
Acute anti-inflammatory potential of P.cerasoides fractions were investigated at its minimal dose producing analgesia against carrageenan induced rat paw edema. The [Figure 3]a shows the edema volume in control and treated groups. The standard drug Diclofenac (5 mg/kg i.p.) showed 75.4% inhibition of edema whereas the corresponding values for PEPCF fraction at 100 and 200 mg/kg were 26.22% and 36.06% respectively and for EAPCF, they were 57.38% and 68.85%. EAPCF extract exhibits significant anti-inflammatory activity with respect to control (P < 0.001) and is comparable to the drug. However, the inhibitory effect of CFPCF fraction was <15% and is not significant. The results were shown in [Figure 3]b. | Figure 3: Inhibitory effect of P. cerasoides stem bark on carageenan induced paw edema model in rats. (a) P. cerasoides stem bark fractions (PEPCF, CFPCF, EAPCF, 100 and 200 mg/kg p.o.). Control distilled water (10 ml/kg, p.o.), standard drug diclofenac sodium (5 mg/kg. i.p.). (b) Percentage reduction of paw volume (ml) in treated groups. PEPCF and EAPCF at 200 mg/kg had exhibited 36.06% and 68.85% inhibition respectively. One way ANOVA followed by multiple Tukey's comparison test. Values are presented as the mean ± SEM (standard error); n = 6 for all groups, (statistically significant values are *P < 0.05; **P < 0.01)
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Antioxidant property of EAFPC
The antioxidant potential of ethyl acetate fraction of P. cerasoides stem bark (EAFPC) was evaluated and was found to be effective in reducing nociception and inflammation. The in vitro and in vivo antioxidant potential of EAFPC is shown in [Figure 4] and [Table 3] respectively. | Figure 4: Free radical scavenging ability of P. cerasoides stem bark (EAPCF) by DPPH radical, Hydroxyl radical and Lipid Peroxidation by in-vitro assays. IC50 value of EAPCF on ROS scavenging potential is compared to the respective standards. Values are mean ± SEM, n = 6, one way ANOVA followed by Tukey's multiple comparison test. Significant values are *P < 0.05; **P < 0.01)
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 | Table 3: The in vivo effect of P. cerasoides stem bark extracts (EAPCF) on liver antioxidant enzymes in CCl4 induced hepatotoxicity in rats
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In vitro antioxidant potential of EAPCF
The in vitro antioxidant activity is measured as the ability to scavenge free radicals as compared to the standard antioxidant molecule. The method is represented in [Figure 4]. EAFPC demonstrated DPPH radical (1-100 µg/mL) scavenging activity with IC50 levels of 42.43 µg/ml, whereas the IC50 value of the standard ascorbic acid (1-5 µg/mL)
was 3.19 µg/ml. The ability to scavenge hydroxyl radicals (4-200 µg/mL) with IC50 value of 89.88 µg/ml and standard Mannitol (1-5 µg/mL) was found to be 4.89 mg/ml. The inhibition of Lipid peroxidation (LPO; 1-200 µg/mL) by EAFPC was found to be IC50 = 75.64 µg/ml as compared to that of standard trolox (IC50 = 8.75 µg/ml). The EAFPC exhibited significant antioxidant potential with IC50 value much greater compared to their respective pure standard components. The purified active component from the EAFPC fraction may be potential with lower IC50 value.
In vivo antioxidant potential of EAPCF
Pretreatment of rats with suspensions of ethyl acetate extract of P. cerasoides at a dose of 250 and 500 mg/kg was compared with control group administered with PBS (Negative control) and Silymarin (positive control) for the activity of liver enzymes, Catalase, SOD, and Peroxidase. Results showed a significant improvement in the activity of enzymes in EPC treated group compared to the hepatotoxic group (Group injected with CCl4 alone) which showed decreased levels of hepatic enzymes induced by CCl4 [Table 3]. These findings indicate that P. cerasoides stem bark extracts could significantly protect against hepatic injury by normalizing the endogenous antioxidant enzymes that are involved in combating ROS.
| Discussion | | |
Reactive oxygen species (ROS) and their intermediates have been found to be the mediators of inflammation and also responsible for the pathogenesis of various inflammatory diseases and tissue damage, which is associated with the stimulation of pain.[4],[6] Pain is a complex process mediated by many physiological mediators e.g., prostaglandins, bradykinins, substance-p etc., The petroleum ether, chloroform and ethyl acetate extracts of P. cerasoides stem bark was evaluated for its analgesic potential in both peripheral (non-narcotic) and central (narcotic) type pain models. Pretreatment with pet ether, and ethyl acetate extracts (100 and 200 mg/kg) markedly reduced the pain response produced by acetic acid, manifested as writhing at the employed doses. In the acetic acid induced writhing model the contractions induced by acetic acid in the mice results from an acute inflammatory reaction with production of PGE2 and PGF2α in the peritoneal fluid.[24] Therefore, it is likely that pet ether, and ethyl acetate extracts at both the tested doses might suppress the formation of these substances or antagonize their action, thus exerting analgesic activity. The hot-plate test is commonly used to assess narcotic analgesics or other centrally acting drugs [25],[26] and the present results showed that pet ether, and ethyl acetate extracts also significantly elevate the response latency period suggesting centrally mediated analgesic effect.
P. cerasoides stem bark extracts have also exhibited a moderate anti-inflammatory potential at the employed doses. It has been proposed that inflammatory reaction occurs in two phases: Via release of histamine, serotonin and bradykinin in the early or first phase, followed by the release of prostaglandin in the late or second phase.[3],[27] A significant anti-inflammatory activity against carrageenan induced inflammation was also observed in pet ether, and ethyl acetate extracts, suggesting influence of the fraction on release, synthesis or action of the inflammatory mediators. We conclude that the pet ether, and ethyl acetate extracts of the plant contains active herbal principles which are moderately polar in nature and possess potential analgesic and anti-inflammatory activities.
Free radical induced lipid peroxidation is believed to be one of the major causes of cell membrane damage leading to a number of pathological situations. The hepatic damage induced by CCl4 is well known to be mediated by its free radical metabolites such as CCl -3 and CCl3 COO -, which interact with unsaturated lipid membrane to produce lipid peroxidation and other cellular macromolecules leading to cell damage and decreased activity of hepatic antioxidant enzymes such as catalase, Super oxide dismutase (SOD) and peroxidase.[28],[29] The results of the present work indicate that ethyl acetate extract of P. cerasoids (250 mg/kg, 500 mg/kg, p.o) increased the depleted antioxidant enzyme levels induced by CCl4, thus protecting the structural integrity of hepatocyte cell membrane or by causing regeneration of damaged liver cells. The extract was found to be capable of enhancing or maintaining the activity of hepatic enzymes which are involved in combating ROS.[30] The P. cerasoides extracts' effect on liver antioxidant enzymes can also be confirmed by correlating it with itsinhibitory effect on the lipid peroxidation in vitro. Further preliminary phytochemical investigation of ethyl acetate extract revealed the presence of polyphenols, flavonoids and coumarins. The mode of action of ethyl acetate extract in affording the potential antioxidant activity against CCl4 may be due to cell membrane stabilization, hepatic cell regeneration and activation of antioxidant enzymes such as SOD, catalase and peroxidase by these active principles. Taken together, our findings indicate that P. cerasoides stem bark extracts apart from alleviating pain may also significantly protect against hepatic injury by normalizing the endogenous antioxidant enzymes that are involved in combating ROS [Figure 5]. | Figure 5: Schematic representation of the pharmacological activities of P. cerasoides stem bark extracts
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| Conclusion | | |
Ethyl acetate extract of P. cerasoides stem bark demonstrated a significant analgesic and anti-inflammatory activity with ROS scavenging potential. This study indicates a positive correlation between the ROS scavenging potential and anti-inflammatory activity. Considering the results of experimental parameters, our study provides strong scientific evidence for considering the P. cerasoides stem bark extracts as natural antioxidants targeting algesia and inflammation. Hence we conclude that P. cerasoides stem bark could be used as a therapeutic drug in oxidative stress induced pathological conditions. But an in depth investigation with the pure compounds is necessary to understand the mechanism behind its significant medicinal importance.
Acknowledgements
The authors gratefully acknowledge the Sahyadri Science College, Shimoga (A constituent College of Kuvempu University) for the supporting this study. Dr. Siddanakoppalu N. Pramod and V. Vigneshwaran acknowledges the University Grants Commission, Government of India.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]
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