|Year : 2014 | Volume
| Issue : 4 | Page : 386-390
Anti-psoriatic activity of Givotia rottleriformis in rats
A Vijayalakshmi, Madhira Geetha
Department of Pharmacognosy, Sri Ramaswamy Memorial College of Pharmacy, Sri Ramaswamy Memorial University, Kattankulathur, Chengalpet, Tamil Nadu, India
|Date of Submission||02-Dec-2013|
|Date of Decision||14-Feb-2014|
|Date of Acceptance||19-May-2014|
|Date of Web Publication||4-Jul-2014|
Department of Pharmacognosy, Sri Ramaswamy Memorial College of Pharmacy, Sri Ramaswamy Memorial University, Kattankulathur, Chengalpet, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Objectives: To evaluate the antipsoriatic activity of Givotia rottleriformis bark in rats.
Materials and Methods: The antipsoriatic activity of the ethanol extract was investigated using ultraviolet B (UV-B)-induced photodermatitis model in rats. The animals were divided into four groups (6/groups). The vehicle-control group animals received normal saline (10 ml/kg, p.o.) and standard group received retinoic acid (0.5 mg/kg, p.o.). Remaining groups were treated orally with the ethanolic extract of bark of Givotia rottleriformis (200 and 400 mg/kg, p.o.) and data were analyzed using one-way analysis of variance (ANOVA). The extract was standardized using chemical markers by high-performance liquid chromatography (HPLC) analysis.
Results: In case of psoriasis model, histopathological analysis revealed that in the section, there were absence of Munro's microabscess, elongation of rete ridges, and capillary loop dilation in ethanol extract (400 mg/Kg) and standard group. The ethanolic extract (200 and 400 mg/Kg) exhibited significant reduction (P < 0.01) in percentage of relative epidermal thickness as compared with positive control. In the HPLC analysis, 4 flavonoids were quantified by comparison to a calibration curve derived from the standard, rutin (0.215 mg/gm) quercetin (1.36 mg/gm), kaempferol (6.36 mg/gm) and luteolin (8.64 mg/gm).
Conclusion: The crude extract containing ethanolic extract of Givotia rottleriformis bark have potent antipsoriatic activity in UV-B-induced psoriasis in rat.
Keywords: Anti-psoriatic, flavonoids, Givotia rottleriformis, UV-B photodermatits model
|How to cite this article:|
Vijayalakshmi A, Geetha M. Anti-psoriatic activity of Givotia rottleriformis in rats. Indian J Pharmacol 2014;46:386-90
| » Introduction|| |
Psoriasis is a genetically determined chronic inflammatory skin disease characterized by red, scaly and raised patches that affects 2.3% of the population worldwide. It occurs when the immune system of body sends out faulty signals resulting in speed up of the growth cycle of skin cells.  Psoriasis has important social, psychological and economic consequences. Its impact on the quality of life is found to be nearly similar to that associated with other chronic medical conditions such as diabetes and depression.  In psoriasis, epidermal hyperproliferation, abnormal keratinocyte differentiation, angiogenesis with blood vessel dilatation and excess Th-1 and Th-17 inflammation have been observed.  Treatment of psoriasis includes topical, systemic, biological, and phototherapy. However, these therapies are associated with side effects.
Givotia rottleriformis Griff. Ex Wight is a moderately sized tree of the family Euphorbiaceae distributed in limited areas of the forests of Tamil Nadu, Andhra, Karnataka, West Bengal and coastal Sri Lanka. The bark and seeds of the tree are used in indigenous medicine in the treatment of rheumatism, dandruff and psoriasis.  However, there is no established scientific study available for its antipsoriatic activity. The aim of this study was to investigate the role of G. rottleriformis in the treatment of psoriasis using UV-B-induced photodermatitis model to establish scientific evidence for its ethnobotanical uses.
| » Materials and Methods|| |
The plant specimen for the proposed study was collected in the forest of Attur, Salem district, Tamil Nadu. It was identified and authenticated by Dr. P. Jayaraman, Director, Plant Anatomy Research Centre (PARC), Tambaram, Chennai. A voucher specimen No. PARC/2011/2140 has been deposited for further references.
About 500 gm of the bark of G.a rottleriformis powder was extracted using a soxhlet apparatus with ethanol (70% v/v) (18 h). The extracted solution was filtered and concentrated in a rotary evaporator under reduced pressure (rotary vacuum flash evaporator). The yield of ethanol extract was about 6.4% w/w.
Concentrated ethanol (70% v/v) extract was screened for the presence of various phytoconstituents using standard procedure. 
Fingerprint Analysis by HPLC
The qualitative and quantitative analysis of the sample was performed according to the method of Boligon et al., 2012.  The HPLC system of Jasco consists of a pump (model Jasco PU2080, intelligent HPLC pump) with injecting facility programed at 20 μL capacity per injection was used. The detector consists of a UV/VIS (Jasco UV 2075) model operated at a wavelength of 270 nm. The software used was Jasco Borwin version 1.5, LC-Net II/ADC system. The column was Thermo ODS Hypersil C18 (250 × 4.6 mm, 5 μm) in isocratic mode. The separation was achieved using a mobile phase of methanol, water and phosphoric acid (100: 100: 1, v/v/v) at a flow rate of 1.5 ml/min. The effluent was monitored using UV detection at a wavelength of 270 nm. The mobile phase was filtered through 0.45 μm nylon filter prior to use.
Powdered drug of G. rottleriformis bark was weighed and transferred to a 250 mL flask fitted with a reflux condenser. About 78 mL of extraction solvent (Alcohol: Water and Hydrochloric acid (50:20:8)) was added, refluxed on a hot water bath for 135 minutes, cooled at room temperature and transferred to a 100 mL volumetric flask. About 20 mL of methanol was added to the 250 mL flask, sonicated for 30 min, filtered and the filtrate was transferred to the 100 mL volumetric flask, the residue was washed on the filter with methanol. The washing was collected in the same 100 mL volumetric flask and diluted to volume.
Identification was based on retention times and on-line spectral data in comparison with authentic standards. Quantification was performed by establishing calibration curves for each determined compound, using the standards.
Acute toxicity studies were carried out using rats as per Organization for Economic Co-operation and Development (OECD) 425 guidelines.  The Institutional Animal Ethics Committee (IAEC) approved the protocol (Registration no. 52 CPCSEA dated 12.8.2012).
Rats were divided into three groups of six rats each. A limit test at a dose of 2000 mg/kg body weight was carried out of ethanol extract. The animals were observed for clinical signs and mortality for 15 days. Body weight was recorded every week. The tested sample was found to be safe and did not produce any mortality after 15 days.
Rat UV Ray Photo Dermatitis Model for Psoriasis
Irradiation of the depilated rat skin was carried out with UV radiation. Immediately after irradiation, initial faint erythema appears that last for 30 min. The second phase of erythema starts after 6 h and gradually increases, peaking between 24 and 48 h. This reaction is confined to the exposed area and has a sharp boundary, and develops a brownish-red color. By 48 to 72 h, silvery white scale appears on the erythematous lesion. These scales are relatively thick and begin to fall beyond 72 h. Although the erythematic reaction is induced artificially, many of the pathological features resemble those seen in psoriasis vulgaris. The close resemblance of inflammatory process produced by UV radiation to the one exhibited in psoriasis provides a good model to investigate drugs that have a potential to reduce the inflammatory reaction associated with psoriasis. 
The hairs of the rat skin, on one side of the flank, were depilated by clipping with a scissors followed by careful shaving taking precaution to avoid injury to the skin. The rats were then placed on a curved wooden block and their legs tied around it, to avoid contact with the floor. This arrangement prevented the movement of the animal during its subsequent exposure UV radiation. Except for an area of 1.5 × 2.5 cm on the depilated skin, the entire animal was covered with a UV-resistant film. The uncovered area of 1.5 × 2.5 cm was then irradiated for 20 min with a UV-B lamp kept at a vertical distance of 20 cm from the skin. The rats were divided into four groups (6 rats/groups). The vehicle-control group rats received normal saline (10 ml/kg, p.o.) and standard group received retinoic acid (0.5 mg/kg, p.o.). Remaining groups were treated orally with the ethanolic extract of bark of G. rottleriformis (200 and 400 mg/kg BW) once daily, 5 times a week, 12 h after irradiation for 2 weeks. Two hours after the last treatment, animals were sacrificed under ether anesthesia; longitudinal sections of the tail skin were made and prepared for histological examination with hematoxylin-eosin staining. ,
Sections were examined for presence of Munro's microabscesss, elongation of rete ridges, and capillary loop dilation by direct microscopy.
The vertical epidermal thickness between the dermoepidermal junction and the lowest part of the stratum corneum (n = 3 measurements per scale, n = 3 scales per animal, n = 6) were examined. The percentage relative epidermal thickness of all the groups was calculated in comparison with the positive control group (100%; n = 54 measurements per treatment).
It was also examined for mean thickness of stratum corneum and stratum granulosum.
All measurements were made at a magnification of 400X using OLYMPUS microscope having a digital camera attachment and software to take measurements.
Values were represented as mean ± SEM. Data were analyzed using one-way analysis of variance (ANOVA) and group means were compared using the Tukey-Kramer Multiple Comparison test using Instat-V3 software. P < 0.05 was considered significant.
| » Results|| |
Preliminary phytochemical screening of ethanol (70%v/v) extract showed presence of alkaloids, steroids, flavonoids, tannins, terpenoids and saponins.
Fingerprint Analysis of HPLC
The HPLC chromatograms of the bark of G. rottleriformis showed 7 components. The main difference was in peaks eluted at 3.96 min, 11.52 min, 16.68 min and 30.76 min, respectively. Four flavonoids were quantified at 254 nm using peak area by comparison to a calibration curve derived from the standard, rutin (0.215 mg/gm) quercetin
(1.36 mg/gm), kaempferol (6.36 mg/gm) and luteolin (8.64 mg/gm) [Figure 1].
|Figure 1: HPLC Chromatogram of ethanol extract of Givotia rottleriformis bark|
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Acute Toxicity Study
During the acute toxicity study, the ethanolic extract was administered orally and animals were observed for mortality and behavioral responses. No mortality was observed in rats treated with 2000 mg/kg of ethanolic extract. All the rats were normal and no gross behavioral changes were observed till the end of the study period.
Rat UV-ray Photo Dermatitis Model for Psoriasis
Histopathological examination revealed fully developed Munro's microabscess, elongation of rete ridges, and capillary loop dilation [Figure 2]. The mean thickness of the epidermis, mean thickness of the stratum corneum and mean thickness of the stratum granulosum in control- and drug-treated animals were tabulated [Table 1]. There was significant decrease in epidermal thickness (P < 0.05) as compared with control group. The section of vehicle-control group showed regular elongation of rete ridges, capillary loop dilation with minimal grade lesion of diagnostic Munro's microabscess and marked increase in relative epidermal thickness as compared with other groups. In ethanol extract (200 mg/kg) treated group, there was a minimal grade lesion of elongation of rete ridges along with capillary loop dilation in the section and absence of Munro's microabscess. In ethanol extract (400 mg/kg) treated group, there was no lesion of Munro's microabscess, capillary loop dilation along with elongation of rete ridges in the section of skin of rats. In the standard group, there was absence of Munro's microabscess, capillary loop dilation along with elongation of rete ridges in the section showing significant therapeutic effects when compared with test-treated groups.
|Table 1: Histopathological features on UV-B-induced psoriasis in rats treated with ethanolic extract of bark of G. rottleriformis|
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|Figure 2: Histopathological features on UV B induced psoriasis in control and drug treated rat|
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| » Discussion|| |
Skin is the largest exposed organ of body and easy target for allergic and immunologic reactions. Skin ailments viz. dermatitis, urticaria, angioedema, psoriasis, etc., are immune mediated chronic, inflammatory disorder.  Psoriasis severely affects patients with the quality of life and the treatment being expensive.  Medicinal plants are considered safe, as for the human health and are widely employed by the traditional healers for the treatment of various diseases including psoriasis. Medicinal plants are known to be a rich citadel of variety of chemical compounds and have attracted researcher's attention to find new treatment for psoriasis. 
Screening of antipsoriatic activity of ethanol extract of G. rottleriformis was carried out using UV-B-induced psoriasis in rat. The irradiated rat skin treated with ethanol extract of Givotia rottleriformis (400 mg/kg) has shown a significant reduction in the total epidermal thickness indicating that it has an influence to retard the hyper proliferation of the keratinocytes that occurs when the skin is exposed to UV radiation. The significant retention of the stratum granulosum is probably due to its ability to enhance the keratinization process, which is a protective strategy adopted by the skin when exposed to penetrating radiation. Further, ethanol extract of G. rottleriformis (400 mg/kg) produced useful changes in the epidermis of the irradiated skin, showing its potential use in psoriasis treatment.
The results of HPLC analysis showed that the bark of G. rottleriformis
is a rich source of the important biologically active flavonoids, rutin, quercetin, kaempferol and luteolin. Flavonoids, a natural polyphenols, recognized as potent antioxidants are multifunctional molecules that can act as anti-inflammatory and antiproliferative agents through the modulation of multiple-signaling pathways. This characteristic could be advantageous for the treatment of multi-causal diseases caused by oxidative stress, including psoriasis. In an earlier work, Vijayalakshmi et al.,  reported that the flavonoid quercetin showed significant reduction in epidermal thickness with respect to control in Perry's mouse tail model. Jadranka Skuric et al.,  reported that the flavonoids from propolis offer some protection against psoriatic complications through their roles as inhibitors of inflammation and as free-radical scavengers on animal model psoriasis, induced by the di-n-propyl disulfide irritant (PPD). The observed antipsoriatic activity may be through the inhibition of keratinocyte proliferation, which may be attributed to the presence of various phytoconstituents, especially flavonoids in the extract.
| » Conclusion|| |
The result of the study showed that the ethanol extract of G. rottleriformis bark exhibited significant antipsoriatic activity. The study implies that G. rottleriformis bark is a promising research for further investigations to prove its antipsoriatic activity.
| » Acknowledgment|| |
Authors acknowledge sincere thanks to the management for the facilities granted for the research work.
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[Figure 1], [Figure 2]