Evaluation of Anti-leucodermal activity of medicinal plant in rats

 

Pankaj M. Chaudhari*, Dr. Dheeraj T. Baviskar

K.V.P.S., Institute of Pharmaceutical Education, B. pharm, Boradi, Dist – Dhule, Maharashtra.

 

ABSTRACT:

The present study designed to evaluate anti-leucoderma activity of medicinal herb in experiment model of C57BL/6 mice. leucoderma is mainly depigmentary disorder affecting very small population of word. C57BL/6 mice were divided into four groups. 40% Monobenzone cream applied daily for induction leucoderma in mice. Euphorbia hirta extract and Punica granatum extract administered for 65 days. At the end of study 6 mm circles of ear tissue were collected. All tail and back skin were removed. Some samples were stored at 80^oC for further tests. Same dorsal skin samples were collected and placed in 10% neutral buffered formalin to histological analysis. Samples also were used to evaluate depigmentation, epidermal melanin levels, measurement of TNF-α level and histological analyses. Monobenzone applied group showed decreased level of melanin mean while plant extract treated group showed increased level and TNF-alpha level increased in control group and test groups shows the reverse action. From this result we conclude that both plants have ant-leucoderma activity.

 

 

 

INTRODUCTION:

Vitiligo (leucoderma) is the most frequently occurring depigmentary disorder affecting approximately 0.5–1% of individual world wides. It can develop at any age, although half of the patients have vitiligo before the age of 20 years. There are no differences in prevalence according to sex, skin type, or race. Vitiligo develops in women at an earlier age and there are marked geographic differences since prevalence rates. The exact pathophysiology of vitiligo is still unknown. In many countries where whitening of the skin is associated with infectious diseases such as leprosy vitiligo has carried many misconceptions. Vitiligo is associated with patients’ lower self-esteem and experience of feelings of embarrassment, humiliation and fear of rejection, leading to social isolation¹.

 

Euphorbia is employed as a constituent of certain asthma preparation, including antihistaminic Elixir N.F. Dr. M.C. Koman says “it has been found by me very beneficial in case of asthma. Tincture of the drug was given in 15 to 30 drop doses in case of asthma and bronchitis in the hospital with very beneficial effect”.

 

It is also an antidote to poisons. Locally it is applied for the cure of ringworms. This is popular remedy for cough, asthma, bronchial affections and disease of the respiratory passages generally. The juice of the plant is given in dysentery and colic, and milk applied to destroy warts. It is sometimes prescribed in gonorrhea. The plant is widely used in West Africa as a medicine. According to Dikshit and Kameshwar Rao experiment conducted on cats, dogs, and rabbits, with and without anesthesia, show that the drug has got a fairly marked action on the respiratory system. Depressing the respiration and producing a well-marked dilation of the bronchioles. The drug produces a local irritation of the stomach when given by mouth in large doses and produce nausea and vomiting. It has depressant action on the movement of intestine^2-4. The present study gives the systematic evaluation of anti-leucoderma activity of medicinal plants.

 

MATERIAL AND METHODS:

Animals:

C57BL/6 mice of 4 weeks age were used for this study. The entire animal was housed in well ventilated condition of 12:12 hr light and dark cycle. They were free access of feed and water during before starting the experiment. The experiments were carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India, and approved by the Institutional Animal Ethical Committee at our institute registration no. 1268/PO/E/09/CPCSEA.

 

Preparation of E. hirta extract:

A hundred grams of Euphorbia hirta leaf powder was extracted by maceration in 400mL of methanol for 14 days with frequent agitation. The mixture was filtered through clean muslin cloth followed by double filtration with Whatman No. 1 filter paper and the filtrate was concentrated by rotary evaporator with vacuum at 50℃, poured in glass Petri dishes and brought to dryness at 60 ℃ oven. The percentage yield of the crude extract was determined as 11.1%. An appropriate concentration of the extracts was made in distilled water and with Vaseline for studying antihypertensive effect and effect in treatment of vitiligo respectively⁵.

 

Experimental protocol:

C57BL/6 mice at 4 weeks of age were divided into six groups, 4–7 mice in each group.

Group 1. Group I: Control group topically applied daily respective application of Vaseline,

Group 2. Group II: 40% Monobenzone cream + Vaseline on the 2ҳ2 cm shaved abdomen

Group 3. Group III: Euphorbia hirta extract + Vaseline topically applied

Group 4. Group IV: Punica granatum extract + Vaseline topically applied

 

Animals had the dorsal region shaved, approximately 24 h before start of experiment. Monobenzone 40% was freshly prepared in non-ionic cream was daily applied (50mL, for 50 days) to the dorsal region (2 x 2 cm2) on the same site near the tail. Creams were massaged until completely absorption using a spatula. In a different dorsal region (near the neck, 2 x 2 cm2), Vaseline, or extracts of Punica granatum and Euphorbia hirta 10% (daily, twice a day) was applied and massaged until completely absorption. All treatments were carried out for 65 days. Animals were euthanized after completion of treatment of 65 days, and 6 mm circles of ear tissue were collected. All tail and back skin were removed. Some samples were stored at - 80⁰C for further tests. Same dorsal skin samples were collected and placed in 10% neutral buffered formalin to histological analysis. Samples also were used to evaluate depigmentation, epidermal melanin levels, measurement of TNF-α level and histological analyses (Hematoxylin–eosin and Fontana-Masson staining)⁶.

 

Determination of epidermal melanin levels:

Tail samples were submitted to melanin assessment. Previously, epidermis was separated of dermis with 20m Methylene diamine tetraacetic acid (EDTA) solution (37 1C, overnight). It was added 0.5mL of RIPA buffer (1M Tris buffer, 4M sodium chloride, 20mM EDTA, 1% triton, 0.1% sodium dodecyl sulfate, 0.5% deoxycolate sodium and H2O Milli-Q), homogenized and centrifuged (12,000 x g, 20 min, 4 1C). Supernatants were separated and 20% dimethyl sulfoxide (DMSO) in 1 N sodium hydroxide (NaOH) solution was added to pellet and kept boiling until the pellets dissolved. The melanin levels were determined colorimetrically using a plate reader (EL808; BioTech Instruments, INC) set to measure absorbance at 470nm. Curve of synthetic melanin was used as reference. Concentrations were normalized to total protein content in supernatants, which was determined using Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Results were expressed as melanin concentration (mg/mL)/mg epidermis.⁷

 

Measurement of TNF-α levels:

Amounts of TNF-α in the dorsal tissue homogenates were quantified using ELISA kit (eBioscience, San Diego, USA) according to the manufacturer instructions. Levels of these cytokines in each supernatant were normalized to total protein content, which was determined using Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA)⁷

 

Fontana-Masson staining:

Histological sections of back skin were deparaffinized and hydrated in successive baths of xylene and alcohol and stained with Fontana-Masson. Samples were soaked in the solution of silver nitrate for 1 h in an oven at 56 0C and then washed with distilled water. Next, slices were subjected to gold chloride solution for 10 min and then washed in distilled water. Samples were then reduced in 5% sodium thiosulfate solution for 5 min, washed and dipped in nuclear fast red solution for 5 min. finally they were rinsed in distilled water, dehydrated, and mounted with Canada balsam followed by coverslip⁸.

 

Statistical analysis:

The mean ± SEM values were calculated for each group using GraphPad Prism 5.0. One-way ANOVA followed by Dunnett’s multiple comparison tests were used for statistical analysis. p<0.05 value was considered statistically significant.

 

RESULT:

Effect on melanin content:

Melanin concentration was evaluated on samples from tail skin and all the groups show differences. There was no alteration in melanin content in control group and mice treated with extracts of Punica granatum and Euphorbia hirta 10% topically. 40% monobenzene treated mice showed significant decrease in melanin levels as compared to control group. (Figure-1).

 

Figure: 1 effect of extracts of Punica granatum and Euphorbia hirta 10% topical treatment on melanin content

Values are expressed as mean ± SEM, n=5. All data are subjected to One Way ANOVA followed by Dunnett’s test. * p<0.05 when compared to sham control.

 

Figure: 2 Effect of extracts of Punica granatum and Euphorbia hirta 10% topical treatment on TNF- alpha content

Values are expressed as mean ± SEM, n=5. All data are subjected to One Way ANOVA followed by Dunnett’s test. * p<0.05 when compared to sham control

 

Effect on Effect on TNF alpha content:

Mice treated with extracts of Punica granatum and Euphorbia hirta 10% topically showed significant decrease in TNF alpha levels as compared to control group. 40% monobenzene treated mice showed significant increase in TNF alpha levels. (Figure-2).

 

Effect on Histopathology:

Histology of back skin samples stained with Fontana-Masson and melanin was marked in brown color inside the hair follicles. As observed in representative figures, monobenzone control group presented low melanin levels (A), P. granatum 10% (B), E. hirta topically (C), and tacrolimus (D) groups showed higher amounts of melanin pigment. (Figure-3).

 

Figure 3: Histological evaluation of melanin trough Fontana-Masson staining.

Representative pictures of melanin contents between, control (A), P. granatum 10% (C), E. hirta 10% and tacrolimus (D) groups were evaluated on 65th day. Representative pictures of histological sections from back stained with Fontana-Masson indicated that melanin levels were evidently elevated on P. granatum 10% (B), E. hirta (C) and on reference drug. (200 x, scale 200)

 

DISCUSSION:

Topical treatment with extracts of Punica granatum and Euphorbia hirta (10%) had no effect on melanin levels while TNF-α levels were significantly decreased. Monobenzone-induced vitiligo model has been explained by the structural homology of monobenzone with tyrosine, the natural substrate of melanogenesis. Monobenzone is then converted into a highly reactive quinone by tyrosinase, and the sub products react with surrounding enzymatic compounds within the confines of the melanosomal organelle.⁹ This reaction induces modifications in the melanosomal enzymes and alters their recognition by T cells, which can lead to the break of the immune tolerance¹⁰. Besides, monobenzone is capable to synergize autoimmune and reactive oxygen species mediating melanocyte destruction and vitiligo-like depigmentation.⁸ In the animal model of topical application of monobenzone there is a systemic immune response to autologous melanocytes and similar vitiligo histological features.¹¹

 

Monobenzone induced TNF-α release is a significant tool to study vitiligo. Therefore, Punica granatum and Euphorbia hirta could avoid this enhancement and probably prevent recruitment of T cells in the skin, IL-8 up regulation, apoptosis of keratinocytes, and melanocytes; contributing to preventing the progression of vitiligo. Our results showed that monobenzone enhanced TNF-α level and evaluated Punica granatum and Euphorbia hirta preparations protected from this effect¹². Melanin content and histo-pathology studies also support our results.

 

Oxidative stress is considered one of the key pathogenic events in the loss of melanocytes¹³. Punica granatum and Euphorbia hirta have shown to posses powerful antioxidant activities in vivo and in vitro^14,15. The antioxidant activity of Punica granatum and Euphorbia hirta may be one of the possible mechanisms for their effect in vitiligo. Over the past years, many studies relate between inflammation and vitiligo.¹⁶ There is presence of low intensity inflammatory infiltrate of mononuclear cells between the upper layer of the dermis and epidermis–dermis interface.¹⁷ Micro inflammatory reaction may be the responsible for triggering local warnings of the innate immune system and precedes adaptive immune responses directed to melanocytes, like release of many substances, including pro-inflammatory mediators and proteolytic enzymes, including ROS.^18,19,20

 

REFERENCES:

1.      Badyal DK, Lata H, Dadhich AP. Animal Models of Hypertension and Effect of Drugs. Indian Journal of Pharmacology 2003;35:349-362.

2.      Gharzouli K, Khennouf S, Amira S, Gharzouli A. Effects of aqueous extracts from Quercus ilex L. root bark, Punica granatum L. fruit peel and Artemisia herba-alba Asso leaves on ethanol-induced gastric damage in rats. Phytotherapy Research 1999;13(1):42-45.

3.      Gracious Ross R, Selvasubramanian S, Jayasundar S. Immunomodulatory activity of Punica granatum in rabbits preliminary study. Journal of Ethnopharmacology 2001;78(1):85-87.

4.      Bhowmik D, Gopinath H, Kumar BP, Duraivel S, Aravind G, Kumar KPS. Medicinal Uses of Punica granatum and Its Health Benefits. Journal of Pharmacognosy and Phytochemistry 2013;1(5):30-35.

5.      Gopalasathees kumar K, Ariharasiva Kumar G, Sengottuvel T, Sanish Devan V, Srividhya V. Quantification of Total Phenolic and Flavonoid content in leaves of Cucumis melo var agrestis using UV- spectrophotometer. Asian Journal of Research in Chemistry 2019; 12(06): 1-10.

6.      Zainab I. Mohammed, Ahmed J. Hassan. Effect of Bee venom on gene expression of HSP70 and IL-1β in male rats induced by arthritis in comparison with Prednisolone drug. Research Journal of Pharmacy and Technology 2019;12(11):1-10.

7.      Moreira CG, Carrenho LZB, Pawloski PL, Soley BS, Cabrini DA, Otuki MF. Pre-clinical evidences of Pyrostegia venusta in the treatment of vitiligo. Journal of Ethnopharmacology 2015;168:315-325

8.      Carriel VS, Aneiros-Fernandez J, Arias-Santiago S, Garzón IJ, Alaminos M, Campos A. A Novel Histochemical Method for a Simultaneous Staining of Melanin and Collagen Fibers. Journal of Histochemistry and Cytochemistry 2011;59(3):270-277.

9.      P A Patil, Monika Kolekar, Vikranti Koli, Amruta Kamble^. Evaluation of Sun Protective Formula (SPF) of Indian Cow Dung as Organic Sunscreen Agent. Research Journal of Topical and Cosmetic Sciences 2019; 10(01): 17-25.

10.   Hariharan V, Toole T, Klarquist J, Longley JB, Mosenson J, Le Poole1 IC. Topical Application of Bleaching Phenols; in-vivo Studies and Mechanism of Action Relevant to Melanoma Treatment. Melanoma Research 2011;21(2):115-126.

11.   Van Den Boorn JG, Melief CJ, Luiten RM. Monobenzone-induced depigmentation: From enzymatic blockade to autoimmunity. Pigment Cell and Melanoma Research 2011;24(4):673-679

12.   Miniati A, Weng Z, Zhang B, Therianou A, Vasiadi M, Nicolaidou E. Stimulated human melanocytes express and release interleukin-8, which is inhibited by luteolin: relevance to early vitiligo. Clinical And Experimental Dermatology 2014;39(1):54-57.

13.   Norgauer J, Dichmann S, Peters F, Mockenhaupt M, Schraufst tter I, Herouy Y. Tumor necrosis factor alpha induces upregulation of CXC-chemokine receptor type II expression and magnifies the proliferative activity of CXC-chemokines in human melanocytes European Journal of Dermatology 2003;13(2):124-129.

14.   Colucci R, Dragoni F, Conti R, Pisaneschi L, Lazzeri L, Moretti S. Evaluation of an oral supplement containing Phyllanthus emblica fruit extracts, vitamin E, and carotenoids in vitiligo treatment. Dermatology Therapy 2015;28(1):17-21.

15.   Nataliya N Borisenko, Inna V Bushueva, Volodymyr V Parchenko, Inna Ya Gubenko, Yevhenii О Mykhailiuk, Olha I Riznyk, Oleksandr G Aleksieiev. Anti-Inflammatory, Antiviral Veterinary Medicine with Immuno-Modulating Activity. Research Journal of Pharmacy and Technology. 2019; 12(11): 25-30.

16.   Al-Olayan EM, El-Khadragy MF, Metwally DM, Abdel Moneim AE. Protective effects of pomegranate (Punica granatum) juice on testes against carbon tetrachloride intoxication in rats. BMC Complementary and Alternative Medicine 2014;14:164:1-9.

17.   Bekir J, Mars M, Vicendo P, Fterrich A, Bouajila J. Chemical composition and antioxidant, anti-inflammatory, and antiproliferation activities of pomegranate (Punica granatum) flowers. Journal of Medicinal Food 2013;16(6):544-550.

18.   Tilottama M. Gatkine, Vaishnavi S. Shete , Nilesh M. Mahajan, Ujwala N. Mahajan. Potential of phyto-constituents as a skin tanning agents. Research Journal of Topical and Cosmetic Sciences 2019; 10(01): 10-15.

19.   Wang H., Pan Y., Tang X., Huang Z. Isolation and characterization of melanin from Osmanthus fragrans seeds. Lebensmittel-Wissenschaft and Technologie 2006; 496-502.

20.   P.Kannan and D.Ganjewala. Preliminary Charaterization of Melanin Isolated form Fruits and Seeds of Nyctanthes arbor-tristis. Journal of Scientific Research 2009; 1(3): 655-661.

 

 

 

Received on 31.12.2019           Modified on 16.02.2020

Accepted on 29.03.2020         © RJPT All right reserved