Objective Glycyrrhiza glabra is well known herb in Indian and Chinese traditional medicines.In our previous study it shows hair grwth promoting property in female rats so here experiments were performed for evaluating its effects in androgenic alopecia in males.MethodIn present study alopecia is induced in three groups(each contain six animals) of male wistar albino rats by intramuscular dose(0.1mL) of testosterone .One group was rendered devoid of any other treatment while other two groups animals are Â treated with finasteride and petroleum ether extract of G. glabra root Â topically once daily. The animals were observed during treatment period of 21 days then one animal from each group wasÂ  euthanized for histoarchitecture Â study.Results and discussionThe study revealed that petroleum ether and finasteride treated animals do not developed alopecia while alopecia was observed in only testosterone treated animals.ConclusionThus it is concluded from this study that petroleum ether extract of G. glabra possesÂ  anti androgenic alopecia activity which is comparable toÂ  that of standard drug finasteride

SUKIRTI, UPADHYAY

VIJENDER, SINGH

English

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   Research Article  POTENTIALITY OF PETROLEUM ETHER (60-80) °C EXTRACT OF GLYCYRRHIZA GLABRA ON ANDROGENIC ALOPECIA 1School of Pharmaceutical Sciences, IFTM University, Moradabad, UP. 2 BBS Institute of Pharmacy and Allied Sciences,Greater Noida India            Email: sukirtiupadhyay@gmail.com Received: 14 June 2013, Revised and Accepted: 31 July 2013 ABSTRACT Objective: Glycyrrhiza glabra is well known herb in Indian and Chinese traditional medicines. In our previous study it shows hair growth promoting property in female rats so here experiments were performed for evaluating its effects in androgenic alopecia in males.  Methods: In present study alopecia is induced in three groups(each contain six animals) of male wistar albino rats by intramuscular dose(0.1mL) of testosterone .One group was rendered devoid of any other treatment while other two groups animals are  treated with finasteride and petroleum ether extract of G. glabra root  topically once daily. The animals were observed during treatment period of 21 days then one animal from each group was  euthanized for histoarchitecture  study.  Results: The study revealed that petroleum ether and finasteride treated animals do not developed alopecia while alopecia was observed in only testosterone treated animals.  Conclusion: Thus it is concluded from this study that petroleum ether extract of G. glabra  posses  anti androgenic alopecia activity which is comparable to  that of standard drug recommended for androgenic alopecia finasteride. Keywords: Hair, alopecia,G.glabraINTRODUCTION Male pattern baldness or Androgenetic alopecia (AGA) term consists of two terms Androgen (Andro) and genes (genetic).It run in families. It is inherited condition associated with a gene or genes. Both the testosterone metabolite DiHydroxyTestosterone and the genes for hair loss must be present for AGA to occur. The hair loss is heritable, androgen-dependent, and occurs in a defined pattern. It is assumed that the genetically predisposed hair follicles are the target for androgen-stimulated hair follicle miniaturization, leading to gradual replacement of large, pigmented hairs (terminal hairs) by barely visible, depigmented hairs (vellus hairs) in affected areas. [1] In US 35 to 40 million are affected by AGA. It affects at least 50% of men by the age of 50 years, and up to 70% of all males in later life. [2] Glycyrrhiza glabra, licorice is a well known herb in traditional Indian and Chinese medicine cultivated in Jammu and Kashmir, Punjab and Sub-Himalayan tracts. It is known as  Yasthi-madhu in Sanskrit, Jethi-madh in Hindi,  Jashtimadhu in Bengali, and Liquorice in English. It is a hardy herb or undershurb, attaining a height of 1.8m. Roots are thick, having many branches with red or lemon colour outside and yellowish or pale inside.  The main chemical constituents found are Glycyrrhizin, glycyrrhizic and glycyrrhetinic acids, liquiritin, isoliquitirin, neoisoliquiritin, liquiritigenin, isoliquiritigenin, rhamnoliquiritin, glabrine, glabranine, formononetin, licuraside, lichalcones a and b, hispaglabridin A and B, licoricidin, glabrene, pinocembrin, prunetin, saponeretin, 11-deoxoglycyrrhetic and 24-hydroxyglycyrrhetic acids, 24-hydroxyliquiritic and liquiridolic acids, olean-12-en-3beta-ol-3o-oic and olean-11,13(18)-dien-3beta-ol-3o-oic acids, methylolean-11,13 (18)-diene-3, 24-diol-3o-oate, glycyrrhetol2, 21alpha-hydroxy-isoglabrolide, glabrolide, deoxoglabrolide, deoxoglabrolide, isoglabrolidde, licoflavonol. kumatakenin, glycyrol, licoricone, glabridin, glabrol, 7-acetoxy-2-methylisoflavone, 7-methoxy-2-methylisoflavone, 7-hydroxy-2-methylisoflavone, glyzarin, glyzaglabrin, licoisoflavones A, B and licoisoflavone, glycyrin, sugars and aspaargin are also reported in this plant. [3] The roots are prescribed in coughs, hoarseness and in respiratory trouble, mixed with citrus juice efficacious in catarrhal affections and with honey in jaundice; in combination with ginger and milk, act as a good tonic during convalescence, infusion, decoction and extract is laxative and a useful medicine in urinary disease, bronchial and gastric troubles. [4] G.glabra is also known for promoting hair growth in traditional and folklore medicines .It is one of the ingredient of Sesa oil which is recommended for hair growth promotion. [5] Hence, experiments were performed for evaluating effects of G.glabra in androgen induced alopecia. MATERIAL AND METHOD  Plant material   The plant material was collected by Jim corbett national park, Ramnagar, Uttra   khand , India. It was authenticated by Dr DV Amla Scientist G, NBRI,   and voucher no NBRI-SOP-202 was provide   to it and it was kept there for further references. Extraction  Plant material was extracted  in soxhlet apparatus with petroleum ether (60-80) °C for eighteen hours. Then the extract was concentrated under vaccum. The yield was 1.6% Phytochemical screening The extract was evaluated for presence of   various phytochemicals by standard procedures.  Preparation of various dosage forms Marketed preparation of testosterone Testoviron depot (German Remedies) (1 mL) was diluted up to 5 mL with water for injection this was able to produce the concentration of 5 mg/mL. The 2% standard  solution of crushed finasteride tablet (Fincar, Cipla Ltd. )solution and 1% extract solutions was prepared in vehicle (ethanol: propylene glycol: water in ratio of 8:1:1). Animals   Vol 6, Suppl 3, 2013                                                                                                                                                                                                         ISSN - 0974-2441                                                                                                                                                                                                 UPADHYAY SUKIRTI *1, SINGH VIJENDER 2 Upadhyay et al. Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013,  52-55 53  Male albino rats were used of weight 120-150 g, 3-4 month age were used for studies. The animals were handled according to CPCSEA Guidelines of Good Laboratory Practice. [6] The research protocol of the animal experimentation (Reg no. 837/ac/04/CPCSEA; Resolution no. 05/837ac/PH/10 of December 12, 2010) was approved by the ‘Institutional Animal Ethical Committee’ of College of Pharmacy, IFTM, Moradabad- 244001, Uttar Pradesh, India. The rats were placed in cages and kept in standard environmental conditions of 12h light and 12h dark cycle, 23 ± 2 oC and 35 – 60 %RH. They were fed with standard diet ad libitum with free access to water. Preliminary skin irritation test This test was carried out by protocol mention in ASTM. [7] The petroleum ether  extract of G. glabra applied in a concentration of up to 10%for seven days on shaved skin surface of wistar rats, didnot show any irritation or erythema on skin surface. Thus the prepared extracts were considered safe for topical administration . [8] Experimentation  The method reported by Matias et al. [9] was followed with slight modification. In brief, the rats were divided in four groups of six rats each. Rats of all the groups were administered testosterone dose (0.1 mL) intramuscularly. Animals of group 2, 3  were also given topical application of 0.4 mL finasteride and petroleum ether extract of root respectively on dorsal skin surface once a day for 21 days . After this period, one rat from each group were selected randomly and sacrificed. The difference in growth of hair in each group was noticed by visual observations and was recorded by photographs. Skin biopsy was also undertaken from balding site and the cyclic phase of hair follicles (Anagen,Telogen) and follicular density was determined with the help of ocular micrometer. RESULTS AND DISCUSSIONS  Phytochemical screening The petroleum ether extract of G. glabra  shows  presence of Glycosides, terpenoid, phenolics and flavonoids.Its Chromatographic characterization shows presence of twelve compounds using mobile phase Toluene: Ethyl acetate in 97:03 ratio and derived with Libbermenn burchard reagent.(Fig 1A)  Fig.1A:  Separation bands of Petroleum ether extract on thin layer chromatogram In vivo hair growth studies against testosterone induced alopecia Qualitative study Alopecia proceeds via cranial to caudal region in rodents. [10]The animals of group 1 showed diffuse alopecia. Loss of hair from dorsal portion of rat was clearly visible after 15 days treatment with testosterone. The observation was better in animals of group 2; no signs of alopecia were developed. In animals of group 3 the alopecic conditions was not visible and showing that the extract successfully prevented and blocked testosterone induced hair loss. ( Figure 1, 2 and 3).   Fig 1  Fig 2  Fig 3 Fig 1. . The animal shows hair loss from cranial reason and hair become thin on dorsal skin, i.e. alopecia condition is visible in group 1 animal due to testosterone administration; Fig 2. Animals of group 2 do not develop alopecia on 21 days treatment with testosterone due to simultaneous treatment with finasteride;  Fig 3. Animals of group 3 do not become alopecic as petroleum ether extract of root combat testosterone effect;  Quantitative study In the present experiment alopecia was induced in rat by administration of testosterone. Testosterone is required, along with a genetic predisposition, for androgenetic alopecia to develop in men. [11] Microscopic examination of skin sections of group 1 animals revealed that testosterone treatment cause miniaturization of hair follicles. The follicles had bulbous appearance and were short (Figure 4). Several hair follicles were in telogen phase. In skin sections of group 2 and 3 animals the effect of testosterone on miniaturization of hair follicle was blocked by administration of topical finasteride and petroleum ether extract of root. The number of follicles in anagen phase was considerable and fewer follicles in telogen phase were observed. A/T ratio was significantly affected by finasteride and extracts which was observed in skin sections of various groups (Table 1)(Figure 4, 5 and 6).The follicular density i.e. no of follicles in mm of skin surface. The follicular density also showed that both seed extracts treated animal had denser hair follicle (Table 1).  Upadhyay et al. Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013,  52-55 54   Fig 4   Fig 5  Fig 6 Fig 4. Skin section of Group 1 animals showed maximum telogenic hair follicle leading to alopecia; Fig. 5. Skin section of Group 2animal shows hair follicles in anagenic (hair growing stage); Fig 6. Skin section of Group 3 animal shows more hair follicles in anagenic stage than finasteride treated group 2 animals; Table 1 Grouping and treatment of animals and variation in there Anagenic and Telogenic hair percent Group  Telogen Anagen  A/T Follicle density 1 83.1±0.5667 16.9±0.5667 0.2034 1.0±0.3371 2 52.4±0.6864*** 47.6±0.6864*** 0.9084 2.5±0.3128 3 34.0±0.4714*** 66.0±0.4714*** 1.9412 2.9±0.2599 Values are mean ± SEM, n=10, *p<0.05, **p<0.01, ***p<0.001, significance Vs control DISCUSSIONS  Roots of G.glabra have there value in traditional system of medicines since ancient time . Recently hair growth promoting activity of roots are explored. [12] Here it was investigated for its effect in antagonizing testosterone effect    in testosterone induced alopecia. Previously studies were conducted which shows that herbs able to reduce serum testosterone level are used in androgenic alopecia. [13]  The roots of  G.glabra is able to reduce serum testosterone level.[14,15] Previous studies shows that testosterone along with 5 Dihydroxy Testosterone is required for androgenic alopecia to occur. Phytosterols that may inhibit the conversion of testosterone into the more active dihydrotestosterone via inhibition of some17ß-HSD isozymes. Licorice (Glycyrrhiza glabra) is reported to inhibit some of the steroid dehydrogenases. [16] So it is useful in androgen driven disorders like androgenic alopecia, acne, Benin prostrate hyperplasia and prostrate cancer and polycystic ovary syndrome etc. The topical application of extract on scalp is able to combat the androgenic  alopecia .  Acknowledgements One of the author, SU, is thankful to Prof R Somvansi of IVRI, Izatnagar for help in histopathological studies and Managing director R M Dubey for allowing part of this work to performed in IFTM. REFERENCES 1. Paus R, Costeralis G. The biology of hair follicles. N Engl J Med 1999 341: 493-7. 2. Norwood  T. Male-pattern baldness. Classification and incidence. South Med. J 1975; 68: 1359–70. 3. Chopra R, Nair S, Chopra I. Glossary of Ind Med. Plants. Council of scientific and industrial  research New Delhi 1956:126.  4. Chatterjee A , Pakrashi S. The  treatise of Indian medicinal plants. National Institute of Sci Commun, New Delhi 1992; pp 67-180.  5. Shrivastava M . Efficacy and safety evaluation of sesa oil vs coconut oil in different hair & scalp ailments: prospective, open label, randomized comparative study Asian J of Pharm Clin Res 2012; 5( 4):62-64.  6. Ministry of Environment and Forests, Government of India Public information: Good Practices:CPCSEA guidelines [Cited 2012 May 26].Available from: http://moef.nic.in 7. A.S.T.M. Standard Practice for testing biomaterials in rabbits for primary skin irritation, A.S.T.M. designation F719-81; Philadelphia: American Society for Testing of Materials 1998; pp 178–179. 8. Roy  R, Thakur M, Dixit VK . Hair growth promoting activity of Eclipta alba in male albino rats. Arch Dermatol Res 2008; 300: 357-364. 9. Matias J, Malloy V, Orentreich N . Animal models of androgen-dependent disorders of the pilosebaceous apparatus. 1. The androchronogenetic alopecia (AGA)mouse as a model for male-pattern baldness. Arch Dermatol Res 1989 281: 247-53. 10. Parthasarathy S, Malloy V, Matias J, Massardo S, Bariether L,Kotes J, Orentreich N. The in-vitro transformation of testosterone and the ability of its 5α-reduced 17 beta hydroxylated metabolites, to induce hair loss in the androchronogenetic alopecia (AGA) mouse (abstract). J Invest Dermatol  1992:98: 4. 11. Hamilton JB. Male hormone stimulation is a prerequisite and an incitant in common baldness. Am J Anat   1942:71: 451-480. 12. Upadhyay S, Ghosh A K and  Singh V. Hair Growth Promotant Activity of Petroleum Ether Root Extract of Glycyrrhiza Glabra L (Fabaceae) in Female Rats Trop J Pharm Res, October 2012;11 (5): 753-58. 13.  Josephs RA, Guinn JS, Harper LM, Askari F.Liquorice consumption and salivary testosterone concentrations. Lancet 2001;358: 1613–1614. 14. Upadhyay S ,Ghosh A K , Singh V and Dixit V K Effect of petroleum ether and ethanol fractions of Abrus precatorius on androgenic alopecia. Revista Brasileira de armacognosia,Brazilian Journal of Pharmacognosy 2012.22(2): 359-363. Upadhyay et al. Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013,  52-55 55  15. Yaginuma T, Izumi R, Yasui H, Arai T, Kawabata M.Effect of traditional herbal medicine on serumtestosterone levels and its induction of regular ovulation in hyperandrogenic and oligomenorrheic women (Article in Japanese).Nippon Sanka Fujinka Gakkai Zasshi 1982;34(7): 939-944.  16. Spires TE, Fink BE, Kick EK, You D, Rizzo CA, Takenaka I, Lawrence RM, Ruan Z, Salvati ME, Vite GD, Weinmann R, Attar RM, Gottardis MM, Lorenzi MV. Identification of novel functional inhibitors of 17beta-hydroxysteroid dehydrogenase type III (17beta-HSD3). .Prostate 2005 ; 65(2):159-70.                                       

POTENTIALITY OF PETROLEUM ETHER (60-80) Â°C EXTRACT OF GLYCYRRHIZA GLABRA ON ANDROGENIC ALOPECIA

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POTENTIALITY OF PETROLEUM ETHER (60-80) Â°C EXTRACT OF GLYCYRRHIZA GLABRA ON ANDROGENIC ALOPECIA

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