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C4f127fd24e13834f7c45260eaa Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 8438101, 8 pages https://doi.org/10.1155/2018/8438101 Research Article Inhibitory Effects of Glycyrrhiza glabra and Its Major Constituent Glycyrrhizin on Inflammation-Associated Corneal Neovascularization Syed Luqman Shah,1 Fazli Wahid ,2 Noorullah Khan,1 Umar Farooq,1 Abdul Jabbar Shah ,1 Shah Tareen,2 Fiaz Ahmad,3 and Taous Khan 1 1 Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan 2Biotechnology Program, Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan 3Department of Pathology, Ayub Medical College, Abbottabad 22060, Pakistan Correspondence should be addressed to Fazli Wahid; [email protected] and Taous Khan; [email protected] Received 11 January 2018; Accepted 14 March 2018; Published 23 April 2018 Academic Editor: Jae Youl Cho Copyright © 2018 Syed Luqman Shah et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Glycyrrhiza glabra L. (Leguminosae) is widely used in folk medicines. Glycyrrhizin, an active compound of G. glabra,possesses anti-infammatory activity. Tis study investigates the G. glabra methanol extract and glycyrrhizin for the treatment of corneal neovascularization (CNV). G. glabra was extracted in 70% aqueous methanol. Phytochemical tests, thin layer chromatography (TLC), and high performance liquid chromatography (HPLC) were used for the analysis of chemical composition. Te topical solution of G. glabra methanol extract (2% w/v) and glycyrrhizin (1% w/v) was prepared in normal saline. Afer corneal burn (1 N NaOH), animals were lef untreated for a week so that neovascularization appears in all groups. Treatments started on day 7 and continued for next 21 consecutive days. Te animals were treated with 3 drops of various topical solutions thrice a day. Digital photograph analysis and histological studies were used for the evaluation of CNV. Phytochemical analysis of the G. glabra methanol extract showed the presence of saponins, phenols, carbohydrates, favonoids, and proteins. TLC and HPLC confrmed the presence of glycyrrhizin. Photograph analysis of the extract and glycyrrhizin treated group showed a considerable decrease in CNV. Histological study of G. glabra andglycyrrhizintreatedgroupsshowednobloodvesselswithproperlyarrangedcollagen fbers. Tis study showed that G. glabra and glycyrrhizin can be used for the treatment of CNV. Bioassay guided isolation can lead to preparation of ophthalmic solutions for the treatment of CNV. 1. Introduction treatment of diferent diseases [4]. G. glabra leaves are used for the treatment of wounds [5] roots for diabetes, Graves’ dis- Glycyrrhiza glabra L. (Leguminosae) [1] is native to the Medi- ease, and fatulence and stem for the treatment of tuberculosis terranean region, central to Southern Russia and Asia, and [6]. It is also used as an aphrodisiac [7]. Te methanol extract now is widely cultivated throughout Europe and Middle East. of the aerial parts of G. glabra showed antimicrobial activity Licorice has a high nutritional value and was used in foods against several bacterial species [8]. Te aqueous methanol since ancient times. In food, it is mainly used as a sweetening extract of G. glabra inhibited in vitro and in vivo proliferation agent. It also has properties to inhibit the sensation of thirst ofEhrlichascitestumorcellsandshowedantiangiogenic [2]. Licorice oil is approved by the Food and Drug Adminis- activity in in vivo, peritoneal, and chorioallantoic membrane tration (FDA) and has applications in various food products assays[9].Licorice also showed antiplatelet aggregation efects such as beverages, toothpaste, chewing gum, and cosmetics [10] and has also been used as a cough-relieving medicinal [3]. G. glabra has been widely used in folk medicine for the herb from ancient times [4]. Chinese licorice roots were 2 Evidence-Based Complementary and Alternative Medicine found to inhibit the growth of Plasmodium falciparum and treatment of CNV. It was found that the G. glabra extract Leishmania donovani in in vitro studies [11]. Moreover, lico- efectively halted CNV but glycyrrhizin was found to be rice has been reported for having antibacterial and antiviral relatively less efective in stopping the development of CNV activities [12]. in animal model. G. glabra contains several chemical constituents, such as saponin, favonoids, isofavonoids, stilbenoids, and coumar- 2. Materials and Methods ins. Te active constituents in saponins are glycyrrhizin, liquiritic acid, and glycyrretol. In favonoids, the active con- 2.1. Plant Material. G. glabra roots were purchased in June stituents include liquirtin, liquiritigenin, and neoliquiritin. In 2015 from an authentic herbal store in Abbottabad, Pakistan. isofavonoids, they are glabridin, glabrone, glyzarin, and gal- Te identifcation of the specimen (voucher number gg-09- brene. Te active constituents in coumarins are liqcoumarin R/15) was confrmed by Dr. Abdul Nazir, Assistant Professor, andumbelliferone[13].Finally,instilbenoids,theactivecon- COMSATS Institute of Information Technology, Abbottabad, stituent is dihydrostilbenes [14]. Glycyrrhizin is a potassium Pakistan, and plant specimen was deposited to Department of and calcium salt of glycyrrhizinic acid. It is a saponin glyco- Pharmacy, COMSATS Institute of Information Technology, side which upon hydrolysis yields glycyrrhetinic acid [15]. Abbottabad, Pakistan. Glycyrrhizin, also known as glycyrrhizic acid, is the major (10 to 25%) active constituent of G. glabra root extract [16]. Gly- 2.2. Chemicals. Glycyrrhizin (purity ≥ 75%) was purchased cyrrhizin helps in inhibition of lung cancer and fbro sarco- from Sigma Aldrich, USA. Ketamine HCl (Indus Pharma, mas[17].InJapan,ithasbeenusedforthetreatmentofHep- Pakistan),xylazineHCl(FARVET,Peru),proparacaineHCl atitis C for more than 60 years [18]. Furthermore, glycyrrhizin (Alcon Laboratories, Inc., USA), and normal saline (Otsuka exhibited proapoptotic properties in a hepatocyte model of Pakistan Ltd.) were purchased from a local pharmacy store. cholestatic liver injury. Glycyrrhetinic acid was found to be a potent inhibitor of bile acid-induced apoptosis and necrosis [19]. Members of the favonoids like isoliquiritigenin and lic- 2.3. Processing and Extraction. Te dried plant material was ochalcone have been found to possess antioxidant, antitumor, ground and extracted by soaking powdered material (745 g) anti-infammatory, and antiangiogenic activities [20, 21]. in 70% aqueous methanol (3 L) at ambient temperature. Te Te cornea is a transparent, avascular, and outer tissue mixture was occasionally stirred with a stainless-steel rod of the eye. Its transparency is necessary for clarity of vision. for two weeks to get the maximum extract. Te extract was In normal conditions, corneal avascularity is maintained by a fltered through a muslin cloth followed by Whatman flter balance between angiogenic and antiangiogenic factors. Te papernumber42(125mm).Teextractwasconcentrated using a vacuum rotary evaporator (Yamato Rotary Evapora- shif of this balance towards angiogenic factors causes corneal ∘ neovascularization (CNV) [22]. CNV is a pathologic condi- tor, RE 801; South Korea) with the water bath set at 40 C. Te tion in which the transparency of cornea is lost due to the fnal percentage yield of the extract was 12.2%. ingrowth of new blood vessel from the limbus region of the eye. Development of CNV leads to a reduction in corneal 2.4. Phytochemical Analysis and Fingerprinting. Te crude transparency and consequently causes visual impairment extract was subjected to preliminary phytochemical analysis usually loss of the central vision or can even result in blind- forallmajorchemicalconstituentsusingstandardchemical ness [23]. tests [29, 30]. For alkaloids, the extract (500 mg) was shaken Although the exact cause of CNV is not yet completely with5mLof1%HClandheatedgentlyfor1minusingawater identifed, several pathological conditions such as infamma- bath. Next, 1 mL of this solution was taken and 0.5 mL of tion, infection, degeneration, and traumatic disorders may Wagner’s reagent was added. Te development of turbidity or induce CNV. Uses of contact lenses also induce hypoxia, precipitates indicated the presence of alkaloids. For steroids, which may ultimately leads to CNV. Among these factors, Salkwoski’s test was used in which 2 mL chloroform was infectious diseases of the cornea are the most important cause added to dissolve 100 mg of extract in a test tube. Ten, 2 mL of CNV [24]. Currently, the treatment options for CNV in- of the concentrated H2SO4 was carefully added to form the clude medications like nonsteroidal anti-infammatory drugs lower layer. A green color formed on the upper layer indicat- (NSAIDs), steroids, and cyclosporine [25, 26], laser therapies ing the presence of steroids. For saponins (froth test), 3 mg of like thermal argon laser photocoagulation [27], amniotic extract was dissolved in 10 mL distilled water and vigorously membrane transplantation, and limbal transplantation [28]. shaken in a test tube and allowed to stand for 1 min. Te However, all the available treatments have disadvantages such development of consistent froth indicated the presence of as high cost, low efcacy, and severe side efects. saponins. For favonoids (lead acetate
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