Journal of Dermatological Treatment (2003) 14, 1–3
`# 2003 Journal of Dermatological Treatment. All rights reserved. ISSN 0954-6634
`
`1
`
`The treatment of atopic dermatitis with licorice gel
`
`M Saeedi1, K Morteza-
`Semnani2 and M-R Ghoreishi3
`
`Departments of 1Pharmaceutics and
`2Medicinal Chemistry, Faculty of
`Pharmacy, Mazandaran University of
`Medical Sciences, Sari, Iran;
`3Dermatologist, Sari, Iran
`
`Glycyrrhiza glabra L. has been
`used in herbal medicine for skin
`eruptions,
`including dermatitis,
`eczema, pruritus and cysts. The
`effect of licorice extract as topi-
`cal preparation was evaluated on
`atopic dermatitis. The plant was
`collected and extracted by per-
`colation with suitable solvent.
`The extract was standardized,
`based on Glycyrrhizinic acid by
`using
`a
`titrimetry method.
`Different topical gels were for-
`mulated by using different co-
`solvents. After standardizing of
`topical preparations,
`the best
`formulations (1% and 2%) were
`studied in a double–blind clinical
`trial in comparison with base gel
`on atopic dermatitis over two
`
`in each
`(30 patients
`weeks
`group). Propylene glycol was
`the best
`co-solvent
`for
`the
`extract and Carbopol 934P as
`gelling agent showed the best
`results in final formulations. The
`quantity of glycyrrhizinic acid
`was determined 20.3% in the
`extract and 19.6% in the topical
`preparation. Two percent licor-
`ice topical gel was more effective
`than 1% in reducing the scores
`for erythema, oedema and itch-
`ing over two weeks (p,0.05).
`The results showed that licorice
`extract could be considered as
`an effective agent for treatment
`of atopic dermatitis. (J Dermatol
`Treat (2003) 14: 000–000)
`
`Received 20 September 2002
`Accepted 3 June 2003
`
`Keywords: Atopic dermatitis — Glycyrrhiza glabra — Oedema —
`Erythema — Itching
`
`Introduction
`
`Atopic dermatitis is the cutaneous expression of the atopic
`state, characterized by a family history of asthma, hay
`fever, or dermatitis in up to 70% of patients.1 Hanifin
`summarized four current hypotheses concerning the cause
`of atopic dermatitis in his review: cyclic nucleotide
`dysfunction, the role of superantigen, Ig-E mediated allergy
`to airborne and food allergens, and autoimmunity to self-
`antigen .2 Topical corticosteroid creams or ointments are
`the mainstay of therapy to control the acute flares of atopic
`dermatitis, probably due to their broad immunomodula-
`tory effects.3,4 As these and other therapies have side effects
`it is necessary to evaluate new therapeutic methods. A
`traditional Chinese medication consisting of a mixture of
`several herbs, especially Glycyrrhiza uralensis (a Chinese
`species of licorice) has provided a therapeutic option for
`children with extensive atopic dermatitis.5,6 Chinese
`licorice root (G. Uralensis) which is a staple herb for skin
`disease in east Asia, contains steroid-like substances,
`
`Correspondence:
`M. Saeedi, Faculty of Pharmacy, Taleghani Blvd., P.O.Box: 48175-861,
`Sari, Iran; Tel/Faxz151-3243109. E-mail: saeedi_m_2001@yahoo.co.uk
`
`which, when taken internally, or even applied topically,
`rapidly provide relief.7
`to Eurasia and
`is native
`Glycyrrhiza
`glabra L.
`cultivated in Europe (e.g. Spain,
`Italy, France),
`the
`Middle East (e.g. Syria, Iran, Turkey, Iraq), and Asia.
`Those parts used are the dried roots collected in the
`autumn.8 G. glabra contains substantial amounts of
`flavones,
`such as
`liquiritigenin and liquiritin, and
`triterpenoids, such as glycyrrhetinic acid and glycyr-
`rhizin. Liquiritigenin, disodium glycyrrhetinic acid and
`glycyrrhizin
`have
`been
`shown
`to
`have
`anti-
`inflammatory activity.9–11 Glycyrrhiza glabra has signi-
`ficant anti-inflammatory and anti-allergic activity.
`Glycyrrhizin reinforces cortisol’s inhibition of antibody
`formation, stress reaction, and inflammation. Alcohol
`extract of G. glabra has displayed anti-microbial activity
`in vitro against Staphylococcus aureus, Streptococcus
`mutans, Mycobacterium smegmatis, and Candida albicans.
`The majority of the antimicrobial effects are due to
`soflavenoid components, with the saponins having a
`lesser antibacterial effect.12 This study was designed to
`compare the clinical efficacy of licorice gel (1% and 2%)
`with that of placebo, in patients with atopic dermatitis.
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`

`

`2
`
`M Saeedi et al
`
`The treatment of atopic dermatitis
`
`Materials and methods
`
`Materials
`
`The following chemicals were used as received from the
`suppliers. Methyl and propyl paraben, propylene glycol,
`PEG 200, PEG 300, PEG 400,
`isopropyl alcohol,
`glycerin, triethanolamine, methanol, acetone, ethanol,
`NaOH, NH3,
`formalin (Merck), Carbopol 940 (BF
`Goodrich).
`
`Plant material
`
`Glycyrrhiza glabra L. roots were collected from Shiraz, in
`the south of Iran, in spring 2001. The dried roots were
`powdered so that all
`the material could be passed
`through a mesh size no larger than 0.5 mm.
`
`Extraction procedure
`
`(600 g) were macerated in 1 l of
`Powdered roots
`methanol
`for one day, and the step was repeated
`twice, following by filtration through filter paper. The
`filtrate was evaporated to dryness under
`reduced
`pressure and weighed (122 g, 20.33%).
`
`Glycyrrhizic acid determination
`
`The licorice extract (2 g) was stirred with 20 ml of 3%
`HNO3 in acetone for 1 hour, then filtered and washed
`with 10 ml of acetone. The residue was refluxed with
`20 ml of acetone, filtered again, and this operation
`repeated three times. The combined acetone extracts
`were diluted to 100 ml, and washed down with 40 ml of
`ethanol; 0.9 ml of 30% NH3 was added dropwise to the
`mixture. Ammonium glycyrrhizate was filtered, washed
`2–3 time with acetone (50 ml total), and dissolved in
`25 ml of water; 20 ml of formalin was added and after
`1 min the mixture was titrated with 0.1 N NaOH in the
`presence of phenolphthalein as indicator.13 This method
`was also used for determination of glycyrrhizic acid in
`licorice preparations. The assay was repeated three
`times.
`
`Preparation of the formulations
`
`PEG 200, 300, 400, isopropyl alcohol and propylene
`glycol were used as co-solvent for dried extract and
`propylene glycol was chosen as the best
`levigator.
`Table I shows the constituents of the selected prepara-
`tions. Carbopol 940 was dispersed in preserved water
`(methyl paraben 0.18% and propyl paraben 0.02%) and
`glycerin overnight. The
`extract was dissolved in
`propylene glycol and was added to the polymer
`dispersion and stirred with a double bladed mixer
`(Ika-werk, Germany) at 500 rpm for 10 min, and
`neutralized by triethanolamine to pH 6.4 and then
`
`Formulation*
`
`Composition % (w/w)
`
`Licorice
`extract
`
`Carbopol
`
`Propylene
`glycol
`
`Glycerin
`
`F1
`F2
`F3
`F4
`F5
`F6
`F7
`F8
`F9
`F10
`F11
`F12
`F13
`F14
`F15
`F16
`F17
`F18
`F19
`Placebo
`
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`2
`2
`2
`2
`–
`
`0.30
`0.30
`0.30
`0.40
`0.40
`0.50
`0.50
`0.50
`0.50
`0.75
`0.75
`0.75
`1.00
`1.00
`1.00
`0.25
`0.50
`0.75
`1.00
`0.50
`
`10
`15
`20
`15
`20
`10
`15
`20
`20
`10
`15
`20
`10
`15
`20
`20
`20
`20
`20
`20
`
`5
`5
`5
`5
`5
`5
`5
`–
`5
`5
`5
`5
`5
`5
`5
`5
`5
`5
`5
`5
`
`*Each formulation consists of preserved water (propyl paraben
`0.02% w/w and methyl paraben 0.18% w/w) to 100 g. The
`formulations was neutralized by triethanolamine to pH=6.8.
`
`Table I
`Formulations composition
`
`mixed at 300 rpm for 10 min. The formulations were
`stored at 4, 25, and 40‡C to ensure physical stability
`evaluation for two weeks. Final
`formulations for the
`clinical trial were controlled microbiologically based on
`USP XXIV.
`
`Clinical trial and statistical analysis
`
`The study was a randomized (simple-random sampling),
`double blind, prospective, placebo-controlled trial. The
`primary endpoint of
`the clinical
`trial
`is severity in
`oedema, itching and erythema. On the assumption of an
`overall mean difference of 0.5 units and a standard
`deviation of 0.5 units, 78 patients (26 in each group)
`were required to achieve a power of 95% to reject a null
`hypothesis of equality, applying a two-sided test at the
`5% significance level. With an estimated fraction of 35%
`of the patients being not evaluable, a total of 108
`patients, aged over 15 years, with clinically diagnosed
`mild to moderate degrees of atopic dermatitis
`(1.
`pruritus and scratching, 2. course marked by exacer-
`bation and remissions, 3. lesions typical of eczematous
`dermatitis, 4. personal or family history of atopy, 5.
`clinical course lasting longer than 6 weeks) were
`recruited.1 Before enrollment
`to the study, written
`informed consent was obtained from all patients or
`the parents of those under the age of 18 years. Patients
`receiving systemic or topical steroids, antibiotics, or
`
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`

`M Saeedi et al
`
`The treatment of atopic dermatitis
`
`3
`
`other effective topical therapy within the previous 7
`days, pregnant women, nursing mothers, and patients
`with other
`skin disorders were excluded. Lesions
`location was classified as head and neck,
`trunk,
`hands, and feet.
`The topical preparations (1% and 2% licorice gels and
`placebo) were administered to patients, in three groups,
`three times a day for two weeks. The overall clinical
`response was assessed by the investigator based on effect
`on oedema, itching, erythema and scaling, according to
`the following 4 -point
`scale: absent=0, mild=1,
`moderate=2, and severe=3. Follow up of patients
`ceased after two weeks. ANOVA, followed by Student-
`Newman-Keuls test, was used to determine significant
`differences between groups and p,0.05 was considered
`significant.14
`
`Results and discussion
`
`the
`Licorice contains as its major active ingredient
`triterpene glycoside glycyrrhizin (also known as glycyr-
`rhizic or glycyrrhizinic acid) in concentrations ranging
`from 1% to 24%, depending on sources and methods of
`assay. Glycyrrhizin on hydrolysis yields glycyrrhetinic
`(or glycyrrhetic) acid and two molecules of glucoronic
`acid.8 Thus the standardization of licorice extract and its
`preparations were performed by determination of glycyr-
`rhizinic acid. From the glycyrrhizic acid determination
`conducted according to the previously described
`method, the quantity of glycyrrhizic acid obtained was
`20.3±0.81% and 19.6±0.74% in licorice extract and
`licorice gel respectively. All investigated formulations in
`the clinical trial used the criteria of USP microbial
`limitation.
`On observing the clinical features of atopic dermatitis,
`some of them showed resistance to topical steroids and
`were even exacerbated by application of steroids, so it is
`likely that
`long term topical use of steroids may
`modulate the barrier-immunity function of the skin.
`The present study compared the efficacy of up to two
`weeks treatment with 1% and 2% licorice extract as a
`herbal gel with placebo in patients with a clinical
`diagnosis of atopic dermatitis.
`A total of 108 patients were recruited in the present
`study by a single investigator. 18 patients were
`excluded from the efficacy analyses. Nine patients
`were given systemic antibiotics, five patients were
`pregnant, one patient was a nursing mother and
`three patients were suffering from other skin disorders.
`All of the 90 evaluable subjects complete two weeks
`treatment. Table II
`shows patient characteristics
`in
`several groups.
`the overall clinical responses,
`The assessment of
`relative to baseline, at the end of treatment after one
`and two weeks is shown in Figure 1. Treatment with 1%
`and 2% licorice gel resulted in a statistically significant
`
`Licorice extract
`(1%)
`
`Licorice extract
`(2%)
`
`Placebo
`
`32.7
`16–51
`
`Age
`Mean
`Range
`Sex
`13
`Male
`17
`Female
`Duration of eczema (years)
`Mean
`3.8
`Range
`0.01–25
`Area of eczema
`Head and neck
`Trunk
`Hands
`Feet
`
`8
`3
`18
`1
`
`34.1
`16–49
`
`35.3
`17–53
`
`12
`18
`
`10
`20
`
`3.5
`0.01–21
`
`3.6
`0.01–25
`
`9
`2
`19
`–
`
`7
`2
`19
`2
`
`Table II
`The comparison of patient characteristics
`
`reduction in the scores for erythema after two weeks
`(p,0.05). This effect was not observed for 1% extract
`gel after one week (pw0.05). The 2% licorice gel
`showed more reduction in the scores for erythema than
`1% extract at
`the end of first and second weeks
`(p,0.05). The licorice extract treatment was signifi-
`cantly more effective than baseline in reducing the
`scores for oedema and itching after one week (p,0.05)
`and two weeks (p,0.01), and 2% licorice gel was more
`effective than 1% extract at the end of first and second
`weeks (p,0.05). Treatment with licorice extract was
`not significantly effective in reducing the scores of
`scaling (pw0.05). The effect of 1% and 2% licorice gel
`in reducing in reducing the scores for erythema, oedema
`and itching were significantly more than placebo after
`one and two weeks (p,0.01).
`Baseline evaluation showed that treatment groups
`were well balanced in respect of number per group, age
`and sex distribution, previous eczema treatment and
`severity of signs and symptoms of eczema.
`At
`the end of
`the treatments,
`the reduction of
`erythema scores was 35.02% for 1% licorice extract
`and 60.76% for 2% licorice gel. The reduction of
`oedema scores was 56.64% and 83.76% for 1% and 2%
`licorice gel treatment after two weeks respectively. At
`the end of the treatments, the reduction of
`itching
`scores was 44.1% and 72.53% for 1% and 2% licorice
`gel respectively. A study carried out in 1994 reinforced
`the excellent reputation of Glycyrrhiza glabra in atopic
`dermatitis. Thirty-seven children were given a Chinese
`herbal medicine containing licorice (and some other
`plants) orally. After one year, 18 of the children had
`experienced at least a 90% reduction in their symp-
`toms.6 Another study showed that
`topical steroids
`exacerbated the dermatitis in about one third of the
`patients15 but no side effects were observed in treatment
`with licorice topical gel.
`
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`The Charlesworth Group, Huddersfield 01484 517077
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`
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`

`

`4
`
`M Saeedi et al
`
`The treatment of atopic dermatitis
`
`Figure 1
`Investigator assessment of overall clinical response, A: Erythema scores, B: Oedema scores, C: Scaling scores, D: Itching scores;
`
`Conclusion
`
`Finally we would like to summarize the current approach
`with regard to the management of atopic dermatitis. The
`final goal of
`the treatment
`is to give patients the
`opportunity for improved social activities in their daily
`
`life. Skin care, control of pruritus and exclusion of the
`exacerbating factors are three facets of the therapy and the
`appropriate use of topical licorice preparations in consider-
`ing the patients’ response to this excellent therapeutic tool.
`The use of 2% licorice extract gives satisfactory effects in
`treatment of atopic dermatitis.
`
`References
`1. Swerlick RA, Lawley TJ, Eczema, psoriasis, cutaneous
`infection, acne, and other common skin disorders. In:
`Fauci AS, Braunwald E,
`Isselbacher KJ, Wilson JD,
`Martin JB, Kasper DL, Hauser SL, Longo DL (eds).
`Harrison’s principles of internal medicine, Mc. Grow-Hill:
`New York, 1998, pp: 298.
`
`2. Hanifin JM, Critical evaluation of food and mite allergy
`in the management of atopic dermatitis. J. Dermatol.
`(1997) 24: 495–503.
`topical steroids in
`3. Lane AT, Efficacy and safety of
`pediatric atopic dermatitis.
`J. Eur. Acad. Dermatol.
`Venerol. (1997) 8(suppl.): S24–S27.
`
`JDT: (gamma) JDT24951.3d 3/7/03 13:08:33 Rev 7.51n/W (Jan 20 2003)
`
`The Charlesworth Group, Huddersfield 01484 517077
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`

`M Saeedi et al
`
`The treatment of atopic dermatitis
`
`5
`
`4. Ramsay CA, Savoie JM, Gilbert M, Gidon M, Kidson P,
`The treatment of atopic dermatitis with topical fusidic
`acid and hydrocortisone acetate. J. Eur. Acad. Dermatol.
`Venerol. (1996) 7(suppl. 1): S15–S22.
`5. Sheehan MP, Atherton DJ, A controlled trial of
`traditional Chinese medicinal plants in wide spread
`non-exudative atopic eczema. Br. J. Dermatol. (1992)
`126: 179–184.
`follow up to
`6. Sheehan MP, Atherton DJ, One-year
`children treated with Chinese medicinal herbs
`for
`atopic eczema. Br. J. Dermatol. (1994) 130: 488–493.
`7. Tamir S, Eizenberg M, Somjen D,
`Izrael S, Vaya J,
`Estrogen-like activity of glabrene and other constituents
`isolated from licorice root. J. Steroid Biochem. Mol. Bio.
`(2001) 78: 291–298.
`8. Leung AY, Foster S., Encyclopedia of common natural
`ingredients used in food, drugs and cosmetics, Wiley
`Interscience: USA, 1996, pp: 346–348.
`9. Yuan R, Lin Y, Traditional Chinese medicine: an
`approach to scientific proof and clinical validation.
`Pharmacology & Therapeutics. (2000) 86: 191–198.
`
`10. Khaksa G, Zolfaghari ME, Dehpour AR, Samadian T,
`Anti-inflammatory and anti-nociceptive activity of dis-
`odium glycyrrhetinic acid and hemiphtalate. Planta Med.
`(1996) 62: 326–328.
`11. Pisanty S, Segal R, Glycyrrhizin as a vehicle for
`iodoxuridine.
`J. Clin. Pharm. Ther.
`(1987) 12(Jun):
`165–171.
`of natural medicine. 2nd
`12. Pizzorno J, Textbook
`Macmillan Press: London, 1999, pp: 767–772.
`13. Znakov T, Determination of glycyrrhizic acid in licorice
`root extracts. Med. Prom. SSSR. (1963) 4: 35.
`14. Atakan N, Erdem C, The efficacy, tolerability and safety
`of a new oral
`formulation of Sandimum– Sandimum
`Neoral
`in severe refractory atopic dermatitis. J. Eur.
`Acad. Dermatol. Venerol. (1998) 240–246.
`15. Katayama I, Taniguchi H, Matsunaga T, Yokozeki H,
`Nishioka K, Evaluation of non-steroidal ointment
`therapy for adult type atopic dermatitis. J. Dermatol.
`Sci. (1997) 14: 37–44.
`
`edn.
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