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Effect of cream containing Melia azedarach flowers on skin diseases in children *.

Introduction

The occurrence of skin diseases in developing countries is highly correlated with poor hygiene, over crowding, malnourishment, non-availability of potable water, high temperature and humidity. Further, drugs used to treat them are antibiotics, steroids and sulfonamides, which are not only out of reach of local population in remote areas but also associated with adverse effects like atrophy, telangiectasia, hirsutism and sensitizing dermatitis which are far more troublesome.

Indigenous medicinal plants have been a readily available source of drugs since ancient times and even today almost 50% new drugs have been patterned after phytochemicals. Majority of the population in developing countries and approximately 25% people indeveloped regions use herbal medicine for prevention and treatment of diseases. Recognizing the medicinal significance of indigenous plants, World Health Organization (WHO) in its 1997-guideline states that "effective locally available plants be used as substitutes for drugs. Research work on medicinal plants and exchange of informations obtained will go a long way in scientific exploration of medicinal plants for the benefit of man and is likely to decrease dependence on imported drugs" (Veerappan et al., 2007).

Hence, keeping the miseries of skin patients in view and WHO's recommendations for the use of indigenous medicinal plants as an efficient and readily available substitute of conventional drugs, the present work deals with the clinical trials of newly formulated herbal cream of Melia azedarach L., a plant which has a long history of its utilization to cure skin diseases at folk level.

Melia azedarach Linn., known in vernacular as Bakayn, is native of West Asia and has been naturalized in Pakistan and almost all warm regions of the world (Sastari, 1998). It has been reported to treat dermato-logical disorders like leprosy, scrofula and eruptive skin diseases in folkloric system of medicine (Nadkarni, 1976). Phytochemical imvestigations of plant have resulted in the isolation of hydroxtigaions of plant have resulted in the isolation of hydroxycoumarins possessing synergistic antifungal activity (Carpinella et al., 2003a, b, 2005), [beta]-carboline alkaloids with anti-inflammatory activity (Lee et al., 2000), monoterpens having bacteriostatic properties (Geris dos Santos and Rodrigues-Fo, 2002) and limonoids bearing antifeed ant (Carpinella et al., 2002) and insecticidal (Carpinella et al., 2003a, b) activities. Meliacarpin (Alche et al., 2003) and meliacin (Alche et al., 2002) have been reported as antiviral components of the plant. Antifungal (Paula et al., 2002), (Carpinella et al., 2003a, b) and larvicidal (Wandscheer et al., 2004) activities of M. azedarach extract, along with its utilization in pharmaceutical preparations like disinfecting solution (Pan and Lian, 2005), antimicrobial cream (Prabhudesai et al., 1999) and an ointment for the treatment of tinea pedis (Li and Li, 2004) have recently been published. Present work is the first report of clinical trials with a cream containing extract of M. azedarach flowers against bacterial infections in children. Earlier, there is a report of antibacterial effect of extract of M. azedarach flowers on rabbits (Saleem et al., 2002).

Material and methods

Plant material

M. azedarach flowers were collected from Karachi and identified by plant taxonomist Dr. Surriya and a voucher specimen (63495 KUH) was deposited in the Department of Botany, University of Karachi, Pakistan.

Extraction of plant

Flowers (2.5 kg) were extracted three times with MeOH (61). Extracts obtained were combined and evaporated under reduced pressure to give residue (311g, 12.44%).

HPLC-fingerprint analysis of the MeOH-extract (Fig. 1)

Sample preparation: 0.1 g of the MeOH-extract residue was dissolved under heating in 10 ml MeOH, the insoluble part filtered off and 10 [mu].I of the soluble extract injected into the HPLC-apparatus.

HPLC-data:

Apparatus: Merck HITACHI D-6000A Interface

Merck HITACHI L-4500A Diode Array Detector

Merck HITACHI AS-2000 Autosampler

Merck HITACHI L-6200A Intelligent Pump

Separation LiChroCart[R] 125-4 with LiChrospher[R] column: 60 RP-select B (5 um), Merck

Precolumn: LiChroCart[R] 4-4 with LiChrospher[R] 60 RP-select B, Merck

Solvent A: 10ml 0.1% [H(sub.)3P0(sub.4] (Merck)/11 water

system: (Millipore Ultra Clear UV plus[R] filtered) B: acetonitrile (Merck)

Gradient: 0-5% B in 30min, 5-25% B in lOmin,

total runtime: 40min

Flow: 0.8ml/min

Detection: 205nm

Preparation of cream

Herbal cream was prepared by using 0.2% preservative (citric acid, methyl and propyl parabens), 10% humectant (propylene glycol), 10% emulsifying agent (cetamacragol and cetosteryl alcohol) and 10% methanolic extract of M. azedarach flowers in 234g of distilled water with stirring and heating up to 70 (Degree.[degrees]C. Cetosteryl alcohol was dissolved separately in 130g liquid paraffin under similar conditions. Both aqueous and oily phases were mixed at 70 (degree.) C with continuous stirring and then methanolic extract of M. azedarach flowers was added. Reaction mixture was homogenized by Silverson Mixer (at 2000 rpm) into a creamy form.

Clinical trials

Trials were conducted in four villages surrounding Hamdard University campus and at Shifa-ul-Mulk Memorial Hospital of Hamdard University. Subjects/patients were children up to the age of 10 years. None was admitted to hospital. They were divided into three groups (I-III) for treatment with cream, placebo and neomycin, respectively. Each group comprises 35 patients (either sex) having bacterial infection on any part of body (arms, legs, feet, chin, back, etc.). Infection sizes measured were 105-150 and 450-600 [mm.Sup.2] (Table 1). Each drug was applied twice a day for 15 days. Placebo (containing same composition as that of cream except the extract of M. azedarach) was used as a negative control, while neomycin skin ointment (0.5% w/w Dong Shin Pharmaceuticals Co., Ltd. Korea) as positive control.

[FIGURE 1 OMITTED]

Inclusion criteria

1. Male and female subjects up to the age of 10 years with bacterial skin diseases on any part of body.

2. Suffering with no systemic disease.

3. Strictly follow the instructions and take no other medicine.

Exclusion criteria

1. Subjects with unconfirmed diagnosis.

2. Subjects who could not respond on due date.

3 Subjects older than 10 years.

4. Subject with fungal, viral and other skin disease.

Measurement of infection

Size of infection was measured in [mm.Sup.2] by tracing infection boundaries on a transparent paper before and after treatment. Healing of infection was expressed as percentage reduction of original infection (i.e. size of infection before start of the treatment).

Basis of identifications

Clinical history/observation, available data and laboratory testing of infectious material were used for the diagnosis of infection. For examination, specimens were taken on sterile swab provided by Dr. Essa's Laboratory and Diagnostic Center, D-16, Block-H, North Nazimabad Karachi, Pakistan, where specimen were submitted for culture and sensitivity test.

Statistical analysis

The data are expressed as mean [+ or -] SEM using analysis of variance followed by Student's t-test and one-way ANOVA. Significance was calculated by comparing percentage of healing in group-I (treated with cream) versus group-II (treated with placebo) and group-III (treated with neomycin); and group-III against group-II. The value of p<0.01 and 0.001 were considered significant. The percentage of healing was calculated as a percentage of the corresponding 0 day's (before treatment) infected area ([mm.Sup.2])
Table 1. Healing effect of cream containing Melia azedarach on
bacterial skin infections in children

Group--I (treated with cream)

Infected area ([mm.sup.2])

Before treatment After treatment % of No. of
 healing subject

127.57 [+ or -] 16.59 00 100 15
567.07 [+ or -] 55.37 00 100 12
573.07 [+ or -] 88.34 143.27 [+ or -] 15.27 75 5
503.06 [+ or -] 56.71 265.50 [+ or -] 30.17 50 3

Group--II (treated with placebo)

Infected area ([mm.sup.2])

Before treatment After treatment % Of healing No.of
 subject

130.61 [+ or -] 21.67 00 100 10
545.21 [+ or -] 5.67 00 100 11
500.20 [+ or -] 21.20 400 [+ or -] 30.63 20 7
590.25 [+ or -] 25.12 590.25 [+ or -] 25.12 0 7

Group--III (treated with neomycin)

Infected area ([mm.sup.2])

Before treatment After treatment % Of healing No. of
 subject

129.71 [+ or -] 25.23 00 100 15
560.25 [+ or -] 40.75 00 100 12
587.75 [+ or -] 30.15 146.73 [+ or -] 20.76 75 5
575.2 [+ or -] 38.19 575.21 [+ or -] 38.19 0 3

Singificance is calculated by comparing % of healing in group -1
versus group-II and III; and group III against II. Results were
found significant in Group -1 versus group II (p<0.001) and
group III versus group II (p<0.01).


Results and discussion

Goths (villages) surrounding Hamdard University are extramely deprived of basic facilities. Poor hygiene, non-availability of potable water, malnutrition and illiteracy exist here. This has affected the lives of population especially children badly. poor sanitary conditions have led the youngers to have skin infections mainly due to decteria. However, some funfal infections along with secondary bacterial infections were also observed. Laboratory testing of spectimen showed that in most of the cases the organism responsible for infection was Staphylococcus aureus, while in some cases Escherichia coil, Streptococcus and Klebsiella species were also reported. Bacterial infections treated during present investigations included cellulitis (figs.2a c), pyogenic, chronic pustules and secondary bacterial infections (Figs. 3a--c).

All three drugs, cream placebo and neomyern, showed 100% cure i several cases (Table 1). But, was compared, cream and neomycin were found significantly potent than placebo. student's t-tesst revealed that % of curing infection in group-I (treated with cream) and group-III (treated with neomycin) is significantly higher than group-II (treated with placebo) with p<0.0001 and 0.01, respectively. Though level of significance for healing the bacterial infection appears greater in cream as compared to neomycin however, when percentages of cure between group-I and group-III were compared by student's t-test, no significant difference was seen. One-way ANOVA also supported t-test analysis and showed that at least two of the tested druge have similar potency to treat infections. Hence on the basis of statistical analysis it may be hypothesized that this newly formulated cream has a strong potential to cure bacterial infections in young children; comparable to conventional neomycin skin ointment.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

It would be interesting to note that when antibacterial study of cream, placebom neomycin and methanolic ectract of M. azedarach flowers was conducted against 15 Gram-positive and 12 Gram-negative organisms including staphyloceus arreus, using dise deffusion method no zone of inhibition was observed in case of cream and placebo. However, extract of M. azedarach and neomycin showed potency (data not included here). Since all the infections healed by the cream were caused by some bacterial pathogen and in spite of inactivity in antibacterial testing clinical trials showed potency of the cream comparable to neomycin it may there fore be hypothesized that the cream may stimulate the immune system as an antibacterial agent. However, detailed work to evaluate the immunostimulating activity of the cream and isolation of the active principles of the extractresponsible for the antiinfectious activity are required for future studies.

Acknowledgement

Authors gratefully acknowledge the Hamdard University Karachi, Pakistan for the award of research grant to support research work.

References

Alche, L.E., Barquero, A.A., Sanjuan, N.A., Coto, C.E., 2002. An antiviral principle present in a purified fraction from Melia azedarach L. Leaf aqueous extract retrains herpes simplex virus type 1 propagation. Phytother. Res. 16, 348-352.

Alche, L.E., Ferek, G.A., Meo, M.C., Celia, E., Maier, M.S., 2003. An antiviral meliacarpin from leaves of Melia azedarach L. J. Biosci. 58, 215-219 Chem. Abstr., 2003, 139, 49815.

Carpinella, C., Ferrayoli, C., Valladares, G., Defago, M., Palacios, S., 2002. Potent limonoid insect antifeedant from Melia azedarach. Biosci. Biotech. Biochem. 66, 1731-1736.

Carpinella, M.C., Defago, M.T., Valladares, G., Palacios, S.M., 2003a. Antifeedant and insecticide properties of a limonoid from Melia azedarach (Meliaceae) with potential use for pest management. J. Agric. Food Chem. 51, 369-374.

Carpinella, M.C., Giorda, L.M., Ferrayoli, C.G., Palacious, S.M., 2003b. Antifungal effects of different organic extracts from Melia azedarach L. on phytopathogenic fungi and their isolated active components. J. Agric. Food Chem. 51, 2506-2511.

Carpinella, M.C., Ferrayoli, C.G., Palacios, S.M., 2005. Antifungal synergistic effect of scopoletin. A hydroxycoumarin isolated from Melia azedarach L. fruits. J. Agric. Food Chem. 53, 2922-2927.

Geris dos Santos, R.M., Rodriguese-Fo, E., 2002. Meroterpenes from penicillium sp found in association with Melia azedarach. Phytochemistry, 907-912.

Lee, B.G., Kim, S.H., Zee, O.P., Lee K.R., Lee, H.Y., Han, J.W., Lee H.W., 2000. Suppression of inducible nitric oxide synthase expression in RAW 264.7 macrophages by two [beta-carboline] alkaloids extracted from Melia azedarach. Eur. J. Pharmacol. 406, 301-309 Chem. Abstr., 2001, 134, 80795.

Li, X., Li, M., 2004. External use of inunctum for treating tinea pedis and its preparation. Faming Zhuanli Shenqing Gongkai Shuomingshu, CN 1,355,040. Chem. Abstr. 140, 380610.

Nadkarni, K.M., 1976. The Indian Materia Medica. Popular Parkashan, Bombay, pp. 784-785.

Pan, X., Lian, Y., 2005. Disinfecting cleaning solution made from Chinese medicine extract. Faming Zhuanli Shenqing Gongkai Shuomingshu, CN 1,458,257. Chem. Abstr. 142, 341880.

Paula, P.M., Berra, A., Coto, C.E., Alche, L.E., 2002. Therapeutic action of Meliacine, a plant derived antiviral on HSV-induced ocular disease in mice. Exp. Eye Res. 75, 327-334. Chem. Abstr., 2003, 138, 313971.

Prabhudesai, V.R., Bijlani, N.S., Nambudiary, M.E.N., Gore, V.K., Sinkar, V.P., 1999. A process for preparing antimicrobial creams of plant extract with a-hydroxy acids. Indian IN 183-156 (CI. A61K31/00) 25 September, Chem. Abstr., 2004, 140, 327038.

Saleem, R., Ahmad, S.I., Shamim, S.M., Faizi, S., Siddiqui, B.S., 2002. Antibacterial effect of Melia azedarach flowers on rabbits. Phytother. Res. 16, 762-764.

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Rubeena Saleem (a),*, Rizwana Rani (b), Muhammad Ahmed (c), Farzana Sadaf (c), Syed Iqbal Ahmad (c), Navaid ul Zafar (d) Sobia Sajida Khan (e) Bina Shaheen Siddiqui (e) Lubna (e) Farheen Ansari (f) Shakeel Ahmed Khan (f) Shaheen Faizi (e)

(a) Faculty of Pharmacy, Hamdard University, Karachi 74600, Pakistan

(b) Hamdard Al-Majeed College of Eastern Medicine and Surgery, Pakistan

(c) Dr. HMI Institute of Pharmacology & Herbal Sciences, Hamdard University, Karachi 74600, Pakistan

(d) Hamdard Laboratories, Nazimabad No.3, Karachi, Pakistan

(e) HEJ Research Institute of Chemistry, University of Karachi, Pakistan

(f) Department of Microbiology, University of Karachi, Karachi-75270, Pakistan

* Ethical declaration: It is stated that studies on human subjects have been conducted strictly according to the internationally accepted principles and taking into consideration the ethical values mentioned in Helsinki Declaration of 1964 (revised in 2004). Since all the volunteers were up to the age of 10, their parents were informed about the efficacy and safety of drugs under trial. However, herbal practitioner involved in this project was fully aware of her responsibilities as a doctor for the welfare and benefit of human subjects and accepted to work mainly due to two reasons. First, plant used in the formulation of cream is already used to treat skin diseases in herbal system of medicine, and chemicals used in preparation of cream and placebo are commonly used items in such preparations and are generally harmless, Second, WHO in its 1997-guidelines, permitted the use of appropriate medicinal plant as a substitute drug.

* Corresponding author.

E-mail address: rs127pk@yahoo.com (R. Saleem).
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Author:Saleem, Rubeena; Rani, Rizwana; Ahmed, Muhammad; Sadaf, Farzana; Ahmad, Syed Iqbal; Zafar, Navaid ul
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Apr 1, 2008
Words:2611
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