Clinical Evaluation of Melatonin Alone and in Combination with Pizotifen in the Prophylaxis of Migraine Iraqi J.Pharm.Sci., Vol.16 (1) ,2007 Anise-Thyme Lotion for Chicken Pox. 8 Preparation of Anise and Thyme Lotion for Topical Use Issraa R. Abed Al- Rahman* ,1 , Fatima J. Jawad* , Ekbal H.Alkateeb* and Alaa A. Abdulrasool* *Department of Pharmaceutics, College of Pharmacy, Baghdad University, Baghdad- Iraq. Abstract : Anise and thyme crude extract were used to prepare a lotion for topical application due to their antimicrobial, germicidal and antifungal effects. Two formulas were prepared using the mentioned natural plants, formula 2 (selected lotion) was the most acceptable one which contained veegum and xanthan gum as suspending agents in addition to other exceipients providing it good properties with high physical stability because of its flocculating, pouring, resuspending easily with sedimentation volume (F) 0.96. In addition to unchangeable odor and color with expiration date of one year. While the Preliminary clinical study was done using this lotion on 10 patients with infecious viral skin diseases, it was found that this lotion was successful as a topical preparation for allergy and mostly chicken pox. Key words: Anise, Thyme, Crude Extract, Antimicrobial Effect. ة:ـــــالخالص أسخخذو انًسخخهض انخاو نًادحٙ انُٛسٌٕ ٔانضعخش نخحضٛش غسٕل نالسخعًال انخاسجٙ بسبب فعانٛت حهك انًٕاد فٙ قخم انبكخشٚا، انجشارٛى غسٕنٍٛ باسخخذاو َفس انًٕاد انُباحٛت انخاو، كاٌ انغسٕل انزاَٙ انًحضش )انظٛغت انًخخاسة( ْٕ األكزش قبٕالً حى ححضٛش ٔانفطشٚاث. الحخٕائّ عهٗ يادحٙ انضاَزاٌ ٔانفٛكاو يع يٕاد اخشٖ يضافت ٔانخٙ اعطج طفاث جٛذة نٓزا انغسٕل حٛذ كاٌ راث رباحٛت فٛضٚأٚت عانٛت . اضافت انٗ عذو حذٔد حغٛش فٙ نَّٕ ٔسائحخّ يع يذٖ طالحٛخّ 0..6دِ بانشس ٔنّ حجى حشسٛب نكَّٕ ال ٚخكخم ٔٚعاد حٕصٚع يٕا ايا بانُسبت نهذساست انسشٚشٚت االٔنٛت ٔانخٙ حًج باسخخذاو ْزا انغسٕل عهٗ عششة اشخاص يظابٍٛ بانخٓاباث نالسخعًال نًذة سُت ٔاحذة. ل انخاسجٙ نهحكت ٔخظٕطاً يشع انجذس٘ انًائٙ.جهذٚت فاٚشٔسٛت. فقذ اربج َجاحّ كًسخحضش نالسخعًا Introduction : Volatile oils are contained largely in the plant, they are secreted in oil cells, in secretion duct or cavities or in glanular bairs. (1) These oils are used for their non-therapeutic action; flavoring e.g oil of lemon, in perfummry e.g oil of rose or as starting materials for the synthesis of other coumpounds e.g oil of turpentine. For therapeutic purposes they are administered externally as inhalation for catarrhs of the respiratory tract e.g thyme, eucalyptus and anise; as gargles and mouth washes e.g thymol and as transdermally for example many essential oils including those of lavender, rose mary and bergamot are employed in the practice of aromatherapy (1, 2) . It was found that the oils with high phenol content like clove and thyme have antiseptic, antibacterial and antifungal actions, while those with ether as chief constituent produce mild antispasmodic, mild insecticidal in addition to antibacterial action e.g funnel and anise (2, 3). The constituents of many volatile oils are started to interfere with respiration and electron transport in a variety of bacteria, hence accounting for their use in food preservative and in cosmetic preparations (1) . Pharmaceutically these volatile oils can be used as extract, tincture or using the dried parts of the plant itself to be prepared for external use e.g thyme (thymi herba), cresping thyme (serpyili herba) wild oregano (origanie herba) and eucalyptus (oleum eucalypti) (4,5) Many formulations can be used locally (external use) which include: collodions, liniments, lotions, paints, ointments and creams. (6) Lotions which are liquid preparation are easier to apply and less messy than many semisolid external preparation, they frequently contain suspended particles or emulsified liquid droplets. Depending on wether they are solid-liquid or liquid-liquid dispersions, therefore lotions can be classified as suspensions or emulsions (6) . Medicinal lotions are antiseptic and germicidal, they are used in the treatment of skin diseases and as cooling and mildly anesthetic applications for skin irritations. e.g calamine lotion and Benzyl benzoate lotions N. F. (6, 7) . Aim of the work to prepare topical pharmaceutical liquid dosage form (lotion) from natural plants; arial part of thyme and fruit part of anise for topical applications. 1 Corresponding author : E-mail issraapharm @yahoo.com Received : 4/6/2006 Accepted : 4/3/2007 mailto:suhadsami04@yahoo.com 9 Materials and Methods :- Materials and Instruments Anise (Pimpinella anisum) dried fruits plant. Thyme (Thymus vulgaris) the stripped and dried leaves and flowers (aerial plant) (2) earial plant .Diethyl ether, Ethanol 96%, Anisaldehyde spraying reagent (BDH chemical Ltd, England), Propyl paraben, Methyl paraben, Veegum powder, Sodium carboxy methylcellulose, Xanthan powder (E-Merk, Darmstadt, west Germany), Rose mary spirit, (Judex laboratory reagents, England). Electrical stirrer or mixer, Voss instrument, Meldon, Essex, England ,TLC silica gel plate , Oven. Preparation of stock dispersion of suspending agents .(8) Stock dispersion of each suspending agent used (sodium carboxy methyl cellulose, sodium lauryl sulfate, veegum and xanthan gum) was prepared by dispersing (0.8,1,2.5 and 5gm) of the suspending agents, respectively in 75 ml of distilled water (D.W) using an electric mixer at 150 r.p.m. The volume of dispersion was made up to 100ml with D.W, the resultant dispersions were allowed to hydrate for 24 hrs. Preparation of lotion. (9,10) The preparation of lotions were shown in table (1) Table (1): Preparation of Lotions Formula 1 Formula 2 Materials Quantities Materials Quantities Thyme and anise powder (1:1) 1 gm Thyme and anise powder (1:1) 1gm Sodium lauryl sulfate 0.25% w/v Veegum 2.2%w/v Sodium carboxy methyl cellulose 0.5% w/v Xanthan gum 0.24% w/v Glycerine 5 ml Glycerine 5 ml Alcohol 96% 15 ml Alcohol 96% 10 ml Rose mary spirit 0.05ml Rose mary spirit 0.01 ml Methyl paraben 0.18gm Methyl paraben 0.18 gm Propyl paraben 0.02gm Propyl paraben 0.02 gm D. W. Q. S to 100 ml D. W. Q. S to 100 ml The dried fruits of anis and the dried aerial parts (leaves and flowers) of Thyme (1:1) were grinded in electrical grinder then passed through sieve 0.315inch /pore. Fine powder for these parts, methyl paraben and propyl paraben were levigated with glycerine, alcohol and a part of prepared dispersions. The remaining amount of dispersion was added in divided portions to the mixture. The mortar was rinsed several times with D. W and the rinsed volume of dispersion was added to cylinder. Finally, rose mary spirit was added, followed by sufficient quantity of D. W. to make up to the final volume (100 ml). - Sedimentation volume (F) Fifty ml of each formula 1 and 2 was diluted with D.W. to a volume of 100 ml in a stoppered graduated cylinder. The lotions were shaken vigoursly to ensure uniformly then left undisturbed. The sedimentation volume which occurred under the influence of gravity was measured after a period of 48 hours. (11) F is the ratio of ultimate height of the sediment as lotion settles in a cylinder (H) to the initial height of the total lotion (Ho) (11) . H Thus F= H0 When the ultimate volume of the sediment is smaller than the original volume of lotion, the sedimentation volume is less than 1. If this value can be made to approach unity, the product becomes in flocculation equilibrium and show no supernatant on standing so it is pharmaceutically acceptable. Therefore 50 ml of each the selected formulas should be diluted with distilled water to a volume of 100 ml in a stoppered cylinder. The lotion was shaken vigorously to ensure uniformity, then left undisturbed. The sedimentation volume which occurred under the influence of gravity was measured every 4 hours for a period of 48 hours. (10) Resuspendability of lotions (12, 13) :- The resuspendability of formula 1 and 2 was evaluated quantitatively. The efforts of redispersion required to convert the sedimented system to homogenous lotion by shaking the cylinder manually rated as resuspendable, resuspendable with difficulty or not resuspendable. Rheogram (11,13) :- Rheogram was obtained at 40 0 C with Brook field DV-II+pro viscometer which read viscosity versus shear rate. Assay of the volatile oils (14, 15) : TLC technique was used to determine the active ingredients present in the prepared lotion in comparison with standard powder of 2 plants (1:1) which was done by diluting the lotion to 200 ml using D.W, filtering, then the powder on filter paper was macerated in ether for 3 days. On the other hand 0.5 gm of standard powder (1:1 dried from of anise and dried aerial parts of thyme was also macerated in ether over night, then the products of both were concentrated by evaporating of ether, then TLC technique was done using benzene and chloroform (1:1) as mobile phase, while anisaldehyde was used as spraying reagent to calculate the retarded factor (RF) value of spots produced of both lotion and standard powder. RF value is directly propotional to the remaining quantities of active ingredients. Stability and Shelf life (13) : Iraqi J.Pharm.Sci., Vol.16 (1) ,2007 Anise-Thyme Lotion for Chicken Pox. 10 The same method mentioned above was used in wich the total amount of stored lotion (50 ml) was diluted, filtered and macerated in ether then TLC technique was done to determine the stability of prepared lotion stored at 40 0 C, 50 0 C and 60 0 C for four months by measuring the RF values of all samples by calculating their quantities in comparison with standard lotion stored at room temperature. Preliminary clinical study: The prepared lotion was applied topically to 10 patients twice daily those with skin disease (allergy, infection, chicken pox …) (1). Statistical Analysis: Analysis of variance ANOVA table and X 2 test (chi-square) was used to examine the effect of some parameters. Results and Discussions :- Formulation of the lotion: Lotions are usually suspensions of solids in an aqueous medium. A few lotions are, in fact, emulsions or solutions (6) . A wide variety of ingredients may be added to the preparation to produce better dispersions (6) . Methyl cellulose or sodium carboxy methyl cellulose will localize and hold the active ingredient in contact with the affected site and at the same time be rinsed off easily with water. However they are usually added to lotion, cataplasms and burn medications to produce tough and flexible films. Surfactants as sodium Lauryl sulfate can also be used in proparation of lotions to induce the flocculation of particles in dispersion (i.e formation of a loose aggregation of particles). (7,13). Therfore formula 1 was prepared using sodium lauryl sulfate and sodium carboxy methyl cellulose as a suspending agents at concentrations of 0.25% w/v and 0.5% w/v respectively (16). While in formula 2, the veegum and xanthan gum combination was used to control flocculation (17) The following excipients additives were also added to the prepared lotions: alcohols for its drying and cooling effect, glycerine to keep the skin moist for a considerable period of time, rose mary spirit as an odouring agent, methyl paraben and propyl paraben as preservatives (7, 18). Sedimentation volume (F) and Resuspendability: Formula 1 had F=0.2 and was resuspended while formula 2 had F=0.96 and was easily resuspended (19) . Depending on physical properties, formula 2was selected to study its rheogram, shelf life and clinical effect. Rheogram: Rheogram of formula 2 is represented in figure (1). The profile showed that the viscosity of formula 2 lotion decreases with increasing rate of shear, therefore, the results illustrated that the selected formula exhibited pseudoplastic flow due to the blend of veegum and xanthan gum. This is resulted to get a formula with good viscosity, physical stability and smooth flow characteristics. Such blend was well suited to stabilizing all types of fluid suspensions and lotions (17, 19, 20) . Figure (1): Rheogram of formula 2 Study of shelf life (T 10%) (9, 8, 13): The accelerated studies at higher temperatures (40 0 C, 50 0 C, and 60 0 C) were employed to predict the breakdown of active ingredients that may occur over prolonged periods of storage at normal shelf condition as shown in figures 2 and 3. The loss of anise and thyme active ingredients is directly propotional to the concentrations remaining with respect to time, it is called a first order reaction. Figure (2): Accelerated stability study of anise crude extract in prepared lotion at elevated temperatures 1.955 1.96 1.965 1.97 1.975 1.98 1.985 1.99 1.995 2 0 2 4 6 Time (months) L o g % r e m a in in g 40 o C 50 o C 60 o C 0 100 200 300 400 500 600 700 0 50 100 150 Viscosity (CP) S h e a r r a t e ( R P M ) Iraqi J.Pharm.Sci., Vol.16 (1) ,2007 Anise-Thyme Lotion for Chicken Pox. 11 Figure (3): Accelerated stability study of thyme crude extract in prepared lotion at elevated temperatures. The mathematical expression is : -dc = KC dt where C is the concentration of intact active ingredients remaining. t is time (dc/dt) is the rate at which the intact active ingredients degrades, and K is the specific reaction rate constant. The integrated and more useful form of equation: - Kt log C = + log Co 2.303 The degredation rate constants (k) were calculated from slopes of straight lines. These degradation rate constants are summarized in table(2). Table (2): Degradation Rate Constants (K) and RF Values of Both Anise and Thyme Crude Extracts in Formula 2 at Different Temperatures. Tempera- tures (C 0 ) K(month -1 ) RF Anise Thyme Anise Thyme Degree Active ingredients of crude extracts 40 17 x 10 -3 10.6 x 10 -3 0.93 0.95 50 25.5 x 10 -3 15.6 x 10 -3 0.89 0.93 60 38 x 10 -3 24 x 10 -3 0.85 0.91 25 8.6 x 10 -3 6.2 x 10 -3 - - To determine t 10%, Arrhenius plots were constructed to predict the degredation rate constants at 25 0 C (K25) for both anise and thyme crude extracts. Figures 4 and 5 predicted the rate constants of formula 2 which were found to be equal to 8.6 x 10 -3 and 6.2 x 10 -3 month -1 respectively. 0.104 Thus T10% = . K25ºC The shelf lives of both anise and thyme crude extracts in formula 2 were 1.0 and 1.3 years respectively. Therefore the expiration date of formula 2 was about 1 year. Figure (4): Arrhenious plot for shelf life estimation of anise crude extract in the lotion. 1.92 1.93 1.94 1.95 1.96 1.97 1.98 1.99 2 0 2 4 6 Time (months) L o g % r e m a in in g 40 o C 50 o C 60 o C 1 10 100 2.9 3 3.1 3.2 3.3 3.4 1/T x 10 3 (Kelvin -1 ) K x1 0- 3 (m on th -1 ) 3.35 1 10 100 2.9 3 3.1 3.2 3.3 3.4 1/T x 10 3 (Kelvin -1 ) K x1 0- 3 (m on th -1 ) 3.35 Iraqi J.Pharm.Sci., Vol.16 (1) ,2007 Anise-Thyme Lotion for Chicken Pox. 12 Figure (5): Arrhenious plot for shelf life estimation of thyme crude extract in the lotion. As shown in scheme 1 (21) , thyme extract contains chemical materials thymol and carvacrol which known as phenol types that are held responsible for antiseptic properties of thyme. (3) Thyme extracts according to the results shown in table (2) were significantly (P