ISSN 2229 6859

IJPIs Journal of Pharmacology and Toxicology

Visit www.ijpijournals.com

Safety Evaluation of the Essential Oil from the Dried Leaves of Pogestemon cablin (Blanco) Benth. (Lamiaceae)
1,2,3

Aleth Therese L.Dacanay*, 3Marina O. Osi

University of Santo Tomas Faculty of Pharmacy, University of Santo Tomas Research Center for Natural and Applied Sciences, 3 University of Santo Tomas Graduate School, Manila, PHILIPPINES

Corresponding Author: Dacanay ATL.

Email address: aldacanay_ust@yahoo.com

ABSTRACT:
Essential Oils are widely used in pharmaceutical, cosmetic, and perfumery industry for their aesthetic and therapeutic properties. Over the past few decades, researchers engaged in formulating dosage forms using the essential oil of Pogostemon cablin (Blanco) Benth. without assessing first its toxicity. The present study assessed and evaluated the dermal toxic characteristics of the essential oil of Pogostemon cablin to resolve this knowledge gap. Pogostemon cablin belongs to the Lamiaceae family and is commonly known as kablin in the Philippines. Fresh leaves of Pogostemon cablin were collected, dried, and extracted using steam distillation method. Dermal toxicity tests were performed based on the guidelines of Organization for Economic Co-operation and Development (OECD). A mixture of the essential oil with Polysorbate 80 at a 1:1 ratio was prepared. The mixture was topically applied to Swiss mice at doses of 2000-, 6309.57-, 19,952.62-, 63,095.7-, and 199,526.12 mg/kg, using a log dose interval of 0.5 to determine the median lethal dose concentration of the oil. Since there was no sign of toxicity and death, the arbitrary dose of 2000 mg/kg was used as the dose level in all the succeeding tests. The risk for systemic absorption of the oil was also addressed by performing median lethal dose administered by oral intubation and at the same dose levels as that for topical application. Since all mice survived, confirmation of its safety was determined through histopathological examination of the necropsied organs. These showed no significant changes. The Dermal Irritation/Corrosion test using Albino rabbits was observed to be non-irritant, with a primary irritation index of 0.00. Acute Dermal Toxicity test and Repeated Dose Dermal Toxicity 21/28Day study on the Sprague-Dawley rats did not manifest any evidence of toxicity. Dermal Sensitization test using Swiss mice was observed to be non-irritant to the ears of the mice and without ear swelling. Histopathological examination of the necropsied organs showed no significant changes. The essential oil of Pogostemon cablin is nontoxic when administered through dermal application and is therefore safe to use for future dosage form formulations.

Keywords: Dermal toxicity, Essential oils, Pogostemon cablin (Blanco) Benth. (Lamiaceae), Organization for Economic Co-operation and Development (OECD)

Vol 2:10 (2012) 1. INTRODUCTION

IJPIS Journal of Pharmacology and Toxicology

Science teaches that all things recycle and now is another era of resurgent interest in plants as medicine. Although botanicals are enjoying widespread use for treatment of several ailments, little is known about their toxicity and safety issue is always a concern (Teshome, et al. 2008). According to Croteau (as cited in Hamid, 2010), plants produce primary and secondary metabolites, which encompass a whole array of function. Essential oil is one of the compounds that can be obtained from plants and is responsible for the odor of many aromatic plants. Essential oils are known for their therapeutic properties hence, are widely used in pharmaceutical industry as raw materials. It has been traditionally used as medicinal product over a long period of time despite lack of toxicological data (Australian Pesticides and Veterinary Medicines Authority, 2005). Chemical substances in contact with the skin may have toxic effects. These can occur locally, the compound causing irritation and damage to the skin, or the compound may be absorbed into the general circulation, leading to systemic toxic effects. As in all toxicological works, initial studies of dermal toxicity should be carried out in animals to detect actual toxic and irritant substances (Somers, 1964). Dermal toxicity tests determine the potential for an agent to cause irritation and inflammation of the skin. This may be the result of direct damage to the skin cells by a substance or indirect response due to sensitization from prior exposure. The essential oil of Pogostemon cablin was subjected to dermal toxicity study in order to assess and evaluate its dermal toxic characteristics. 2. MATERIALS AND METHODS 2.1 Preparation of the Plant Material: Fresh leaves of Pogostemon cablin were harvested from one area in Barangay Del Rosario, Igbac, Buhi, Camarines Sur. Plant specimen consisting of the whole plant of Pogostemon cablin was submitted to the Herbarium of the National Museum for authentication. After garbling, the leaves were subjected to air-drying for three days and were weighed using a triple beam balance. The air-dried leaves were comminuted for volatile oil extraction by steam distillation method. 2.2 Extraction of the Essential Oil: Pre-weighed airdried leaves were loaded one at a time in a laboratory steam distillation flask filled with water up to the plate. The steam distillation set-up was provided with a Clavenger tube receiver attached to a condenser. The distillation chamber was heated to about 120 C. The distillation was carried out for at least six hours, or until distillation was complete as manifested by a constant oil output in the receiver. The volume of the oil was measured on a wet basis and separated from aqueous distillate. Then it was dried with addition of 2 grams of anhydrous sodium sulfate until no more clumping of the salt was observed. This was done by using a funnel and a loose pledget of cotton placed before the stem. Sodium sulfate was added and the oil was filtered. To determine complete dryness, the oil was dropped into anhydrous copper sulfate crystals. Since the color did not change to blue, then it was considered dried and assured that moisture has been removed. Two batches of the clear and moisture-free oil was placed in an amber bottle and stored at 0C until required (Omolo M.O., 2004). The mean percentage yield was 2.3518%. 2.3 Preparation and Handling of Test Animals: Albino Mice and Albino Rabbits were procured from the University of the Philippines Manila National Institute of Health, and acclimatized in the Animal House for a week prior to the experiment. During this period, the test animals were observed daily to confirm suitability for study. The test animals were kept under ambient light/dark cycle, room temperature and relative humidity. They were also given free access to food and water all throughout the observation period. All procedures and techniques used in these studies were in accordance with the protocol approved by the University of Santo Tomas Institutional Animal Care and Use Committee (IACUC) with Bureau of Animal Industry Dacanay ATL. et al Page 2

Vol 2:10 (2012) (BAI) Number LAF-017.

IJPIS Journal of Pharmacology and Toxicology

2.4 Experimental Protocol: 2.4.1 Median Lethal Dose Determination- Topical Application Ten randomized, specified and properly identified female nulliparous and non-pregnant Swiss mice were used in this test. Mice were caged individually and conventional laboratory diet was used with an unlimited supply of drinking water. The 1st animal was applied topically with the essential oil mixture, 1:1 ratio of essential oil and Polysorbate 80 as vehicle using an arbitrary dose of 2000 mg/kg BW. Since the 1 st animal survived, the test was carried on using additional 4 test animals which were topically administered with sequential doses higher than the starting dose of 2000 mg/kg BW using 0.5 intervals. Thus with 4 additional animals treated with as such, a total of 5 animals were used. If three animals die, the approximate LD50 (ALD) is presumed to be between the two consecutive doses- the lower dose that did not produce death and the starting dose that is lethal to the animals. 2.4.2 Median Lethal Dose Determination- Oral Administration The same procedure was carried out by oral administration of the oil mixture using an intubation needle. The number of deaths that occur during the 48 hours interval of each sequential dose was recorded both on the topically applied and orally administered oil mixture. 2.5 Dermal Toxicity Testing: 2.5.1 Dermal Irritation/Corrosion Healthy 4 young albino rabbits, 2 females and 2 males were used in each study. The rabbits were put in a cage individually. Each body of the rabbits was divided from the skin lateral to the spinal groove. The left side of the groove was utilized as the negative control and the right side as the experimental side. 2.5.2 Patch Test The rabbits were shaved (1.5 x 1 cm) and cleaned from the skin lateral to the spinal groove. The sites were cleaned using 70% alcohol. The essential oil mixture was applied appropriately at the right side of the groove. Both sites were covered with TegadermTM and secured using Micropore. The rabbits were left undisturbed for 24 to 72 hours. The patches were removed after 24 hours of exposure and the reactions were evaluated according to scores and were reattached after recording. After 72 hours another scoring was done. 2.5.3 Scratch Test Procedure and scoring method for Scratch test are the same as that of the Patch test, but with slight modification such that the skin of the four rabbits was laterally abraded to the spinal groove by slightly scratching the skin five to seven times with a sterilized 20-gauge hypodermic needle. The essential oil mixture was immediately applied on the abraded skin. The results were observed and recorded in the same manner as the Patch test. The average scores of the 24 hour- and 72 hour-reading were computed by combining the average scores of the Patch and Scratch test. This combined average was used to determine the Primary Irritation Index of the essential oil mixture. 2.5.4 Dermal Sensitization 6 to 9-week old 12 Swiss Mice were used for this test. Test animals were divided into two groups: 8 mice for the experimental group and four mice for the control group. Each mouse in the test group was administered with 20 L (2000 mg/kg) of the test oil mixture on both the ventral (front) and dorsal (back) sides of the ears. Then all mice were individually caged according to their respective groups and were left undisturbed. Ear thickness measurements were taken for both ears after 24 and 48 hours using a micrometer. The percentage (%) ear swelling difference (%) was calculated for each mouse, both in the experimental and control group. The % responder was also calculated, for the test group only, by dividing the number of mice with a positive result (% >20) to the total number of mice in the test group, and % after 24 and 48 h reading, for the purpose of comparison and analysis. In addition, the percent ear

Dacanay ATL. et al

Page 3

Vol 2:10 (2012)

IJPIS Journal of Pharmacology and Toxicology

swelling for the test group were calculated by dividing the total ear swelling measurement results of the left ear of all mice in the group to the same result of the right ear of all mice and multiplying by 100. 2.5.5 Acute Dermal Toxicity Test Twelve male adult Sprague-Dawley rats, weighing 200 to 300 grams each were used in this study. The rats were divided into two groups: experimental and control group. The rats were caged individually. Five days prior to the test proper, the test animals were acclimatize to the laboratory conditions. Twenty-four hours before the test, the fur was removed from the dorsal area of the trunk of the test animals by clipping and shaving. Care was taken to avoid abrading the skin, which could modify its permeability. Not less than 10 percent of the body surface area (BSA) was removed for the application of the essential oil mixture (OECD, 1987). The essential oil mixture (2000 mg/kg) was applied homogenously over the area of the shaved skin. It was held in contact with the skin using TegadermTM and was secured using Micropore for exposure period of 24 hours. After the exposure period, excess experimental substance was removed using distilled water (OECD, 1987). The rats were observed for a period of at least 14 days. There was frequent observation of the rats from the first day and once a day for the succeeding days. Cage side observation such as evaluation of the skin and fur, eyes, respiratory effects (salivation, diarrhea, and urination), and central nervous system effects (tremors and convulsion, changes in the level of activity, gait and posture, reactivity to handling, sensor stimuli, altered strength and stereotyped or bizarre behaviour) were carried out daily. The weights of the rats were taken weekly to determine the changes in weight. Observations seen in the experimental group were compared to that of the control group. After the 14-day observation period, all test animals in the experimental group were euthanized by cervical dislocation. The control group was kept for the Repeated-Dose Dermal Toxicity study. 2.5.6 Repeated Dose Dermal Toxicity 28-Day Study Eighteen adult Sprague-Dawley rats (12 males and 6 females) weighing 200 to 300 grams each were used. The rats were divided into two groups: 12 rats (6 males and 6 females) as the experimental group and 6 rats as the control group. The control group was the same group of rats used in the Acute Dermal Toxicity. The female rats used, were nulliparous and non-pregnant. The rats were individually caged. Cage-side observation was done. Measurements were made upon food consumption daily and the test animals weighed daily. Twenty-four hours before the test, the fur was removed from the dorsal area of the trunk of the test animals by clipping and shaving. Care was done to avoid abrading the skin, which could modify its permeability. Not less than 10 percent of the BSA was removed for the application of the essential oil mixture (OECD, 1987). The essential oil mixture (2000 mg/kg BW), was applied homogenously to the experimental group six hours per day on a five-day-per-week basis for a total exposure of 28 days. It was held in contact with the skin using TegadermTM and secured using Micropore between applications. At the end of the exposure period, excess materials were removed using distilled water (OECD, 1981). Observations were done once every day and signs of toxicity were noted accordingly. The test animals were weighed in a weekly basis to measure if there was weight lost. Consequently, food and water consumptions were also observed. After the 28-day application period, all animals were euthanized by cervical dislocation. Necropsied organs such as the skin, heart, lungs, brain, kidney, liver and gut, were submitted for histopathological analysis at the University of the Philippines Manila. 2.6 Histopathological Analysis: All the sacrificed rats were necropsied. Specimens were collected from different organs and fixed in 10% neutral buffer formalin. Paraffin sections (6-8 microns) were prepared and stained with haematoxylin and eosin. 2.7 Statistical Analysis: Data on dermal irritation was just presented as visual scores based on Draize method of erythema and edemagrading system and PII was calculated, whereas the data of the sensitization test was individual ear swelling

Dacanay ATL. et al

Page 4

Vol 2:10 (2012)

IJPIS Journal of Pharmacology and Toxicology

measurements in millimeters and % differences were computed. Data obtained from body weight measurements were expressed as ANOVA Repeated Measures. 3. RESULTS AND DISCUSSION Median Lethal Dose Determination The Median Lethal Dose was determined using five female Swiss mice. The first mouse was applied topically with the mixture of the oil using the arbitrary dose of 2000 mg/kg BW. The volume of essential oil applied was calculated using its specific gravity (i.e. 0.9294). As death did not occur when administered with the arbitrary dose, 4 additional experimental animals were treated with sequential doses higher than the arbitrary dose using the 0.5 log interval. During the observation period, all test animals survived after the application of the essential oil. The same procedure was applied to the 2nd group of the test animals which were orally administered with the essential oil using an intubation needle. Oral use of the essential oil of Pogostemon cablin was ascertained as non-toxic. The researcher then decided to use the arbitrary dose of 2000 mg/kg BW as the standard dose to all succeeding dermal toxicity tests. The summary of the data and the results, including the different dose levels calculated and used in determining the median lethal dose, is found on Table 1. It is perceived that no test animal died after the application of the essential oil and all throughout the observation period. Dermal Irritation/Corrosion The essential oil of Pogostemon cablin did not produce any erythema and edema formation in all the rabbits within the 24-hr to 72-hr observation period at a dose of 2000 mg/Kg BW using the Draize method of skin irritation. The Primary Irritation Index (PII) was calculated as zero (0.0). This signifies that the essential oil of Pogostemeon cablin is non-irritating to the skin of the test animals. Records of observation are shown on Table 2. Dermal Sensitization MEST (Mouse Ear Swelling Test) was used as tool for this test as it is sensitive, efficient and cost effective alternative to the guinea pig dermal sensitization test (Gad et al., 1986; Gad, 1995). Since it was founded on the principle that age has an impact on the degree of response to sensitization of xenobiotics or chemicals foreign to biologic systems. Thus, 6 to 9-week old mice were selected. Furthermore, chemical method of immune enhancement technique was not employed, with the thought that the essential oils potential of sensitization in a chemical -free environment could give a clear picture. In addition, interpretation of data from tests that employ adjuvant or nonspecific stimulator of immune response of mice is complex and the results are not directly applicable to human use conditions (Kimber, et al., 2001). Ear swelling measurements after the challenge test with the Pogestemon cablin essential oil is presented in Table 3, 24 hours after challenge; % was 0 and 0 for the test and control group, respectively. The % appeared unchanged with time as it remains 0 for test and control group following 48 hours application. The % responder was noted to be 0, as there was no mouse with a % of 20% or above. The dose appropriate for the actual sensitization test was selected by a dermal irritation and toxicity probe study. Probe studies usually indicate that the highest non-irritating concentration on the ear is greater than the justirritant concentration on the belly region (Gad et al., 1986). This might be because the highest non-irritating concentration to the ear is identified by only one (acute) application while there was a repeated application on the abdomen. This applies to this study. The non-irritating concentration on the ear and the non-irritant to the abdomen was 2000 mg/Kg BW. The setback is that the normal ear thickness at the tip of the pinna may vary among mice, chances are, systematic error in measuring the ear thickness may arrive at an inaccurate conclusion. It is therefore, noteworthy to consider not only the ear swelling measurement but making observation on erythema and edema as well to outwit the problem. Conclusion on the presence or absence of sensitization was made based on % between test and control

Dacanay ATL. et al

Page 5

Vol 2:10 (2012)

IJPIS Journal of Pharmacology and Toxicology

ears. Positive sensitization response was considered to have occurred if the test ear of one or more mouse was at least 20% thicker than the control ear. This effect criterion guarantees a level of false positives of less than 1 in 1000 (Gad et al., 1986). Based on this criterion, none of the test results showed 20% thicker test ear than the control ear indicating the essential oil is a non-sensitizer. Acute Dermal Toxicity All (100%) of the test animals did not manifest any toxic effects after the application of the essential oil of Pogostemon cablin. Test animals were healthy and alive all throughout the 14-day observation period and there was no sign of erythema or edema. When the semi-occlusion was removed, rats in the test group showed no signs of irritation and did not appear to be frail. In addition, an increase in weight was observed on the essential oil-treated rats which might be ascribed to the absence of diarrhea which could be a possible evidence of toxicity. After 48 and 72 hours, rats in the test group continued to be active enough to take food and drink as what was previously observed. One of the main purposes of acute toxicity testing is to obtain an appropriate dose for long-term toxicity tests and to find out the most affected organ, if any, so that the long term toxicity study could be designed based on the acute test results. The other role of acute toxicity test is to determine LD50. However, this parameter is no more in use because of animal welfare reasons. In addition, LD50 is not a correct indicator of potential toxic hazards because death may not occur while essential organs remain affected for long time (Teshome, et al., 2008). The fact that there was no death at these doses indicates that the essential oil of Pogostemon cablin is essentially non-toxic. Repeated Dose Dermal Toxicity Repeated dermal application of the essential oil of Pogostemon cablin to rats for 28 days at 2000 mg/Kg BW did not produce mortality. In addition, the daily observations made for each rat showed that there were no explicit clinical signs of toxicity such as pain when touched, frail looking, poor food consumption and reduced locomotion. There was also no evidence of dermal irritation that was noted. Furthermore, there was no statistically significant weight loss that was recorded. These results are in congruence with the acute dermal toxicity test results and may indicate the possibility that the essential oil is in fact non-toxic Histopathological Analysis Physiological functions of vital organs such as the liver, kidney, heart and other major organs may be impaired by toxic agents. Histopathologic analysis of the toxic potential of a particular plant material on target organs may also be significant. It is also accepted that all functional studies in toxicology should be coupled with appropriate morphologic pathology studies (Hothorn and Hajian, 1999). The 28 days repeated dermal toxicity test indicated that the essential oil is non-toxic on rats, the histopathological evaluation confirms that the necropsied samples did not show any significant changes. Moreover, there was no increase in the number of cells and no inflammatory infiltrates were observed. Table 1: Result of the Median Lethal Dose Determination at Different Dose Levels Standard Dose Level (mg/Kg) 2,000 6,309 19,953 63,096 199,526.12 Weight of Swiss Mice (Kg) 0.024 0.026 0.026 0.025 0.024 Actual Dose Level (mg/Kg) 48 164.05 518.77 1,577.39 4,788.63 Volume of Essential Oil (mL) 0.052 0.18 0.56 1.7 5.15 Survived Survived Result (Topical Application) Result (Oral Administration)

Dacanay ATL. et al

Page 6

Vol 2:10 (2012)

IJPIS Journal of Pharmacology and Toxicology Table 2: Irritant/Corrosive Response of each Albino rabbit per observation Rabbit No. 1 Individual Scores Erythema Edema 24 hrs Intact Abraded 2 Intact Abraded 3 Intact Abraded 4 Intact Abraded Total Primary Irritation Index 0 0 0 0 0 0 0 0 72 hrs 0 0 0 0 0 0 0 0 0/32 = 0 24 hrs 0 0 0 0 0 0 0 0 72 hrs 0 0 0 0 0 0 0 0 Average Score 0 0 0 0 0 0 0 0 0

Skin

Table 3: Ear swelling measurement results of actual sensitization test of Pogostemon cablin essential oil on mice; test group (A) and control group Reading after 24 h (mm) Left ear A 1 2 3 4 5 6 7 8 Sum B 1 2 3 4 Sum 0.11 0.13 0.13 0.09 0.11 0.11 0.14 0.13 0.95 0.13 0.13 0.14 0.12 0.52 Right ear 0.11 0.13 0.12 0.1 0.12 0.11 0.14 0.12 1.0 0.12 0.12 0.13 0.11 0.48 0 0 8.33 -10 8.33 0 0 8.33 -5.0 8.33 8.33 7.69 9.09 8.33 % Reading after 48 h (mm) Left ear 0.11 0.13 0.13 0.09 0.11 0.11 0.14 0.13 0.95 0.13 0.13 0.14 0.12 0.52 Right ear 0.11 0.13 0.12 0.1 0.12 0.11 0.14 0.12 1.0 0.12 0.12 0.13 0.11 0.48 0 0 8.33 -10 8.33 0 0 8.33 -5.0 8.33 8.33 7.69 9.09 8.33 %

Mice No.

% ear swelling (after 24 h) = (0.95/1.0) x 100 = 95 % ear swelling (after 48 h) = (0.95/1.0) x 100 = 95 Dacanay ATL. et al Page 7

Vol 2:10 (2012)

IJPIS Journal of Pharmacology and Toxicology

Figure 1: Comparison between the Patch Test site (A); Scratch Test site (B) and Control site (C)

Figure 2: Comparison between the Control site (A) and Test sites (B, C)

4. CONCLUSION The Pogostemon cablin leaves collected from Sta. Cruz, Buhi, Camarines Sur did not manifest any form of toxicity during the entire course of the study when it was applied topically and orally administered to the test animals. The topical application of the essential oil of Pogostemon cablin did not form any erythema and edema during the acute dermal irritation test. The grade for both erythema and edema is 0. The oil, having a score of 0.00, is classified as non-irritating substance based on Primary Irritation Index (PII) by Draize (1959).

Dacanay ATL. et al

Page 8

Vol 2:10 (2012)

IJPIS Journal of Pharmacology and Toxicology

Any noxious effects were not observed during the course of toxicity tests on Acute Dermal Irritation, Acute Dermal toxicity test, Repeated Dose Dermal Toxicity Test and Dermal Sensitization Test. The test animals were observed to have an increase in weight over time. Furthermore, improvement of skin complexion of the test animals was recognized. Therefore, Philippine variety of Pogostemon cablin is non-toxic, as the oil did not demonstrate any form of toxicity upon dermal application and oral administration. This may also conclude that the essential oil has no systemic absorption. However, even if the essential oil has proven on this study to be non-toxic, it is still worthwhile to pursue extensive investigation so that fragmented test results will be further evaluated. 5. ACKNOWLEDGEMENT This research was funded by the Department of Science and Technology Science Education Institute (DOSTSEI), the University of Santo Tomas Office for Grants and Endowment, and the Fund for Assistance to Private Education, Phil. Also, the researcher is very grateful to Dr. Marina O. Osi of the University of Santo Tomas Graduate School, Manila, Philippines for her unwavering support. 6. REFERENCES (1) Active Organics. (2002). Actiphyte of patchouli. 1097 Yates Street Lewisville Texas 75057. (2) Benedict, A.C. (2009). Extraction of the essential oil of Aquilaria malaccensis (Gaharu) using hydrodistillation and solvent extraction methods. Universiti Malaysia Pahang. (3) Bunrathep, S., Lockwood, G.B., Songsak, T., & Ruangrungsi, N. (2006). Chemical constituents from leaves and cell cultures of Pogostemon cablin and use of precursor feeding to improve patchouli alcohol level. Science Asia, 32, 293-296. (4) Burade, K.B., Chopade, A.R., & Kucheckar B.S. (2009). Acute dermal toxicity studies of pitika mardini and esabdamini in rodents. Arch Pharm Sci & Res, 1(2), 171-174. (5) Environmental Protection Agency. (1998). Health effects and guidelines of oppts. 870.1200 Acute Dermal Toxicity. (6) Environmental Protection Agency. (2006). Biopesticides registration action document: Cold pressed neem oil pc code 025006. (7) Evans, W.C. (2009). Trease and Evans Pharmacognosy. 16th ed. Elsevier Saunders. (8) Gad, S.C. (1995). The mouse ear swelling test. In: Burleson, R.G., Dean, H.J., Monson, E.A., (Eds.). Method in Immunotoxicology. Wiley-Liss, New York, pp 357-372. (9) Gad, S.C., Dunn, J.B., Dobbs, W.D., Reilly, C., Walsh, R.D. (1986). Development and validation of an alternative dermal sensitization test: the mouse ear swelling test (MEST). Journal of Toxicology and Applied Pharmacology 84, 93-114. (10) Gaspillo, P.D., Maridable, J.B., Auresenia, J.L., & Tan, R.G.R. (2006). Physico-chemical and antimicrobial properties of essential oil extracted from lemongrass and patchouli using the supercritical carbon dioxide. (11) Hamid, A.A., Aiyelaagbe, O.O., & Usman, L.A. (2010). Essential oils: Its medicinal and pharmacological uses. (12) Henderson, W., Hart, J.W., How, P., & Judge, J. (1970). Chemical and morphological studies on sites and sesquiterpene accumulation in Pogostemon cablin (patchouli). Phytochemistry, 9, 1219-1228. (13) Hothorn, L.A., Haijan, G., (1999). Biostatistics in toxicology. In: Marquardt, H., Schafer, S.G., McCllelan, R.O., Welsh, F. (Eds.) Toxicology. Academic Press, San Diego. P.34. (14) Hussain, A.I. (2009). Characterization and biological activities of essential oils of some species of lamiaceae. Unpublished doctoral dissertation, University of Agriculture, Faisalabad, Pakistan.

Dacanay ATL. et al

Page 9

Vol 2:10 (2012)

IJPIS Journal of Pharmacology and Toxicology

(15) Kimber, L., Basketter, A.D., Berthold, K., Butler, M., Garrigue, J.L., Lea, L., Newsome, C., Roggeband, R., Steiling, W., Stropp, G., Waterman, S., Wiemann, C., (2001). Skin sensitization testing in potency and risk assessment. Toxicological sciences 59, 198-208. (16) Kimmel, C.A., & Francis, E.Z. (1990). Proceedings of the workshop on the acceptability and interpretaion of dermal developmental toxicity studies. Fundamental and Applied Toxicology, 14, 386-398. (17) LeFevre, J.W. & Suny O. (1998). Isolation clove oil from cloves using steam distillation. Chemical Education Resource. (18) Lu, T., Liao, J., Huang, T., Lin, Y., Liu, C., Chiu, Y., & Peng, W. (2009). Analgesic and anti-inflammatory activities of methanol extract from Pogostemon cablin. eCAM Advance Access, 1-9. (19) Murugan, R., & Livingstone, C. (2010). Origin of the name patchouli and its history. Current Science, 99(9), 1274-1275. (20) Mutai, M. (2000). The handbook of experimental animals: The laboratory rat (G.J. Krinke, Ed.). Solvenia: Midas Printing Ltd. Retrieved March 6, 2011, from the Google database: http://books.google.com/books (21) Organization for Economic Co-operation and Development. (1981). Guidelines for testing of chemicals: Repeated dose dermal toxicity 21/28-days study. (22) Organizaiton for Economic Co-operation and Development. (1987). Guidelines for testing of chemicals: Acute dermal toxicity. (23) Organization for Economic Co-operation and Development (2002). Guidelines for testing of chemicals: Acute dermal irritation/ corrosion. (24) Price, L. (2007). Aromatherapy for health professionals (S. Price, & L. Price, Ed.). Elsevier. Retrieved March 6 ,2011, from Google database: http://books.google.com/books (25) Quisumbing, E. (1978). Medicinal plants of the Philippines. Manila: Bureau of Print. (26) Rashid M.F. (2006). Extraction of essential oils from a jasmin flower using solvent extraction method. University College of Engineering & Technology Malaysia. (27) Robin, B., Dunn, D., Sharrer, E., & Lengerich, L. (1988). Primary skin irritation test in the rabbit of water jel burn dressing. North American Science Associates, Inc. (28) Roe, R.M., Donohue, K.V., Raleigh, N.C., Jones, A., Homs, L.C., Clayton, N.C. et al. (2006). Development of a novel all natural tick and insect repellent, bioud, as a deet replacement and for use on cotton fabric. (29) Sadeghi-Nejad, B., & Sadhu Deokule, S. (2010). Antidermatophytic activity of Pogostemon parviflorus Benth. Iranian Journal of Pharmaceutical Research, 9(3), 279-285. (30) Sanders, A. (2008). Acute dermal irritation in the rabbit. (Item 236-0033). (31) Somers, G.F. (1964). Testing drugs for dermal toxicity. J.Soc. Cosmetic Chemists, 15, 385-394. (32) Sulaiman, D.H. (2008). Extraction of patchouli oil using steam distillation. University of Malaysia Pahang. (33) Swamy, M.K., Blasubramanya, S., & Anuradha, M. (2010). In vitro multiplication of Pogostemon cablin Benth. through direct regeneration. African Journal of Biotechnology, 9(14), 2069-2075. (34) Teshome, K., Gebre-Mariam, T., Asres, K., Perry, F., Engidawork, E. (2008).Toxicity studies on dermal application of plant extract of Plumbago zeylanica used in Ethiopian traditional medicine. Journal of Ethnopharmacology, 117 (2008), 236-248. (35) Trongtokit, Y. et al. (2008). Botanical medicine in clinical practice. (R.R. Watson & V.R. Preedy Ed.) Cromwell Press, Trowbridge. Retrieved March 6,2011, from Google database: http://books.google.com/books (36) Winitchai, P., Thanapane, W., Kongtud, W., Ruangmarerng, J., Meewang, C., & Supjarean, S. (2009). Antimicrobial property of the essential oil and crude extract from patchouli leaves.

Dacanay ATL. et al

Page 10