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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

1 University of Santo Tomas Faculty of Pharmacy,

2 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: [email protected]

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 21/28-

Day 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) IJPI’S Journal of Pharmacology and Toxicology

Dacanay ATL. et al Page 2

1. INTRODUCTION

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 air–dried 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 0°C 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



(BAI) Number LAF-017.

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 1st 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 µL

(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



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 edema-

grading system and PII was calculated, whereas the data of the sensitization test was individual ear swelling



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 oil’s 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 just-

irritant 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



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)

Weight of

Swiss Mice

(Kg)

Actual Dose

Level

(mg/Kg)

Volume of

Essential Oil

(mL)

Result

(Topical

Application)

Result

(Oral

Administration)

2,000 0.024 48 0.052

Survived Survived

6,309 0.026 164.05 0.18

19,953 0.026 518.77 0.56

63,096 0.025 1,577.39 1.7

199,526.12 0.024 4,788.63 5.15



Table 2: Irritant/Corrosive Response of each Albino rabbit per observation

Rabbit

No. Skin

Individual Scores Average

Score Erythema Edema

24 hrs 72 hrs 24 hrs 72 hrs

1 Intact 0 0 0 0 0

Abraded 0 0 0 0 0

2 Intact 0 0 0 0 0

Abraded 0 0 0 0 0

3 Intact 0 0 0 0 0

Abraded 0 0 0 0 0

4 Intact 0 0 0 0 0

Abraded 0 0 0 0 0

Total 0

Primary Irritation Index 0/32 = 0

Table 3: Ear swelling measurement results of actual sensitization test of Pogostemon cablin essential oil on mice;

test group (A) and control group

Mice No.

Reading after 24 h

(mm) %

Reading after 48 h

(mm) %

Left ear Right ear Left ear Right ear

A

1 0.11 0.11 0 0.11 0.11 0

2 0.13 0.13 0 0.13 0.13 0

3 0.13 0.12 8.33 0.13 0.12 8.33

4 0.09 0.1 -10 0.09 0.1 -10

5 0.11 0.12 8.33 0.11 0.12 8.33

6 0.11 0.11 0 0.11 0.11 0

7 0.14 0.14 0 0.14 0.14 0

8 0.13 0.12 8.33 0.13 0.12 8.33

Sum 0.95 1.0 -5.0 0.95 1.0 -5.0

B

1 0.13 0.12 8.33 0.13 0.12 8.33

2 0.13 0.12 8.33 0.13 0.12 8.33

3 0.14 0.13 7.69 0.14 0.13 7.69

4 0.12 0.11 9.09 0.12 0.11 9.09

Sum 0.52 0.48 8.33 0.52 0.48 8.33

% ear swelling (after 24 h) = (0.95/1.0) x 100 = 95

% ear swelling (after 48 h) = (0.95/1.0) x 100 = 95



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).



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 (DOST-

SEI), 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.

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