An Experimental Study on the Efficacy and Skin Irritation of the Fruit Extract of Capsicum frutescens (Siling Labuyo) as Compared

to Emla (Lidocaine/Prilocaine) as a Topical Local Anesthetic in Rabbits

Taca, Arturo C., Torres, Neil Stephen A., Sarmiento, Frances May A. Tenorio, Vinna Marie R., Sta. Maria, Arlene C., Santos, Christine Therese A., Santos, Ma. Rosa C., Sarenas, Maria Pamela C., Sulit, Bryan Paul D., Tan, Christopher S., Tiangco, Mark Homer T., Tibar, Jayson P., Tiong, Valerie T., Tirol, Juan Manuel U., Toledo, Strix Sandro S., Torno, Jardine Davies R., Torres, Marilou A. and Dr. Alfaretta Tan - Reyes

I. Abstract The efficacy of the topical local anaesthetic effect of the fruit extract of Capsicum frutescens (Siling labuyo) in comparison to EMLA (Lidocaine or Prilocaine) was studied in rabbits. Different concentrations of the siling labuyo extract were used as the treatment groups. Comparison of the different concentration groups relative to the negative and positive control was then done to determine the concentration that exhibited a local anaesthetic effect. The anaesthetic effect was measured using the skin twitch response. The results were analyzed using via the t-test. Among the different concentrations of the fruit extract, only 0.75 concentration of capsaicin showed a topical local anaesthetic effect. This concentration was then subjected to the skin irritation test using the Draize method. Data analysis was done using one- way ANOVA. No significant skin reaction was observed. This points to the conclusion that 0.75 Capsaicin extract exhibits a topical local anaesthetic effect similar to EMLA, as studied in rabbits.

II. Introduction Capsicum frutescens (Linn) or siling labuyo has been recognized for its palatability as well as its therapeutic effects. It has been known to alleviate chronic leg and foot pain in patients suffering from diabetes mellitus and arthritis. Capsicum frutescens has also afforded relief for sore throat and rheumatism (Villanueva, 1993). The plant is a small, spreading shrub found throughout the Philippines. It is erect, branched, half-woody, with oblong-ovate leaves and flowers that are either solitary or several in each axil. The fruit is commonly red when ripe, oblong-lanceolate in shape, and the seeds are numerous and discoid. (Quisumbing, 1978) The fruit contains the active principle capsaicin (pronounced cap-SAY-shun) 0.14%, and capsicum. It also contains fatty oils, 15-20%; volatile oil; starch 0.8 - 1.2%; pentosans, 8.57%; and pectin, 2.33% (Quisumbing, 1978). Capsaicin, 8-methyl-n-vanilyl-6-nonenamide, is the active substance found in pure extracts of red hot peppers. Capsaicin oleoresin, the active substance found in crude extracts, contains 0.02% capsaicin, 1.5% volatile oils, a fixed oil, and up to 0.2% ascorbic acid. (Tyler, Brodef and Robbers, 1988) Capsaicin is structurally similar to eugenol, the active principle found in the oil of cloves, which has a local anesthetic effect. (The Lancet, 1983) Anesthesia is defined as the loss of sensation. Locally, nerve conduction is blocked directly. Local anesthetics cause the disappearance of sensation in the following order: temperature, pain, touch, joint and pressure sensations. (Ganong, 1988) The therapeutic effects of pepper include its ability to act as a stimulant, its promotion of digestion, and its ability to afford relief from atonic gout, dyspnea with flatulence, tympanitis and paralysis. (Quisumbing, 1978) However, prolonged application to the skin may cause dermatitis, and excessive internal use can cause gastroenteritis and kidney problems. (Sas, 1984) Previous studies conducted using the rat-paw pressure test have shown that capsaicin has an anti-nociceptive effect. (Hayes, et. al., 1984) Capsaicin has been proven to produce neurogenic inflammation, followed by long-lasting diminished

responsiveness to subsequent irritation. It has also been documented that capsaicin disrupts the effects of Substance P, which is the key neurotransmitter for relaying pain messages to the brain. This effect is achieved by high doses of capsaicin, which deplete nerve neurotransmitters. (McCourt, 1991) On local or systemic administration, capsaicin initially causes an intense short-lasting irritation, possibly due to local and / or central release of Substance P from primary afferent fibers. This is followed by a later phase in which the treated animal displays insensitivity to noxious stimuli. Small single doses of capsaicin have a transient anti-nociceptive effect lasting several hours. This is followed by a profound fall in body temperature. (Hayes, et. al., 1984)It may thus be seen that capsaicin will be a useful tool for studying the role of Substance P in nociception because it first mimics Substance P action and then serves as a Substance P antagonist. (The Lancet, 1983) Aside from the postulated effects of capsaicin on pain neurotransmitters, there is also evidence of capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants. Capsaicin pre-treatment in rats induced a long-lasting desensitization of the airways to various exogenous and anaphylactic irritants. (Lundberg and Saria, 1983) Studies have also been made on the effect of antagonists on the action of capsaicin. In 1983, it was reported that, in newborn rats, administration of a protein called nerve growth factor partially antagonized the deleterious effect of capsaicin on Substance P-containing neurons in the dorsal root ganglia. It has been further concluded that capsaicin destroys the perikaryon of primary sensory peptidergic neurons by interfering with the action of nerve growth factor, probably by blocking its retrograde axonal transport. (Otten, Lorez and Businger, 1983) With regard to dose-dependent effects, increasing analgesia has been observed in rats treated with increasing concentrations of capsaicin oleoresin. This is due to the corresponding increase in the number of receptors being bound to the capsaicin molecule. This higher concentration of oleoresin corresponds with a greater amount of capsaicin in solution.(Villanueva, 1993)

The general objectives guiding this study are: 1.) to determine the anesthetic effect of the pepper extract and 2.) to assess the irritability of the topical application of the extract. This would be supported by the specific objectives which include determining the drug response and local anesthetic effect of the pepper extract, and then determining the extent of skin irritability produced by the extract proven to be an effective topical local anesthetic, as evaluated by the Draize method. III. Significance It is imperative that the medical community continually search for alternative approaches to healing and patient care. Low-cost, ready availability, and most importantly the effectiveness of alternative therapies are factors in its widespread use. With its analgesic properties firmly established in past research (McCourt, 1991), this study aims to explore the possibility of using crude, or pure pepper extract as an alternative to the use of synthetic local anesthetizing agents. Should the pepper extract prove to be as efficacious as Emla, it should be a superior alternative to imported topical anesthetic drugs presently used due to the fact that it is cheaper and is widely available. Siling labuyo could also serve as a potential raw material for pharmaceutical companies willing to explore its anesthetic properties.

III. Methods The study design used was an experimental study design. The methods used in this experiment were prepared to evaluate the efficacy of the Capsicum frutescens extract. The experiment proper consisted of two parts. The first part was the determination of the drug response and local anesthetic effect. The anesthetic response of the rabbits to the crude pepper extract was compared to EMLA (positive control) and pure mineral oil (negative control). Drug response is determined by the absence of a skin twitch, which is the desired positive response for the experiment. Efficacy of anesthetic effect was defined as the percentage of negative responses over the total number of times a stimulus was applied. The second part of the experiment was the dermal irritation test patterned after the primary irritation test protocol of Dr. Lim-Navarro. Dermal irritation was determined by the presence of erythema and edema on the test sites treated with the

pepper extract that produced the highest anesthetic effect - as determined from the first part of the experiment. Grading of the extent and severity of erythema and edema formation was also conducted , based on the Draize method, after 24 and 72 hours. Plant Material One and a half kilograms of ripe, red-chili peppers (Capsicum frutescens) were obtained from a farm in Cavite and cut into small pieces. These were then air-dried for seven days and soaked in six liters of acetone for twenty four hours. The pepper bits were then removed from the liquid medium. Extraction of the active component, capsaicin, was done using a rotary evaporator until the acetone solvent had evaporated. The crude pepper extract collected was divided into four equal parts and diluted with mineral oil using the following ratios: (a) 3:1 (25% extract), (b) 1:1 (50% extract), (c) 1:3 (75% extract), (d) A pure 100% extract. The negative control consisted only of mineral oil. The positive control used was Emla, a preparation of Lidocaine and Prilocaine. These preparations were set aside until the experiment proper commenced. Experimental Animals Eleven male rabbits, with weights ranging from 1.2 to 2.0 kilograms were obtained from a rabbit farm in Batangas and quarantined for seven days prior to the experiment proper. Proper feeding and maintenance procedures were observed during this period. A day prior to the experiment, a fifteen square centimeter area on each of the backs of the rabbits was shaved. These were then divided into six marked areas for the topical application of the six prepared solutions. All eleven rabbits were randomized for the distribution of each test concentration and controls. Determination of Anesthetic Response The rabbits were restrained with their shaven backs exposed. The different test solutions and drugs were applied topically in each marked area. These areas were tested for reaction to pain stimuli using a prick apparatus made from cork and tailoring pins. The stimuli was randomly applied by a single investigator for the whole experiment in a consistent manner on the previously marked shaven areas. The rabbits were stimulated

by pricking each area in succession with an interval of five minutes. The sharp point of the needles were thrusted acutely and perpendicular to the marked area. Determination of a Drug Response Time was recorded until a positive response - no skin twitch - was observed. This signified the beginning of the drugs anesthetic effect. end of the anesthetic effect. Dermal Irritation Test Ten male rabbits, as proposed by Dr. Lim-Navarro, weighing at least 1.2-2.0 kilograms and quarantined for seven days were utilized for the dermal irritation test. A day prior to the experiment an area of 15 centimeters by 15 centimeters was shaven off the rabbits backs. The exposed skin was divided into six equal parts. The first three parts were used for the topical application of the test product with the highest anaesthetic effect determined from the previous test. The three remaining areas were abraded and the test drug was applied. Data Processing and Analysis The results were analyzed initially using the one-way ANOVA and the f-test to determine if significant variation existed among the treatment means (capsaicin extract at 0.2, 0.5, 0.75 and 1.0 concentration). Having established that a significant difference existed among the different treatment means, the individual treatment means were then subjected to the t-test against the negative control, mineral oil. This test served to determine whether a significant anesthetic effect was obtained for any of the experimental treatments. Among the different concentrations of the fruit extract, only the 0.75 concentration of capsaicin displayed a topical local anesthetic effect when compared against mineral oil. This effective concentration was then subjected to the skin irritation test using the Draize method. Data analysis for the skin irritation test was done with the t-test. IV. Results Pricking was continued until a negative response - a skin twitch - was observed. This lack of a skin twitch signified the

A listing of the data obtained from the experiment is seen in Appendix A.

A statistical comparison using the t-test was first done between the positive and negative controls (EMLA and MO, respectively) in order to establish the local anesthetic effect of the positive control, EMLA. This comparison was based on the assumption that mineral oil had anesthetic effect.

A t-test comparison of the different treatments (corresponding to the different concentrations of capsaicin used) relative to the negative control was then done in
Means of Treatment Plans

80 70 60 50 40 30 20 10 0 0.25caps 0.50caps 0.75caps MEAN 1.0caps EMLA 1.0caps 38.273 MO EMLA 73.989 MO 38.969

0.25caps MEAN 55.545

0.50caps 52.502

0.75caps 64.909

order to determine if these treatments exhibited a significant local anesthetic effect. A

statistically significant difference with the p-value of 95% was found between the treatments and the negative control group was taken to correspond with a definite local anesthetic effect. The treatment shown to have a statistically significant effect in comparison to the negative control (75% capsaicin extract) was then compared to the positive control (EMLA) in an attempt to demonstrate that the local anesthetic effect of this treatment was significant enough to statistically approximate that of the positive control (EMLA). Data analysis was done on raw scores from the experiment converted into percentages of anesthetic response. The percentages thus obtained were used as frequencies which would then be subjected to the t-test. A true or definite anesthetic effect, as demonstrated these tests, was taken to be one that displayed a statistically significant difference over the negative control at p = 0.05 (95% level of confidence). A test of correlation between the positive and the negative control, and between the effective treatment and the 0.01, respectively). The six treatment groups were first subjected to an analysis of variance (F-ratio) with the level of significance tested at = 0.05. This was done to determine whether or not the treatment groups exhibited the same effect. The computed F ratio (2.981) revealed that a significant difference existed between the six treatment groups (F ratio > , at 0.05 = 2.37). There was therefore a greater than 95% probability that at least a pair of treatment groups exhibited an experimental effect significantly different from the other treatment groups. Each of the treatment groups was then compared to the negative control, mineral oil, to assess its local anesthetic effect. Local anesthetic effect was assayed using Shiffes test (refer to table 1). EMLA vs. Mineral Oil negative control was then done to validate the significance of the t-test at both the 95% and 99% confidence levels (p = 0.05 and p =

The level of significant difference between the positive (EMLA) and negative control (mineral oil) was first established. As shown in Table 1, a highly significant difference existed between the two control groups at both p = 0.05 and p = 0.01. A probability of > 99% exists that the anesthetic effect of Emla and mineral oil greatly differ. 25% capsaicin and Mineral Oil Statistical test results indicate that no significant difference exists between the negative control (mineral oil) and 25% crude capsaicin. The 2 treatment means are statistically equal, and it cannot be said that 25% capsaicin has a significant anesthetic effect. 50 % capsaicin and Mineral Oil From Table 1, it may be seen that no significant difference exists between the treatment means of 50% capsaicin and MO. The effect of these 2 treatment groups are equal. The 50% capsaicin treatment groups has no significant anesthetic effect. At p = 0.05 or a 95% level of confidence, it was seen that the treatment means of the 2 groups (50% capsaicin and MO) were equal. No significant difference existed between the treatment means compared. This meant that 100% crude capsaicin extract did not have any anesthetic effect when compared to the negative control. 75% capsaicin and MO The treatment means of 75% capsaicin and MO tested at p = 0.05, and even at p = 0.01, showed were significantly different. A probability of greater than 99% existed that a statistically significant difference existed between effect when compared against mineral oil. Since it has been statistically established that, among the crude pepper extracts, only the 75% concentration had a significant anesthetic effect when compared against the 2 treatment means compared. It could therefore be said that 75% capsaicin had a very high anesthetic

the negative control (mineral oil), a statistical comparison of the anesthetic effect of the crude capsaicin extract and that of the positive control was made. The anesthetic effect of 75% capsaicin was compared with EMLA at p = 0.05 level. As shown in table 1, the treatment means of these groups were statistically equal. No significant difference existed between the treatment means compared. There was therefore a probability of greater than 95% that the anesthetic effect of 75% capsaicin was statistically comparable to that of the positive control, EMLA. SKIN IRRITATION TEST RESULTS 24 HOURS F-value 0.617143 is less than F-critical 2.386066 which is the minimum value needed to show a significant difference at 5% confidence . Statistical analysis shows no marked difference among all 6 areas which are comprised by 3 areas with intervention and 3 areas with control for skin irritation after 24 hours. 72 HOURS F-value 0.846695 is less than the F-critical 2.386066 which is the minimum value needed to show a significant difference at 5% confidence. Statistical analysis show no marked difference among all 6 areas comprised by 3 areas with intervention (Area 13) and 3 areas with control (Area 4-6) for skin irritation after 72 hours. V. Discussion The local anesthetic effect of crude pepper extract at different concentrations was determined by the statistical analyses noted in the above section on results. The inactivity or inert nature of mineral oil was established when a statistically significant difference resulted from its comparison with Emla, a clinically proven topical anesthetic. This procedure standardized the testing for local anesthetic effect of the different concentrations of crude pepper extract. All the treatments, prepared at 0.25, 0.50, 0.75 and 1.00 were hypothesized to have a local anesthetic effect. Only the 0.75 concentration (75% crude extract with 25% mineral oil), however, yielded a statistically significant result. A topical anesthetic effect

was thus only established for the 0.75 capsaicin treatment. While the other capsaicin treatments ( concentrations of 0.25, 0.50 and 1.00) showed a difference in effect when compared against mineral oil, none of these results were statistically significant enough to warrant a label as a definite local anesthetic effect. It is highly probable that the local anesthetic effect of capsaicin, the active ingredient of the crude pepper extract, is dose related (such that an increase in dose would correspond with an increase in anesthetic effect). This probability is illustrated in Figure 1, which shows the duration of anesthetic effect increasing with an increase in treatment concentration. The trend noted above, however, was not seen in the 1.00 treatment or pure pepper extract. This treatment not only failed to elicit a longer duration of anesthetic effect than the 0.75 concentration, but also failed to demonstrate a statistically significant difference in effect over mineral oil. It thus appears that the local anesthetic effect of 0.75 crude extract did not merely exhibit a dose-related effect. The lipid solubility of the treatment compound is thought to enhance the skin penetration and absorption of the active ingredient of the pepper extract. The factors of capsaicin dose and lipid solubility of the treatment are closely hewed in the ability of a crude pepper extract to produce local anesthesia. The proven local anesthetic effect of 0.75 concentration pepper extract implies that there should be about 3 parts of crude extract in a pepper mixture to produce an effective dose, and about 1 part mineral oil should be used to effect sufficient lipid solubility for the active ingredient to penetrate and be absorbed in the skin. It is probable that the ideal crude extract dose to lipid content ration of the pepper extract mixture approximates 3:1 for the effect of local anesthesia. Comparison of the 0.75 concentration against the clinically proven topical anesthetic Emla yielded no significant difference. This implies that the crude extract and

its active ingredient compare favorably with Emla in terms of ability to produce topical local anesthesia. The Draize Method measures the degree and extent of erythema and edema formation on the abraded and non-abraded area s which was attributed to drug intervention. Seventy-five percent (75%) capsaicin extract, which demonstrated the Erythema and edema formation were 24 and 76 hours after the initial greatest anesthetic efficacy based on the previous portion of the experiment proper, and the (-) control group (no drug) were used. observed and rated based on a four-point scale.

application of both (+) and (-) controls. One-way ANOVA analysis of results of 24-hr. observation set at 95% confidence level revealed no significant difference between (-) control and 75% capsaicin. Repeated testing at 99% confidence level again proved (-) significant difference. The same results were seen from the data gathered in the 72-hr. group. The test results conclusively indicate that capsaicin has very little irritant effect (erythema & edema formation) after both 24 hour and 72 hours. Secondly, it also shows that the irritant effect of 75% is no different from that of the negative control on abraided and non-abraided areas at 24 hours and 72 hours after test application, The experiment indicates that capsaicin , the active ingredient from pepper extract, has a significant anesthetic effect. It is recommended that further studies be done on determining the specific concentrations of capsaicin and mineral oil in mixture that would exhibit optimum local anesthesia. Follow-up experiments should deal with precise measurement of capsaicin and mineral oil needed in a mixture by comparing the anesthetic effect of these test concentrations with the established effective concentration of 3:1. The duration of action of capsaicin- mineral oil mixture should also be determined to ensure comparability with drugs already in the market. More detailed studies on capsaicin of Capsicum frutescens should improve the chances of the emergence of a cheaper but possibly equally effective means of generating a local topical anesthesia.

VII. Bibliography Ganong, W, 1988. Review of Medical Physiology, 14th edition, Appleton and Lange, California. 1983. Hot Peppers and Substance P. Lancet. 1, 1198. Hayes, A., Oxford, A., Reynolds, M., Shingler, A.H., Skingle, M., Smith, C., and Tyers, M.B., 1984. The Effects of a Series of Capsaicin Analogues on Nociception and Body Temperature in the Rat. Life Sciences. 34, 1241-1248. Lundberg and Saria, A. 1983. Capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants. Nature. 302, 251-253. McCourt, R. 1981. Some Like it Hot. Discover. 12, 48-52. Lim-Navarro, Pilar. 1996. Consultation, Skin Irritability Test. Department of Medical Biology, Unilab, Mandaluyong City. Otten, U., Lorez, H.P., and Businger, F, 1983 Nerve growth factor antagonizes the neurotoxic action of capsaicin on primary sensory neurones. Nature. 301, 515517. Quisumbing, E. 1978. Medicinal Plants of the Philippines. Katha Publishing Company, Inc., Manila. Rosenburg, I., ed. 1986. Metabolism and Toxicity of Capsaicin. Nutrition Reviews. 44, 20-22.

Appendix A
0.25caps 0.50caps 0.75caps 1.0 caps EMLA MO 1 82.00 23.33 80.00 16.66 96.55 43.75 2 77.00 82.35 42.00 80.00 100.00 23.08 3 79.00 82.61 82.00 82.35 81.81 66.66 4 75.00 97.22 75.00 48.72 94.12 89.19 5 25.00 21.17 65.00 25.92 34.29 16.13 6 75.00 81.81 65.00 63.63 95.45 63.16 7 31.00 28.57 71.00 3.33 25.00 2.50 8 69.00 40.00 70.00 18.75 80.00 25.00 9 73.00 86.00 59.00 55.55 83.33 35.71 10 19.00 16.60 29.00 26.09 83.33 35.71 11 6.00 17.86 76.00 .00 40.00 27.77 Table ___. Listing of percentages of anesthetic effect obtained for the different treatment groups. --------------------- DESCRIPTIVE STATISTICS --------------------NO. NAME N MEAN STD. DEV. MINIMUM MAXIMUM 1 0.25caps 11 55.5455 28.7728 6.0000 82.0000 2 0.50caps 11 52.5018 32.9073 16.6000 97.2200 3 0.75caps 11 64.9091 16.3000 29.0000 82.0000 4 1.0 caps 11 38.2727 29.2740 .0000 82.3500 5 EMLA 11 73.9891 27.3027 25.0000 100.0000 6 MO 11 38.9691 25.1850 2.5000 89.1900 Table ___. Summary of descriptive statistics obtained for determination of % anesthetic effect of the various treatment groups. ---------------------- ANALYSIS OF VARIANCE ---------------------testing null hypothesis that all 6 treatment means are equal NUMBER OF CASES: 11 NUMBER OF VARIABLES: 6 ONE-WAY ANOVA GROUP 1 2 3 4 5 6 GRAND MEAN SOURCE BETWEEN WITHIN TOTAL MEAN 55.545 52.502 64.909 38.273 73.989 38.969 54.031 N 11 11 11 11 11 11 66 D.F. 5 60 65 MEAN SQUARE 2192.244 735.525 F RATIO 2.981 PROB. .0181 Treatment 0.25caps 0.50caps 0.75caps 1.0caps EMLA MO

SUM OF SQUARES 10961.219 44131.491 55092.711

F ratio needed for significance at 5% point = 2.73 F ratio computed = 2.981 2.981 > 2.73, therefore reject null hypothesis that all 6 treatment means are equal. Probability > than 95% that a statistically significant difference exists between the 6 treatment means. Proceed to compare experimental treatment means versus negative control. ------------------- HYPOTHESIS TESTS FOR MEANS ------------------NUMBER OF CASES: 11 NUMBER OF VARIABLES: 6 DIFFERENCE BETWEEN TWO GROUP MEANS: POOLED ESTIMATE OF VARIANCE GROUP 1 GROUP 2 MEAN = 55.5455 38.9691 STD. DEV. = 28.7728 25.1850 N= 11 11 DIFFERENCE = 16.5764 STD. ERROR OF DIFFERENCE = 11.5292 T= 1.4378 (D.F. = 20) GROUP 1: 0.25caps GROUP 2: MO

PROB. = .0830 insignificant difference ------------------- HYPOTHESIS TESTS FOR MEANS ------------------NUMBER OF CASES: 11 NUMBER OF VARIABLES: 6 DIFFERENCE BETWEEN TWO GROUP MEANS: POOLED ESTIMATE OF VARIANCE GROUP 1 GROUP 2 MEAN = 52.5018 38.9691 STD. DEV. = 32.9073 25.1850 N= 11 11 DIFFERENCE = 13.5327 STD. ERROR OF DIFFERENCE = 12.4943 T= 1.0831 (D.F. = 20) GROUP 1: 0.50caps GROUP 2: MO

PROB. = .1458 insignificant ------------------- HYPOTHESIS TESTS FOR MEANS ------------------NUMBER OF CASES: 11 NUMBER OF VARIABLES: 6

DIFFERENCE BETWEEN TWO GROUP MEANS: POOLED ESTIMATE OF VARIANCE GROUP 1 GROUP 2 MEAN = 64.9091 38.9691 STD. DEV. = 16.3000 25.1850 N= 11 11 DIFFERENCE = 25.9400 STD. ERROR OF DIFFERENCE = 9.0452

T=

2.8678

(D.F. = 20) GROUP 1: 0.75caps GROUP 2: MO

PROB. = 4.756E-03 = 0 .004756 highly significant ------------------- HYPOTHESIS TESTS FOR MEANS ------------------NUMBER OF CASES: 11 NUMBER OF VARIABLES: 6 DIFFERENCE BETWEEN TWO GROUP MEANS: POOLED ESTIMATE OF VARIANCE GROUP 1 GROUP 2 MEAN = 38.2727 38.9691 STD. DEV. = 29.2740 25.1850 N= 11 11 DIFFERENCE = -.6964 STD. ERROR OF DIFFERENCE = 11.6434 T= -.0598 (D.F. = 20) GROUP 1: 1.0 caps GROUP 2: MO

PROB. = .4765 insignificant ------------------- HYPOTHESIS TESTS FOR MEANS ------------------NUMBER OF CASES: 11 NUMBER OF VARIABLES: 6 DIFFERENCE BETWEEN TWO GROUP MEANS: POOLED ESTIMATE OF VARIANCE GROUP 1 GROUP 2 MEAN = 73.9891 38.9691 STD. DEV. = 27.3027 25.1850 N= 11 11 DIFFERENCE = 35.0200 STD. ERROR OF DIFFERENCE = 11.1995 T= 3.1269 (D.F. = 20) GROUP 1: EMLA GROUP 2: MO

PROB. = 2.655E-03 = 0.002655 highly significant

Skin Irritation Test Erythema Grading Erythema Grading with 0.75 capsain on with 0.75 capsain on abraded areas taken non-abraded areas after 24 hours taken after 24 hours Rabbit Erythema Rabbit Erythema Area Grading Area Grading 1 0 1 0 2 0 2 0 3 0 3 0 4 0 4 0 5 0 5 0 6 7 8 9 10 11 12 13 14 15 0 0 0 2 0 0 0 0 1 0 6 7 8 9 10 11 12 13 14 15 0 0 0 1 0 1 0 0 0 0

Erythema Grading Erythema Grading with control on with control on nonabraded areas taken abraded areas taken after 24 hours after 24 hours Rabbit Erythema Erythema Erythema Area Grading Grading Grading 1 0 1 0 2 0 2 0 3 0 3 0 4 0 4 0 5 0 5 0 SUMMARY Groups Erythema Grading A Erythema Grading B Erythema Grading C Erythema Grading D

6 7 8 9 10 11 12 13 14 15

0 1 0 0 0 0 0 0 0 0

6 7 8 9 10 11 12 13 14 15

0 0 0 0 0 0 0 0 0 0

Count 15 15 15 15

Sum 3

Average Variance 0.2 0.314286 0.12381

2 0.133333

1 0.066667 0.066667 0 0 0

ANOVA Source of Variation Between Groups Within Groups Total

SS 0.333333 7.066667 7.4

df

MS

P-value

F crit

3 0.111111 0.880503 0.456808 2.769433 56 59 0.12619

Erythema Grading Erythema Grading with 0.75 capsain on with 0.75 capsain on abraded areas taken non-abraded areas after 72 hours taken after 72 hours Rabbit Erythema Rabbit Erythema Area Grading Area Grading 1 0 1 0 2 0 2 0 3 0 3 0 4 0 4 0 5 0 5 0 6 0 6 0 7 0 7 0 8 0 8 0 9 1 9 0 10 0 10 0 11 0 11 1 12 0 12 0 13 0 13 0 14 1 14 0 15 0 15 0 Anova: Single Factor SUMMARY Groups Erythema Grading A Erythema Grading B Erythema Grading C Erythema Grading D

Erythema Grading Erythema Grading with control on with control on nonabraded areas taken abraded areas taken after 72 hours after 72 hours Rabbit Erythema Rabbi Erythema Area Grading Area Grading 1 0 1 0 2 0 2 0 3 0 3 0 4 0 4 0 5 0 5 0 6 0 6 0 7 1 7 0 8 0 8 0 9 0 9 0 10 0 10 0 11 0 11 0 12 0 12 0 13 0 13 0 14 0 14 0 15 0 15 0

Count 15 15 15 15

Sum

Average Variance 2 0.133333 0.12381 1 0.066667 0.066667 1 0.066667 0.066667 0 0 0

ANOVA Source of Variation Between Groups Within Groups Total

SS 0.133333 3.6 3.733333

df

MS

P-value

F crit

3 0.044444 0.691358 0.561155 2.769433 56 0.064286 59

*** Both Statistical tests for 24 hour and 72 hour periods show no significant difference between the 75% capsaicin and the control group.