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YCTIM-1253; No. of Pages 6 ARTICLE IN PRESS

Complementary Therapies in Medicine (2013) xxx, xxx—xxx

Available online at www.sciencedirect.com

ScienceDirect

jour nal homepage: www.elsevierhealth.com/journals/ctim

Efficacy and safety of topical Trikatu

preparation in, relieving bite

reactions: A randomized controlled trial

a b,c,d,e,∗

Ratree Maenthaisong , Nathorn Chaiyakunapruk ,

f g

Waree Tiyaboonchai , Apiwat Tawatsin ,

h g

Archawin Rojanawiwat , Usavadee Thavara

a

Clinical Pharmacy Research Unit, Department of Clinical Pharmacy, Faculty of Pharmacy, Mahasarakham

University, Mahasarakham, Thailand

b

Discipline of Pharmacy, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway 46150,

Selangor, Malaysia

c

Center of Pharmaceutical Outcomes Research, Faculty of Pharmaceutical Sciences, Naresuan University,

Phitsanulok, Thailand

d

School of Pharmacy, University of Wisconsin, Madison, WI, USA

e

School of Population Health, University of Queensland, Brisbane, QLD, Australia

f

Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University,

Phitsanulok, Thailand

g

The National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand

h

Clinical Research Center, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand

Received 6 June 2012 ; received in revised form 16 August 2013; accepted 20 August 2013

KEYWORDS Summary

Introduction: Trikatu is composed of dried fruits of Piper nigrum L and Piper retrofractum

Trikatu preparation;

Vahl, and dried rhizomes of Zingiber officinale R. Although this preparation has been used to

Mosquito bite;

relieve pruritis, pain, and inflammation for a long time, there is no clinical evidence to confirm

Papule size

reduction; its efficacy and safety. Therefore, we performed a double-blind, within person-randomized

controlled study of 30 healthy volunteers to determine efficacy and safety of topical Trikatu on

Mosquito bite

symptoms; mosquito bite reactions.

Methods: All subjects were bitten by laboratory mosquitoes on their forearms

Eucalyptus oil

and they were randomly assigned arms to apply either Trikatu or reference product on the

mosquito bite papule. The main outcome was the difference of papule size reduction at 30 min,

measured by a caliper, between the Trikatu and reference arms. Pruritis, redness, pain, and

patient satisfaction were assessed at 15, 30, 60, 180, and 360 min as secondary outcomes.

∗

Corresponding author. Tel.: +603 5514 4413; fax: +603 5514 63261.

E-mail addresses: [email protected], [email protected] (N. Chaiyakunapruk).

0965-2299/$ — see front matter © 2013 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ctim.2013.08.014

Please cite this article in press as: Maenthaisong R, et al. Efficacy and safety of topical Trikatu prepa-

ration in, relieving mosquito bite reactions: A randomized controlled trial. Complement Ther Med (2013), http://dx.doi.org/10.1016/j.ctim.2013.08.014

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YCTIM-1253; No. of Pages 6 ARTICLE IN PRESS

2 R. Maenthaisong et al.

Results: There were no significant differences between treatment and reference arms on any

outcome at any time of measurement.

Conclusion: Trikatu did not show additional effects for relieving mosquito bite reaction as com-

pared with the reference product containing camphor, menthol, and eucalyptus. For further

study, it is very important to consider a proper selection of subjects, comparator product, and

concentration of extract when Trikatu preparation is investigated.

© 2013 Elsevier Ltd. All rights reserved.

Introduction

of Medical Sciences, Thailand to compare the effects of a

topical Trikatu product with a reference product. The study

Trikatu is one of the popular Thai traditional preparations

was conducted in compliance with the principles of the Good

comprising of dried fruits of black pepper (Piper nigrum

Clinical Practice, in accordance with Declaration of Helsinki

Linn., family Piperaceae), long pepper (Piper retrofractum

and approved by the Institutional Review Boards of Nare-

Vahl., family Piperaceae) and dried rhizomes of ginger (Zin-

suan University. Thirty subjects participated to the study and

giber officinale Roscoe., family Zingiberaceae) in various

all of them provided informed consent before study partic-

ratio. The various formulas of Trikatu have been used to

ipation. This study was supported by the National Research

adjust patient’s element during rainy season for the treat-

Council of Thailand (NRCT) which had no role in study design,

1,2

ment of illness due to fire, wind, and water, respectively.

data collection and analysis, decision to publish, or prepa-

Trikatu is also used as Ayurvedic in India for a wide range

ration of the manuscript.

of diseases and symptoms such as cold, asthma, pruri-

3,4

tis, pain, inflammation. Dried rizhomes of ginger are

composed of various substances such as essential oils, 6- Subjects

gingerol, 6-shogaol, 6-gingessulfonic acid, gingerglycolipids

5—8

A, B and C. Ethanol extract of ginger has antioxidant,

Healthy person with age ≥18 years who had reactions to

anti-edema, antipyretic, and anti-inflammatory

mosquito bite (had papule and pruritis within 30 min after

8—13

effects. Dried fruits of black pepper are composed of

mosquito bites) were enrolled. Subjects were excluded if

piperine, chavicine, piperamine, piperidine, and volatile

they took oral or apply , corticosteroid, non-

14—16

oils. Ethanol extract and volatile oils of pepper have

steroid anti-inflammatory (NSAIDs) within 2 weeks before

17—21

anti-inflammatory, and anti-edema effects. Dried fruits

enrollment. In addition, drug abuse subjects, pregnant and

of long pepper are composed of piperine, pipernon-

breast-feeding women, as well as subjects who were known

15,22,23

aline, piperundecalidine, and dehydropipernonaline.

against product containing ginger, pepper, long pep-

Its ethanol extract and volatile oils have anti-inflammatory,

per, or other ingredients in study product or history of

14—26

and can decrease abdominal pain and peptic ulcer.

anaphylactic reaction from mosquito bites or insect sting

The main active ingredients of Trikatu are piperine from

were excluded. All included subjects came to participate in

black and long pepper dried fruits, and gingerol from

the experiment get their outcomes measured at the Clinical

5,27

ginger dried rhizomes. Although the Trikatu capsule

Research Unit on a date.

(500 mg) is now available commercially in India and other

preparations of Trikatu have been used in India and

1,2,4,5

Thailand for a long time in various conditions, the Randomization and interventions

majority of research was on pharmacology and toxicity with-

out any studies demonstrating clinical evidence of such

We randomly assigned arms of each subject to apply either

preparation.2,5,28—31

Trikatu product or reference product on the mosquito bite

Mosquito bites frequently cause skin symptoms includ-

papule. Randomized codes were generated by a computer

ing pruritis, redness, and papules. These symptoms are

using block size of 4. To conceal the researcher enrolling

mediated by antisavila IgE and reac-

(AT) and assessing (AR) participants, random allocation

32,33

tion leading to induce inflammatory process. Current

sequences and products were placed in sequentially num-

therapeutic options for treating mosquito bite associated

bered, opaque and sealed envelopes by a research assistant

symptoms are oral and topical steroids and

(PS) who was not involved in patient enrollment, product

antihistamine. Moreover, OTC products containing men-

distribution, or data collection. The Trikatu and reference

thol, eucalyptus have been used for relieving insect bite

products were similar in appearance and odor. Dosage form

34—37

reactions. It was postulated that the Trikatu product

of both products was stick.

exerting anti-inflammatory effects may have efficacy for

The treatment product was topical Trikatu preparation

controlling symptoms of mosquito bites. This study was con-

containing ethanol extract of Trikatu 1%, camphor 2%, men-

ducted to determine the efficacy and safety of this product

thol 2%, and eucalyptus oil 4%. The extract was obtained

on mosquito bite reactions.

from dried fruit of black and long pepper, and dried rhi-

zomes of ginger. The final product composed of piperine

Methods and gingerol of 0.074% and 0.046% w/w, respectively, as

determined by HPLC method. The reference product was

a stick preparation containing the same ingredients of the

A double blinded, within person-randomized, controlled

treatment product except Trikatu extract.

trial was conducted at Clinical Research Unit, Department

Please cite this article in press as: Maenthaisong R, et al. Efficacy and safety of topical Trikatu prepa-

ration in, relieving mosquito bite reactions: A randomized controlled trial. Complement Ther Med (2013), http://dx.doi.org/10.1016/j.ctim.2013.08.014

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YCTIM-1253; No. of Pages 6 ARTICLE IN PRESS

Efficacy and safety of topical Trikatu preparation in, relieving mosquito bite reactions: A randomized controlled trial 3

Mosquito exposure and measurement of bite

Table 1 Baseline demographic and clinical characteristics reactions

of subjects.

Mosquito-bite reaction was induced by Aedes aegypti labora- Average age; years (SD): 33.17 (6.95)

tory mosquitoes. One mosquito in a cage was allowed to bite Demographic characteristics

on the right or left forearm for 5 min. The reactions including Sex, n (%)

papule size, intensity of pruritis, erythema, and edema were Male 11 (36.7)

measured after biting for 30 min as baseline. The papule Female 19 (63.3)

size was measured (two perpendicular diameters in mm) 3 Occupation, n (%)

times per each arm by a physician using digital caliper, then Officer 7 (23.3)

areas of papules were calculated and average of them were Employee 21 (70.0)

reported. For irregularly shaped papules, the two perpendic- Own business 2 (6.7)

ular diameters were measured and the area was calculated Clinical characteristics

2

based on its multiplication. Symptoms of mosquito bite (pru- Size of papule, mm (SD)

ritis, erythema and edema) and other adverse reactions Treatment arm 22.84 (12.25)

(edema, burning and pain) were evaluated by a physician Reference arm 21.22 (13.66)

and subjects using visual analogue scale (VAS: range from 0 VAS of edema by physician (SD)

to 100 mm) and rating score (range from 0 to 3), respectively, Treatment arm 3.83 (1.34)

35—37

as previous described. Effects of Trikatu and refer- Reference arm 3.55 (1.28)

ence products were measured at 15, 30, 60, 180, 360 min VAS of erythema intensity by physician (SD)

after the applications. Physician measuring outcomes was Treatment arm 3.23 (1.68)

trained as general physicians, had been practicing medicine Reference arm 3.18 (1.78)

more than 10 years, and had attended continuing good

VAS = visual analogue score (mm).

clinical practice of randomized controlled trial training

programs.

The primary outcome was the difference of papule

size reduction, measured by a digital caliper, after appli-

cation Trikatu and reference products for 30 min. The Results

digital caliper (range; 0—300 mm, resolution; 0.01 mm)

has been used to determine the efficacy of drugs for

35—37 Thirty-seven subjects were screened for eligibility but

relieving mosquito bite symptoms in previous studies.

seven of them did not meet inclusion criteria. As a

Secondary outcomes were adverse reactions and other

result, 30 subjects were enrolled and included into the

mosquito bite associated clinical symptoms including pru-

intent-to-treat analysis (ITT analysis). There was no lost

ritis, erythema, and edema which were measured at

follow-up or drop out during study period (Fig. 1). Thirty

all measurement times by physician and subject using

VAS. subjects with average age of 33 years (SD; 6.95) were

included in the study. Sixty-three percents of them were

In addition, subject’s satisfactions were assessed as sec-

female and 70% of them were employee. Both treatment

ondary outcome at the end of study in various aspects

and reference arms had similar baseline characteristics

including overall efficacy, efficacy for reducing papule

(Table 1).

size, erythema, edema and pruritis, product cost, con-

At 30 min after application of each product, papule

tinuous use, and recommendation each product in the

future. size was not changed in both arms. However, reduction

of papule size in both arms was seen after application

of each product for 1 h and this reduction was statisti-

cally significant different from baseline at 3 h after product

Statistical analysis and sample size application (p-value < 0.05). However, there was no statis-

tically significant difference in percentage of papule size

The sample size required in this study was 30 partici- between the treatment and reference arms at all times

pants. The sample size calculation using within-person study of reduction measurement (Table 2). In addition, there

designed was based on a pilot study of 5 participants. The were no statistically significant differences in erythema,

sample size was calculated with type 1 error of 0.05% and edema, and pruritis symptoms between treatment and ref-

80% power to detect 37.94% difference in size reduction erence arms at any time of assessment (data not shown).

of papule (SD; 37.9) after application Trikatu product for Subject’s satisfactions in various aspects such as over-

30 min. This calculation has taken into account the possible all efficacy, efficacy for reducing papule size and reliving

withdrawal rate of 10%. erythema, edema, and pruritis symptoms of Trikatu prod-

All analyses used the intention-to-treat approach. uct were similar to those of reference product (data not

Descriptive statistics were used to describe characteris- shown). In terms of safety, there was no loss to follow

tics of enrolled subjects. Paired simple t-test was used to up or drop out due to adverse effects and no need for

compare size reduction of papule between treatment and additional drug to treat symptoms of mosquito bites dur-

reference arms. McNemar’s test was used to compare the ing study period. No signs and symptoms of allergic reaction

differences of binary outcomes between treatment and ref- related to study preparation at any time of assessment were

erence arms at any time of assessment. reported.

Please cite this article in press as: Maenthaisong R, et al. Efficacy and safety of topical Trikatu prepa-

ration in, relieving mosquito bite reactions: A randomized controlled trial. Complement Ther Med (2013), http://dx.doi.org/10.1016/j.ctim.2013.08.014

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YCTIM-1253; No. of Pages 6 ARTICLE IN PRESS

4 R. Maenthaisong et al.

Assess ed for eligibility (n=37)

Excluded (n=7)

Enroll ment ¨ Not meeting inclusion criteria (n=7)

- 5 persons have no reaction after mosquito bite

- 1 person continuously used NSAIDs

- 1 person received antihistamine during 2 weeks

Randomized side of arm within-person to receive each intervention

(n=30)

Allocatio n

Allocated to Trikatu product (n=30) Allocated to reference (euca lyptus based) product (n=30)

¨ Received allocated treatment product (n=30) ¨ Received allocated reference product (n=30)

Lost to follow-up (n=0) Follow -Up Lost to follow-up (n=0)

Discon tinued intervention (n=0) Discontinued intervention (n=0)

Analysis

Analysed (n= 30) Analysed (n=30)

Fig. 1 CONSORT 2010 flow diagram for Trikatu study.

Discussion insect stings (bees, wasps etc.) may increase chance of

Trikatu product to exert its full benefits. Third, the amount

of Trikatu in the preparation might be too low.

Our finding indicates that the efficacy of topical Trikatu

Current evidence has suggested that all ingredients in

preparation in reducing symptoms of mosquito bites is

2,4,5,13,30,43,44

Trikatu preparation were quite safe. Therefore,

similar to that of reference preparation. However, both

there was no need for performing irritation test before con-

preparations can reduce papule size and relieve erythema

ducting a clinical study. In addition, other ingredients in

intensity, edema and pruritis symptoms from mosquito bite

reaction. Trikatu product had been used in several current topical

38—40

products in the market and safety data of each agent

The absence of difference in efficacy between Trikatu

2,30,45

indicated minimal irritation effect. In this study, we

preparation and reference preparation can be explained

found no adverse events detected at all times of assessment,

by several reasons. First, the reference product contains

38—40 thereby supporting the safety and tolerability of topical use

eucalyptus which is good anti-inflammatory agent. In

Trikatu product.

addition, camphor and menthol have cooling effect that

41,42 The strength of our research work is that we performed

can reduce pruritis symptom of mosquito bites. This

and reported this clinical trial with high standard adhering

might lead to a small room for Trikatu product to exert its

46—48

to CONSORT statement. The sample size was calculated

anti-inflammatory effect. Second, the level of inflammatory

based on the pilot study and all analyses were performed

reaction induced by mosquito bites might be so low that

using intention to treat approach. We believe that our

it does not require the anti-inflammatory effects obtained

good adherence to the protocol ensure the high quality

from the combination of Trikatu and eucalyptus. Other con-

and internal validity of our findings. In addition, we chose

ditions with higher level of inflammatory reaction such as

Please cite this article in press as: Maenthaisong R, et al. Efficacy and safety of topical Trikatu prepa-

ration in, relieving mosquito bite reactions: A randomized controlled trial. Complement Ther Med (2013), http://dx.doi.org/10.1016/j.ctim.2013.08.014

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YCTIM-1253; No. of Pages 6 ARTICLE IN PRESS

Efficacy and safety of topical Trikatu preparation in, relieving mosquito bite reactions: A randomized controlled trial 5

Table 2 Effect of Trikatu and reference preparations on papule size of mosquito bites.

2 a b c

Time of assessment Papule size, mm (SD) Percent reduction (SD) Differences (SD) p-value

Treatment arm Reference arm Treatment arm Reference arm

Baseline 22.84 21.22 — — —

(12.25) (13.66)

15 min 26.75 23.75 −11.88 −4.00 −7.88 0.422

(19.25) (19.24) (30.82) (50.71) (52.98)

30 min 27.62 23.25 −8.85 1.93 −10.78 0.337

(24.48) (20.32) (55.61) (56.27) (60.54)

1 h 22.54 19.18 9.29 29.16 −19.87 0.09 (22.20) (20.51) (60.36) (48.73) (62.09) * **

3 h 10.49 6.93 59.93 67.52 −7.59 0.511

(17.64) (9.59) (55.32) (41.02) (62.46)

**

6 h 11.25 5.71 58.21 67.38 −9.18 0.619

(34.78) (10.53) (101.63) (58.73) (100.03)

*

p-value = 0.001.

**

p-value < 0.001 of each group when compared with baseline.

a

Percent reduction of papule size compared with baseline.

b

Mean differences of papule size reduction between treatment and reference arms.

c

p-value of comparison between treatment and reference arms.

reference product that was similar in appearance and odor Sisopa who generated randomisation codes, allocation

to treatment product to blind both physicians and subjects. sequences and products for this study.

However, the inflammatory and cooling effects of some

ingredients in reference product result in reduction of sen-

Appendix A. Supplementary data

sitivity to detect the specific effects of Trikatu in treatment

product.

Supplementary data associated with this article can be

A key lesson can be drawn from this research work.

found, in the online version, at http://dx.doi.org/10.1016/

Selection of the right comparator and subjects to be investi-

j.ctim.2013.08.014.

gated is the most crucial process. At early stage of product

development, choosing a placebo (without active ingredi-

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Please cite this article in press as: Maenthaisong R, et al. Efficacy and safety of topical Trikatu prepa-

ration in, relieving mosquito bite reactions: A randomized controlled trial. Complement Ther Med (2013), http://dx.doi.org/10.1016/j.ctim.2013.08.014