Senior High School Department

Malabon Campus
 Morphology and Identification of pain-inducing toxins in the stinging hairs

of Dendrocnide meyeniana (Walp.) Chew (Lipang-kalabaw) and Laportea interrupta (L.) Chew

(Lipang-aso)

A Research Proposal

for the course on

Practical Research 1 - STEM

Submitted by

Dizon, Christen Amber

Latonio, Jan Christine

Mangali, Kylie Marie

Ching, Michelle Renee

Teacher

March 27, 2018

 Abstract

Dendrocnide meyeniana (Walp.) Chew (Lipang Kalabaw) and Laportea interrupta (L.)

Chew (Lipang Aso) are two species in the Urticaceae family native to the Philippines. Both

plants, particularly their leaves, are covered by minute stinging trichomes that resemble

hypodermic needles that exude pain-inducing agents. Once human skin contacts the stinging

hairs of either plants, the irritants are released and induce cause contact dermatitis which are

characterized by intense itching, wheals or stinging sensation lasting up to 24 hours or more.

However, the nature and constituents of irritant-inducing toxins in the stinging hairs of D.

meyeniana and L. interrupta are poorly known. Therefore, in this study, the types, morphology

and distribution of trichomes on the leaves of both D. meyeniana and L. interrupta will be

examined. Bioassays and chemical analyses will also be undertaken to characterize the pain-

inducing agents of the stinging hairs of both species.

Keywords:

Dendrocnide meyeniana, dermatitis, Laportea interrupta, morphology, pain-inducing agents,

trichomes
 Table of Contents

Page

Introduction 2

Statement of the Problem 5

Purpose of the Study 6

Scope and Limitations 7

Significance of the Study 8

Materials and Methods 10

 CHAPTER 1

INTRODUCTION

1.1 Background of the study

Stinging trichomes received considerable scientific interest since they were first

described by Robert Hooke in 1665 (Thurston and Lersten, 1969; Pollard and Briggs, 1984; Lin

et al., 1991; Corsi, 1992; Fodor and Cseh, 1993; Tuberville et al., 1996). Each stinging trichome

usually consist of an elongated, tapered, stinging cell with a constriction just below the tip and a

bulbous base embedded in a multicellular sheathing pedestal of the epidermis. Although stinging

trichomes are commonly associated only with the nettle family (Urticaceae), the stinging

trichomes are also known to occur in the families of Euphorbiaceae, Loasaceae, and

Hydrophyllaceae (Thurston and Lersten, 1969).

In addition to their unusual structures, stinging trichomes are of interest because their

contents can cause toxic reactions ranging from mild skin irritation (contact dermatitis) to death,

depending on the contacting plant and animal species involved (Oliver et al., 1991; Taskila et al.,

2000). Toxins from stinging trichomes are known to incapacitate and even kill horses (Bennett,

1880; Bailey, 1906; Cleland, 1912; Aston, 1923). Aside from horses, dogs were reported to

stagger and vomit after contact with the stinging trichomes of an unidentified Urtica sp. (Clark,

1958; Davis, 1958; Hayes, 1958).

Many plants possessing stinging trichomes are considered dangerous. One should take

preventive measures such as learning to recognize the plants, avoiding them and wearing

protective clothing. Current treatments are mostly anecdotal and have varied results. Examples of

these treatments include application of crushed broad-leaved dock plants (Rumex obtusifolius
L.), sage (Sage officinalis L.), peppermint (Mentha x piperita L.), mud and toothpaste on the site

of skin exposed to the toxins of Urtica dioica L.

In Southeast Asian countries, Dendrocnide meyeniana (Walp.) Chew (Lipang-kalabaw)

and Laportea interrupta (L.) Chew (Lipang-aso) in the family Urticaceae are known to instigate

contact dermatitis upon contact of their stinging trichomes. In their natural habitats, the

trichomes hairs are known to contain chemicals that serve as defenses against herbivores (Pollard

and Briggs, 1984). Thus, when an animal brushes against the plant, the tip of stinging trichome

fractures and its broken end (often beveled like a hypodermic needle) penetrates the skin, and the

contents are expelled. The released contents will then induce an immediate and extreme reducing

pain and stinging sensation for some time (Chang, Shen, Wong, & Yen, 2009).

While many potential pain-inducing toxins have been proposed as the causative agents

originating from the stinging trichomes (Thurston and Lersten, 1969), such as histamine,

acetylcholine and serotonin (Emmelin and Feldberg,1947; Collier and Chesher, 1956; Oliver et

al., 1991; Taskila et al., 2000), none of these pain-inducing toxins in the stinging trichomes of D.

meyeniana and L. interrupta have never been systematically demonstrated. Furthermore, little

work has been done on the morphology of stinging trichomes of both species and most

knowledge about these trichomes are based on unsound references which are either superficial or

inaccurate. Due to these uncertainties, a systematic investigation is therefore required to clarify

the morphology and toxic elements in the stinging trichomes of Dendrocnide meyeniana and

Laportea interrupta.
1.2 Statement of the problem

The stinging trichomes have been studied for more than 300 years, however, much of the

work has been fragmentary and questionable, particularly that dealing with morphology and

toxicology. For instance, the morphology of stinging of Dendrocnide meyeniana (Lipang-

kalabaw) and Laportea interrupta (Lipang-aso) are poorly known while their pain-inducing

effects, particularly that of D. meyeniana, on human skins although are known to vary from mild

discomfort and swelling about the area of penetration to much more serious effects, their causes

are not truly understood. To date, most studies of the chemical contents of stinging trichomes

have focused on members of the Urticaceae, particularly Urtica spp., concluding that trichomes

of Urtica and related genera contained histamine, acetylcholine, and serotonin. These findings,

however, are now being questioned by studies using more modem instrumentation. Moreover,

only few researches have considered examining the stinging trichomes of D. meyeniana and L.

interrupta.

1.3 Purpose of the study

The aim of this study is to contribute to understanding of stinging trichomes morphology

and toxicology in general. The goal of this study is to examine the morphology of stinging

trichomes of Dendrocnide meyeniana (Walp.) Chew (Lipang-kalabaw) and Laportea interrupta

(L.) Chew (Lipang-aso), two representative Philippine plants under family Urticaceae and

characterize their pain-inducing contents. The objectives are:

● Study and examine the morphology of stinging trichomes of Dendrocnide meyeniana

(Lipang-kalabaw) and Laportea interrupta (Lipang-aso);

 ● Estimate fluid volume of single stinging hair of Dendrocnide meyeniana and Laportea

interrupta;

● Quantify and identify the molecules from the stinging trichomes of both D. meyeniana

and L. interrupta; and,

● Determine the pain-inducing agents in the stinging trichomes of D. meyeniana and L.

interrupta.

1.4 Scope and limitations

This study will used Dendrocnide meyeniana (Walp.) Chew (Lipang-kalabaw) and

Laportea interrupta (L.) Chew (Lipang-aso), two representative Philippine plants under family

Urticaceae. Both plants will be obtained from the province of Bataan by Mr. Virgilio Linis, a

faculty of De La Salle University-Manila. Only the leaves of these plants will be examined and

analyzed while preparations, data collection, chemical analyses and other necessary laboratory

works will be conducted at St. Joseph Hall’s laboratories located at De La Salle University, Taft

Avenue, Malate, Manila. Relevant literatures and other sources relevant to this study will be

provided by Mr. Linis. Meanwhile, research manuals will be consulted to test the study’s

validity.

This study will only focus on examining the morphologies of stinging trichomes from

leaf samples of D. meyeniana and L. interrupta. Characterization of plant extracts in comparison

with known standards will be attempted using UV-visible spectrophotometry. Since leaves of

both species have high number of large stinging trichomes covering the abaxial and adaxial

surfaces, it is also possible to obtain extracts of chemicals from the stinging trichomes alone. For

this purpose, active agents from stinging trichomes that are responsible for inducing pain will be
isolated and examined. However, results of this study pertaining to the toxicology of these plants

will not be used to addressing medical treatments.

1.5 Significance of the study

This study investigates the structural morphology of stinging trichomes found on these

plants which will lead to further understanding of the mechanism behind the stinging principle of

these structures. In addition, examining the external morphology of these trichomes may lead to

further refinement of their descriptions which could become useful in distinguishing the two

species or even their respective genera.

The existence of pain-inducing agents in the stinging trichomes of both D. meyeniana and

L. interrupta has never been systematically demonstrated. This study will therefore be conducted

to identify the persistent pain-inducing agents in the stinging trichomes of D. meyeniana and L.

interrupta.

The identification for possible long-lasting pain-inducing agents in the stinging trichomes

of D. meyeniana and L. interrupta are relevant, particularly in understanding the mechanism of

skin reaction after being stung. The results will help clarify pain-inducing agent which are

significant not only for evaluating clinical manifestations of symptoms after being stung but also

for treatment of patients with acute exposure.

Finally, all information to be gathered from this study will serve as reference data for the

mechanism underlying the induced pain caused by stinging trichomes. Pain sensations induced

by these trichomes are little understood and are issues which remain to be fully addressed. Same

data will also serve as a valuable information to improve practices among upland farmers

especially those living adjacent to the forest edge. Encounters with D. meyeniana and L.
interrupta are high among upland farmers in this country. Farmers accidentally brush their skins

against these plants and contact the leaves’ stinging trichomes. These inadvertent exposure to D.

meyeniana and L. interrupta cause stinging and burning sensation on the skin which create

unpleasant experience for the farmers. Proper management of farmlands especially along borders

adjacent to the forest edge is crucial to the survival of D. meyeniana and L. interrupta.
 CHAPTER 2

MATERIALS AND METHODS

2.1 Materials and equipment

Summary of materials and equipment below by task or activity is shown in Appendix 1.

2.1.1 Glassware, instruments, and other equipment

High-Performance Liquid Chromatography (HPLC), ultrasonic bath, Ultra-violet

(UV) detector, fine forceps, closed glass vials, test tubes, test tube racks, 200-500 µl

micropipettes, microcentrifuge tubes, stirring rod, 250 ml Erlenmeyer flasks, analytical

balance, magnetic stirrer, hot plate, Tender shoots of Dendrocnide meyeniana (Walp.)

Chew (Lipang Kalabaw) and Laportea interrupta (L.) Chew (Lipang Aso).

2.1.2 Chemicals and reagents

Extraction solvents of different polarity (aqueous, methanol, aqueous ethanol, 0.5

McFarland standard distilled water (dH20)), sodium bicarbonate (Na2O3), calcium oxalate

(CaC2O4), mercuric chloride (HgCl2), sodium formate (HCOONa), sodium hypochlorite

(NaCIO) and potassium permanganate (KMnO4)

2.2 Preparation of plant samples

Three hundred grams of tender shoots from four plants of Dendrocnide meyeniana

(Walp.) Chew (Lipang Kalabaw) and Laportea interrupta (L.) Chew (Lipang Aso) with

trichomes will be obtained. Each plant of Dendrocnide meyeniana and Laportea interrupta will

be sourced from Bataan, Rizal and Laguna provinces, Philippines, respectively. The plant
materials will be cleaned of ectoparasites and debris before being subjected to laboratory

analyses.

One hundred grams of the plant materials will be used for estimating fluid volume of

single stinging hairs of Dendrocnide meyeniana (Walp.) Chew (Lipang Kalabaw) and Laportea

interrupta (L.) Chew (Lipang Aso). Trichomes from these plant materials will be removed using

fine forceps to be able to extract the fluid or toxins from their stinging hairs.

The remaining 200 grams of the plant material will be air-dried for 7-15 days in the shade

at 27-37 degrees Celsius. These will be powdered mechanically using commercial electrical

stainless-steel blender and extracted with ethyl alcohol (400 ml) and acetone (200 ml). The

residue obtained will be stored at 4 degrees Celsius as materials for the HPLC-UV analyses,

determination of formic acid and determination of oxalic acid assays.

2.3 Laboratory procedures

2.3.1 Estimation of fluid volume of single stinging hairs

Thurston (1969) classified nettle stinging hairs which are regarded as trichomes as

sessile glands which secreted an unidentified sap. These saps will be removed from the

trichomes of Dendrocnide meyeniana (Walp.) Chew (Lipang Kalabaw) and Laportea

interrupta (L.) Chew (Lipang Aso) using standard solid-liquid extraction or also known as

maceration as the extraction method. A certain volume of the plant sample will be placed

in a closed glass vial together with extraction solvents of different polarity – aqueous,

methanol, aqueous ethanol or water (Vajic et al., 2015). The sample will then go through

ultrasonic bath in varying extraction times at room temperature and will be filtered

afterwards. A certain reagent will be added in a definite volume of the extract and will be
incubated for a couple number of hours. The total composition of the hair will be then

estimated using a spectrophotometer.

2.3.2 HPLC-UV analysis

In this study, the High-Performance Liquid Chromatography (HPLC) coupled with

Ultra-violet (UV) detector will be used for the quantification and identification of the

molecules from the stinging trichomes of both Dendrocnide meyeniana and Laportea

interrupta. This will be done using chromatographic techniques in the mobile phase based

on the chemical, physical, and biological properties of the plants pain-inducing plants. On

the other hand, the UV light will act as the detection technology by subjecting the known

concentrations of the molecule of interest through the UV detector and measuring the

response afterwards. The various responses that were gathered will then be plotted versus

the concentration of the plants’ molecules and the HPLC-UV chromatograms will be

obtained. Specifically, this method aims to analyze the formic acid and oxalic acid found in

Dendrocnide meyeniana and Laportea interrupta. These acids are usually found and

gathered in the stinging nettles of plants which in this case, from the trichomes of the

plants.

2.3.3 Determination of formic acid assay

Formic acid or HCOOH or CH2O2 is important in the synthetization of chemicals.

Since this study is about the stinging trichomes of Dendrocnide meyeniana (Walp.) Chew

(Lipang Kalabaw) and Laportea interrupta (L.) Chew (Lipang Aso), it aims to establish the

connection between formic acid and the stinging sensation and irritation caused by the
stinging hairs of these plants on people’s skin. Aside from using the HPLC-UV to analyze

this acid, the colorimetric micromethod will also be used. In this method, standard sodium

formate solution with 0 to 30 micrograms per mL will be prepared. This solution would

then be diluted a couple of times to connect the colorimetric readings and the concentration

of the acid. The solution will then be mixed with mercuric chloride reagent and heated on a

steam bath. Afterward this solution will be cooled down and precipitated. The precipitation

of the mercurous chloride from the reaction will be observed to allow for the determination

of the amount of formic acid in the toxins.

2.3.4 Determination of oxalic acid assay

Alongside HPLC-UV analysis, determination of oxalic acid will be carried out

using titration method. Oxalic acid or oxalate will be obtained in the part of the plant with

the highest concentration which is the stinging hairs or trichomes. A standard solution of

oxalic acid will then be prepared by dissolving 100 g of it in distilled water while also

diluting it in a 100 ml of the said water. Another way to extract oxalic acid from the plant

material is by using sodium carbonate (Na2O3) followed by an overnight precipitation with

calcium oxalate. Afterwards, the oxalic acid will be quantified by titration with standard

potassium permanganate. In this process, the concentration of the oxalic acid will be

determined by adding the known amount of base and an indicator, which is

phenolphthalein. When the indicator changes color, the acid changed from acidic to basic

and the concentration and volume of the base added will then be determined. Afterwards,

the concentration of the acid itself can now be calculated.

 2.3.5 Disposal of wastes

To prevent the disposal of pathogens and the arising of infections, glasswares and

laboratory materials will be subjected to autoclaving at 121oC after the experiment. In

addition, all micropipette tips and residues will be immersed in sodium hypochlorite.

2.4 Data analyses

Differences among means of the sample replicates will be determined using one-way

analysis of variance (ANOVA) at 5 % level of significance.

2.5 Research Design

Four laboratories analyses (A1 to A4) will be conducted in this study. These will be

applied to four samples of Dendrocnide meyeniana (DM) and Laportea interrupta (LI) plant

materials collected from the three sites (S1 to S3) totaling to 12 samples overall (Appendix 2).

2.6 Flow Chart

The entire experiment will consist of several major tasks. The workflow of each of this

task is revealed by a flow chart shown in Appendix 3. In this chart, the tasks are depicted in

boxes and their order by connecting the boxes with arrows.

2.7 Work Schedule

This study will have a duration span of one year starting January 2018 and ending by

December 2018. The study’s schedule is revealed by a GANTT chart in Appendix 4. The chart

lists all the major tasks of this project on the vertical axis while the time intervals (in months) are
on the horizontal axis. Extent of the shaded horizontal bars indicate the duration of each task

activity.

2.8 Projected Budget

The projected total budget for this study is estimated at Php 9700.0 (Appendix 5). This

budget estimate is split into components as follows: Equipment and supplies (Php 6600.0);

Operating cost (Php 1700.0) and Miscellaneous cost (Php 1400.0). Most are considered direct

costs which will be shouldered by the proponents except for some items under the operating and

miscellaneous costs which may be requested for funding.

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Appendix 1. List of required materials, equipment and other supplies.

A. Laboratory supplies and equipment

Preparation of plant Estimation of fluid HPLC-UV analyses Determination of Determination of Data analyses
samples volumes of single formic acid assay oxalic acid assay
stinging hairs

1. 1200 g of 1. Closed glass 1. HPLC 1. Steam bath 1. Test tubes 1. computer

Dendrocnide vials equipment: 2. Test tubes 2. Test tube racks 2. IBM SPSS
meyeniana 2. Ultrasonic bath pump; injector; 3. Test tube racks 3. Test tube Statistics ver. 22
2. 1200 g of 3. Filter paper column; detector; 4. Test tube holders
Laportea 4. Funnel and, computer holders 4. Stirring rods
interrupta 5. Spectrophotome 2. Filter paper 5. Stirring rods
3. Collecting bags ter 3. Storage container
4. Weight scale 6. Analytical 4. Aluminum foil
5. forceps balance 5. Aluminum tray
6. Blender 7. Glass storage 6. pH meter
7. Laboratory containers 7. 500 ml Beakers
spatula 8. Stirring rod
8. Erlenmeyer
flasks

B. Chemicals and other materials

1. Ethyl alcohol 1. Extraction 1. Sodium formate 1. Sodium

2. Acetone solvents solution carbonate
(aqueous, 2. Mercuric chloride 2. Potassium
methanol, Permanganate
aqueous ethanol, 3. Penolphthalein
distilled water
Appendix 2. Research design and analyses descriptions per site. Site: S1 = Bataan province; S2

= Rizal province and S3 = Laguna province.

SAMPLES (S1 = Morong)

(DM = Dendrocnide meyeniana (Walp.) Chew;
ANALYSES LI = Laportea interrupta (L.) Chew)

1 2 3 4

Estimation of fluid DM1/S1 DM2/S1 DM3/S1 DM4/S1

volumes of single
stinging hairs (A1) LI1/S1 LI2/S1 LI3/S1 LI4/S1

HPLC-UV analyses DM1/S1 DM2/S1 DM3/S1 DM4/S1

(A2) LI1/S1 LI2/S1 LI3/S1 LI4/S1

Determination of DM1/S1 DM2/S1 DM3/S1 DM4/S1

formic acid assay (A3) LI1/S1 LI2/S1 LI3/S1 LI4/S1

Determination of DM1/S1 DM2/S1 DM3/S1 DM4/S1

oxalic acid assay (A4) LI1/S1 LI2/S1 LI3/S1 LI4/S1

SAMPLES (S2 = Rizal)

(DM = Dendrocnide meyeniana (Walp.) Chew;
ANALYSES LI = Laportea interrupta (L.) Chew)

1 2 3 4

Estimation of fluid DM1/S2 DM2/S2 DM3/S2 DM4/S2

volumes of single
stinging hairs (A1) LI1/S2 LI2/S2 LI3/S2 LI4/S2

HPLC-UV analyses DM1/S2 DM2/S2 DM3/S2 DM4/S2

(A2) LI1/S2 LI2/S2 LI3/S2 LI4/S2

Determination of DM1/S2 DM2/S2 DM3/S2 DM4/S2

formic acid assay (A3) LI1/S2 LI2/S2 LI3/S2 LI4/S2

Determination of DM1/S2 DM2/S2 DM3/S2 DM4/S2

oxalic acid assay (A4)
LI1/S2 LI2/S2 LI3/S2 LI4/S2
 SAMPLES (S3 = Laguna)
(DM = Dendrocnide meyeniana (Walp.) Chew;
ANALYSES LI = Laportea interrupta (L.) Chew)

1 2 3 4

Estimation of fluid DM1/S3 DM2/S3 DM3/S3 DM4/S3

volumes of single
stinging hairs (A1) LI1/S3 LI2/S3 LI3/S3 LI4/S3

HPLC-UV analyses DM1/S3 DM2/S3 DM3/S3 DM4/S3

(A2) LI1/S3 LI2/S3 LI3/S3 LI4/S3

Determination of DM1/S3 DM2/S3 DM3/S3 DM4/S3

formic acid assay (A3) LI1/S3 LI2/S3 LI3/S3 LI4/S3

Determination of DM1/S3 DM2/S3 DM3/S3 DM4/S3

oxalic acid assay (A4) LI1/S3 LI2/S3 LI3/S3 LI4/S3
Appendix 3. Flow chart.

Collection of Dendrocnide
meyeniana and Laportea interrupta
plant samples

Estimation of fluid volumes of

single stinging hairs

HPLC-UV Analyses

Determination of formic acid assay

Determination of oxalic acid assay

Data analysis
Appendix 4. GANTT chart.

Tasks/ Activities 2018

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
1) Literature search/ review

2) Draft writing of the Introduction and RRL

3) Draft writing of methodology

4) Collection of plant samples

5) Estimation of fluid volumes of single

stinging hairs
6) HPLC-UV analyses

7) Determination of formic acid assay

8) Determination of oxalic acid assay

9) Disposal of wastes

10) Data analyses

11) Research paper write-up

12) Final defense

13) Manuscript forwarded for publication

Appendix 5. Budget estimates.

Equipment and supplies

Ultrasonic bath, spectrophotometer, high-performance liquid PhP 1000.0
chromatography, ultra-violet detector rental costs
Chemicals (ethyl alcohol, acetate, aqueous, methanol, distilled water, 3500.0
acetone, methanol, dechlorinated water)
Consumables (plastic bags, aluminum trays, filter paper, etc.) 500.0
General lab supplies 200.0
Sterile sampling supplies 500.0
Fieldwork equipment and materials 200.0
Herbarium kit and supplies 700.0

Operating cost
Cost for extracting of fresh samples at DLSU PhP 1000.0
Cost for collecting trichomes from plant samples 600.0
Transport permit for plant samples 100.0

Miscellaneous cost
Printing costs PhP 400.0
Purchasing books and/ or periodicals 1000.0

TOTAL PhP 9700.0