PROTEIN KINASE INHIBITORY PROPERTIES OF EXTRACTS DERIVED FROMBOCCONIA FRUTESCENS AND GOMPHOCARPUS PHYSOCARPUS
Kūpa‘aikekaiao Thomas1, Mengke Zhang1, Justin Reinicke1, Jimmy Parker3, Donna Ohora2,
Marisa M. Wall4, Thanapat Songsak5, Supakit Wongwiwatthananukit2, and Leng Chee Chang1,*

1

Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, Universityof Hawai‘i at Hilo, 34 Rainbow Drive, Hilo, HI 96720, USA
2
Department of Pharmacy Practice, The Daniel K. Inouye College of Pharmacy, University of
Hawai‘i at Hilo, 34 Rainbow Drive, Hilo, Hilo, HI 96720, USA
3
Early Detection Program Coordinator, Big Island Invasive Species Committee, 23 East Kawili
Street, Hilo, HI 96720, USA
4
United States Department of Agriculture, Agricultural Research Service, Daniel K. Inouye U.S.
Pacific Basin Agricultural Research Center, Hilo, HI 96720, USA
5
Department of Pharmacognosy, Faculty of Pharmacy, Rangsit University, Pathumtani 12000,
Thailand*Corresponding author: E-mail: lengchee@hawaii.edu

1Abstract: Bocconia frutescens is an invasive shrub originally introduced to Hawai’i as an
ornamental plant. Gomphocarpus physocarpus is a shrub that is native to southern Africa, also
introduced to Hawai’i. These two plants were selected for study due to the possibility of alkaloid
contents and biological activities. The objective of this study was to examine the protein kinase
inhibitory activity (PKI) of crude methanol extracts derived from Bocconia frutescens andGomphocarpus physocarpus using hyphae formation inhibition assay against Streptomyces 85E
at concentrations of 20 μg/disk. Extracts that showed protein kinase inhibitory activity at thisconcentration were tested at lower concentrations. Results found that all B. frutescens extracts
showed PKI activity, whereas none of the G. physocarpus showed biological activity. In
addition, the presence of chelerythrine and berberine alkaloids in B. frutescens extracts wereassessed during this study using thin layer chromatography (TLC) and liquid chromatographymassspectrometry (LC-MS) analysis. The results showed that both alkaloids were present in all
B. frutescens extracts.

Keywords: Protein kinase inhibition, hyphae formation inhibition assay, Bocconia frutescens,
Gomphocarpus physocarpus

2

INTRODUCTION
Bocconia frutescens, or plume poppy in English, is native to Mexico, Central America,and South America. Practitioners of traditional Mexican medicine referred to B. frutescens as“gordolobo” or “llorasangre.” The indigenous people of Mexico used various parts of this plant
to treat dermatitis, skin ulcers, tuberculosis, and some respiratory tract infections (SánchezArreola et al., 2006). The sap of this plant has also been used to make anesthetics and treat
bronchitis (Quattrocchi, 2012).
Bocconia frutescens is an invasive shrub that was introduced to Hawai‘i as an ornamental
plant (Sánchez-Arreola et al., 2006). Subsequently the State of Hawai’i classified B. frutescens
as a noxious weed. Infestations exist on the islands of Maui and Hawai’i. The Maui infestation
covers the southern and western slopes of Haleakalā. The Hawai’i island infestations are locatedin the Ka’ū and South Kona districts. Dense B. frutescens populations have been found on
Eucalyptus plantations. Native forests and sugar cane lands have also harbored B. frutescens
populations (Benitez et al., 2007).
Extracts from B. frutescens displayed antimicrobial activity against a number ofpathogenic bacterial species including Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa, and Bacillus subtilis (Sánchez-Arreola et al., 2006). The leaves, stems, and roots
contain phytochemicals that exhibited antibacterial properties against these pathogens (Yu et al.,2014). The compounds with antimicrobial activity include alkaloids such as berberine,
chelerythrine, columbamine, and protopine (Tavares et al., 2014; Stermitz et al., 2000; Liang etal., 2014; Su et al., 2011).
Gomphocarpus physocarpus, or balloon plant, is a shrub that is native to southern Africa
that has spread worldwide. G. physocarpus has been introduced to Australia, China, Hawai’i

3

India, and Mexico among other places. This plant invades pastures and kills livestock in Hawai’iand Australia (Rojas-Sandoval & Acevedo-Rodríguez, 2016).
Africans used all parts of this plant in traditional medicine to treat warts (Munsamy &Naidoo, 2015). They also used the roots to treat stomach aches and the leaves to treat headaches.
G. physocarpus has a variety of African names including balbossie, balmelkbossie,
umsingalwesalukazi, uphuphuma, and usingalwesalukazi (Rojas-Sandoval & AcevedoRodríguez, 2016).
The plant produces a white latex which is toxic when ingested. The laticifers containalkaloids and phenols. Carbohydrates, cardiac glycosides, terpenoids, steroids, phenols, andalkaloids have all been found in crude ethanolic extracts of G. physocarpus. These compounds
could contribute to the toxic properties of the latex (Munsamy & Naidoo, 2015). The methanoland n-hexane extracts from the fruits of the closely related G. fruticosus have shown activity
against P. aeruginosa with a minimum inhibition concentration (MIC) of 31 µg/mL (Madureira
et al., 2011).
It is well known that eukaryotic kinases play a vital role in signal transduction andsignaling pathways by phosphorylating their substrates. Recently, protein phosphorylation oftyrosine has been reported in bacterial species, which might be associated with developmental
processes (Bakal, 2000). Further studies by Bakal (2001) showed that Streptomyces have Signal
Transducer and Activator of Transcription (Stat3)-like proteins similar in amino acid sequencesurrounding a phosphorylated tyrosine which plays a role in cellular regulation in
Streptomycetes. Abnormal phosphorylations by these kinases can lead to diseases (Bakal, 2002;
Shanbhag et al., 2015). Recently, protein phosphorylation kinase inhibitors have been identified
using an assay based on the inhibition of hyphae formation against Streptomyces 85E (Waters et4

al., 2002; Shanbhag et al., 2015). Protein kinases play a role in the formation of aerial hyphae,
spores, and pigmentation in Streptomyces. Additionally, protein kinases have a role in antibiotic
production in some strains. Certain compounds, including eukaryotic kinase inhibitors, exhibit
protein kinase inhibition properties because they can inhibit these kinases (Shanbhag et al., 2015;
Waters et al., 2002). Kinase inhibitors are of significant pharmaceutical interest as potential
breakthroughs for treating diseases including cancers, in part because abnormal phosphorylations
by protein kinases have caused these diseases (Shanbhag et al., 2015). Chelerythrine has shown
cytotoxic activity against human cancer cells because of its ability to inhibit protein kinases(Chmura et al., 2000).
In our continuing search for plant-derived protein kinase inhibitors, a methanol extract ofB. frutescens and G. physocarpus were prepared and selected for further study.

MATERIALS AND METHODS
Plant material
The plant specimens, B. frutescens and G. physocarpus, were collected from Miloli’i,
Hawai’i, USA on July 5, 2016. The plant materials were identified by Jimmy Parker (Early
Detection Program Coordinator, Big Island Invasive Species Committee). The voucher
specimens (No. 0001 and 0002) were deposited at the Natural Products Chemistry Laboratory at
the Daniel K. Inouye College of Pharmacy, University of Hawai’i at Hilo. The plant materials
were air dried for 7 days at the laboratory of DKICP, UH Hilo. The air dried plant materials wereseparated and cut into smaller pieces. Most of these pieces were between one and fivecentimeters long. The plant materials that were used are listed in Table 1.

5Extractions
Samples (50 grams each) of the plant material were individually extracted in 1800 mL of
methanol for 24 h three times. The supernatants were filtered with (VWR Whatman No. 417)
filter paper. The combined filtrates were then concentrated using a rotary evaporator under avacuum at 45° C. Table 1 lists the weight of the dried crude extracts.

Inhibition of hyphae formation assay in Streptomyces
The inhibition of hyphae formation assay in Streptomyces 85E was performed on extracts
through an agar well diffusion method described previously (Waters et al., 2002). The mycelia
fragments of Streptomyces were spread on minimal medium ISP4 agar plates to produce abacterial lawn. Extracts and partitions of known concentration dissolved in DMSO weredispensed onto disks in 20 L aliquots. The air-dried disks were applied directly onto the plates
and incubated at 30o C. After 48 hours of incubation, zones of inhibition were evaluated, and theresults were identified by a clear zone of inhibition and/or bald phenotype around the disk. An
inhibition zone greater than 8 mm is considered active. Samples were tested at 80 g/disk on 7
mm filter disks. Active extracts and compounds were tested at lower concentrations of 40, 20,
10, and 5 g/disk (Waters et al., 2002). DMSO solvent (Sigma-Aldrich Co., St. Louis, MO,
USA) was used as a negative control. Berberine chloride and chelerythrine chloride standards(Sigma-Aldrich Co., St. Louis, MO, USA) were used as positive controls.

Chemicals and Analysis
The analytical thin layer chromatography (TLC) was performed on Merck precoated silica gel 60
F254 plates. The crude organic extracts from B. frutescens and G. physocarpus and compounds

6berberine chloride and chelerythrine chloride were chromatographed on the TLC plate. Plates
were developed in the solvent system chloroform-methanol (10:1). Dragendorff’s reagent wasused to detect the alkaloid contents (Nayeem et al., 2011). High-performance liquid
chromatography (HPLC) grade acetonitrile (Sigma-Aldrich Co, St. Louis, MO, USA) and HPLC
grade water were used for the LC-MS analysis.

Performing LC-MS and MS/MS on extracts that displayed antibacterial activity
In this study, liquid chromatography mass spectrometry (LC-MS) analysis was performed on an
Agilent 6530 Q-TOF mass spectrometer with an Agilent HPLC 1260 binary pump. A Waters
Sunfire C18 column (3.5 m, 2.1  150 mm) was used as the stationary phase. The mobile phase
was run at a gradient with 1% formic acid in water and acetonitrile at a flow rate of 0.2 mL/min.
The mass spectrometer was equipped with an Agilent Jet Stream electrospray ionization (ESI)
source. LC-MS was performed in positive ionization on a mass range of m/z 100 – 1700 at a scanrate of 3 spectra/second. The capillary voltage set at 3500 V. Nebulizer pressure was set at 35
psi. Drying gas temperature was set at 300° C with a flow rate of 8 L/min, and the sheath gas
temperature was 350° C with a flow rate of 11 L/min. The MS/MS conditions were set toautomatically collect at a relative threshold of 0.010%, and the collision energy was ramped witha slope of 5 and an offset of 2.5.

7

RESULTS AND DISCUSSION
All crude extracts were evaluated against protein kinase inhibition activity using hyphaeformation inhibition assay against Streptomyces 85E (Table 1). The negative control, DMSO
solvent, did not inhibit hyphae formation while both of the positive controls, berberine chloride
and chelerythrine chloride, inhibit hyphae formation inhibition. Chelerythrine chloride showed
more potent protein kinase inhibition activity against Streptomyces 85E than berberine chloride.
None of the G. physocarpus extracts showed protein kinase inhibition activity at the highest
concentration that was used during this study (80 μg/disk). Therefore, the G. physocarpus parts
that were tested have very limited, if any, protein kinase inhibition properties. Extracts from thestems and roots of B. frutescens showed biological activity at 80 μg/disk and 40 μg/disk, but theroots showed stronger activity against Streptomyces 85E than the stems. The leaves of B.
frutescens did not display any biological activity after 48 h, but they did show some activity after
24 h. The activity was only measured quantitatively after 48 h.
A preliminary TLC analysis indicated the presence of alkaloids in all crude extracts of B.
frutescens using Dragendorff’s reagent. During TLC analysis spraying with Dragendorff’s
reagent, compounds found in the crude extracted formed spots in similar positions to those foundin the standards providing evidence for the presence of both compounds in the crude extracts.
Other red and orange marks, beside the standards, were present for the crude extracts providing
evidence for the existence of other alkaloids in B. frutescens.
In addition, the compounds of the organic extract of B. frutescens (leaves, roots andstems) were analyzed using HPLC-ESI-QTOF-MS/MS against two standards (chelerythrine andberberine). LC-MS mass spectrometry analysis also supported the presence of both alkaloids in
the B. frutescens crude extracts. Compounds with (m/z) values of the chelerythrine standard,8348.1236, and the berberine standard, 336.1236, were found in all three B. frutescens extracts. Of
note, the same retention time of these respective alkaloids eluted with the same solvent systems
matched with the signals observed with the B. frutescens crude extracts. MS/MS fragmentation
data provided further evidence for the presence of chelerythrine and berberine in the extracts
derived from B. frutescens. The fragmentation profile of the chelerythrine and berberine
standards were also observed in all three extracts. In conclusion, by matching retention time,
mass to charge ratio (m/z) and fragmentation pattern, it was evident that B. frutescens extracts
contain chelerythrine and berberine.

Table 1. Plant Materials and Extracts
SamplesPart

Extract (g)Bocconia frutsecens

Stems

1.8

Leaves

7.5

Roots

5.1

Stems

2.2

Leaves

7.9

Roots

1.7

Seed Capsules*

5.3

Gomphocarpus physocarpus*Seeds were removed from their seed capsules. The antibacterial activity of the seeds was not evaluated during thisstudy due to paucity of materials

9Table 2. Biological Activity of Tested Extracts and Compounds on Growth and Sporulation ofStreptomyces 85E
Zone of Inhibition at Concentrations of Samples (mm) (mean ± SD)
Sample80 μg/disk

40 μg/disk

20 μg/disk

10 μg/disk

5 μg/disk

B. frutescens

B: 17.7 ± 1.5

B: 9.7 ± 1.2

0

NT

NT

B. frutescens

B&C: 20.0 ±B: 13.3 ± 0.6

0

NT

NT

Root Extract

1.0
0

NT

NT

NT

NT

0

NT

NT

NT

NT

0

NT

NT

NT

NT

0

NT

NT

NT

NT

0

NT

NT

NT

NT

B: 8.6 ± 0.3

0

NT

NT

NT

C: 26.6 ± 0.6

B&C: 26.0 ±B: 20.7 ±B: 15.0 ±B:14.7 ±

1.0

0.6

1.0

0.6

Stem Extract

C: 13.7 ± 2.1
B. frutescens
Leaf Extract
G. physocarpus
Stem Extract
G. physocarpus
Root Extract
G. physocarpus
Leaf Extract
G. physocarpus
Seed Pod Extract
Berberine
chloride standard
Chelerythrine
chloride standard

C: 18.7 ± 0.6
Stock solutions were prepared in DMSO. No zone of inhibition was observed with DMSO as a negative control. All
extracts and compounds were tested at 80 μg/disk. Active extracts were tested at lower concentrations (20, 10, 5
μg/disk). B indicates bald phenotype. C indicates clear phenotype. B and C indicates that a clear phenotype waswithin a balding phenotype. NT indicates no test necessary, because substance showed no activity at higher
concentrations.

10

x10 7+ESI TIC MS

Berberine

Abundance (Ion counts)

0
x10 8

+ESI TIC MS

Chelerythrine

0
0
x10 7

+ESI TIC MS

Leaves0x10 8 +ESI TIC MS

Roots

0
x10 8 +ESI TIC MS

Stems

0
1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

Time (min)Figure 1. Total ion chromatogram of berberine, chelerythrine, and extracts derived from B.
frutescens (leaves, roots and stems).

x10 6+ESI EIC (348.1100-348.1350) MS

5Chelerythrine

2.50Abundance (Ion counts)

x10 6
4

+ESI EIC(348.1100-348.1350) MS

Leaves20

x10 6+ESI EIC(348.1100-348.1350) MS

Roots

5

0

x10 6

+ESI EIC(348.1100-348.1300) MS

Stems

4
2
0
1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

Time (min)Figure 2. Extracted ion chromatogram of chelerythrine, and extracts derived from B. frutescens(leaves, roots and stems).

11

Table 3. Accurate Mass (m/z) of Chelerythrine
SampleChelerythrine
Roots
Stems
LeavesObserved m/z
348.1237
348.1224
348.1223
348.1242

Theoretical m/z
348.1236
348.1236
348.1236
348.1236

Difference (ppm)
-0.34
3.40
3.69
-1.77

Table 3 indicates retention time, observed m/z and calculated m/z, relative errors of both thestandards and samples. Standard chelerythrine yielded an observed [M + H]+ peak at a (m/z) of
348.1237 (Table 3). By comparison, crude organic extract of B. frutescens leaves ([M + H]+ peakat a (m/z) of 348.1242], stems ([M + H]+ peak at a (m/z) of 348.1223], and roots ([M + H]+ peakat a (m/z) of 348.1224] produced peaks around that mass. A difference of 10 ppm was the cut off
for homology. These (m/z) values were obtained from the graph in Figure 2. The peakcorresponding to chelerythrine exhibited mass-to-charge (m/z) ratios corresponding to themolecular ions (M + H)+ of standard chelerythrine, confirming the presence of this alkaloid in all
three parts of plant materials.

12

Abundance (Ion counts)

x10 6
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

+ESI Product Ion (rt: 13.112 min) Frag=175.0V CID@20.0 (348.1226[z=1] -> **)333.0977
290.0800

304.0955

318.0753

286 288 290 292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330 332 334 336 338 340 342 344 346 348 350 352 354 356

x10 6
3.75
3.5
3.25
3
2.75
2.5
2.25
2
1.75
1.5
1.25
1
0.75
0.5
0.25
0

+ESI Product Ion (rt: 13.038 min) Frag=175.0V CID@20.0 (348.1213[z=1] -> **)

x10 7
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

+ESI Product Ion (rt: 13.024 min) Frag=175.0V CID@20.0 (348.1396[z=1] -> **)

x10 7
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

348.1223

Chelerythrine

Leaves348.1277
333.1029
290.0835

304.1006

318.0810

286 288 290 292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330 332 334 336 338 340 342 344 346 348 350 352 354 356

Roots* 348.1128

290.0723

333.0883

304.0870

318.0663

286 288 290 292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330 332 334 336 338 340 342 344 346 348 350 352 354 356+ESI Product Ion (rt: 13.072 min) Frag=175.0V CID@20.0 (348.1215[z=1] -> **)Stems

348.1275
290.0839

304.0994

318.0795

333.1022

286 288 290 292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330 332 334 336 338 340 342 344 346 348 350 352 354 356

Mass to Charge (m/z)

Figure 3. MS/MS of of ChelerythrineStandard chelerythrine chloride yielded an [M + H]+ peak at a (m/z) of 348.1223 (Figure 3). The
fragmentation pattern MS/MS spectrum of standard chelerythrine analyzed by ESI-MS/MS
yielded 318.0753 [M –OCH2]+, 304.0995 [M –CO-CH3-H]+, and 290.0800 [M –CH2O-CO]+,respectively. The fragmentation patterns of the samples (leaves, roots, and stems) were similar tochelerythrine’s fragmentation pattern. The ESI-MS/MS results confirm the presence of thealkaloid chelerythrine (Son et al., 2014) in leaves, stems and roots of B. frutescens.

13

x10 6+ESI EIC(336.1100-336.1350) MS(all) Frag=175.0V Berberine

4Berberine

2

Abundance (Ion counts)

0
x10 5
2

+ESI EIC(336.1100-336.1350) MS(all) Frag=175.0V Leaves

Leaves

1
0

x10 5
5

+ESI EIC(336.1100-336.1350) MS(all) Frag=175.0V Roots

Roots

2.5
0

x10 5

+ESI EIC(336.1100-336.1350) MS(all) Frag=175.0V Stems

4

Stems

2
0
1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

Time (min)Figure 4. Extracted ion chromatogram of berberine
Table 4. Accurate Mass of berberine (m/z)
SampleObserved Mass
Berberine
336.1239
Roots
336.1243
Stems
336.1228
Leaves336.1219

Theoretical Mass
336.1236
336.1236
336.1236
336.1236

Difference (ppm)
-0.94
-2.13
2.33
5.00

Standard berberine yielded an observed mass [M + H]+ peak at a (m/z) of 336.1239. Bycomparison, crude organic extract of B. frutescens leaves, stems, and roots produced peaks
around that mass. The peak corresponding to berberine exhibited mass-to-charge (m/z) ratios
corresponding to the molecular ions (M + H)+ of standard berberine, confirming the presence of
the alkaloid in all three plant materials. A difference of 10 ppm was the cut off for homology.
These (m/z) values were obtained from the graph in Figure 4.

14

+ESI Product Ion (rt: 12.763 min) Frag=175.0V CID@19.4 (336.1225[z=1] -> **)

x10 4
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

+ESI Product Ion (rt: 12.832 min) Frag=175.0V CID@19.4 (336.1183[z=1] -> **)Abundance (Ion counts)

x10 6
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

x10 5
1.6

Berberine

336.1256321.1012
292.0992
263.0850
260

265

306.0783

278.0825
270

275

280

285

290

295

300

305

326.7949
310

315

320

325

330

Leaves335

340

336.1218

321.0973
292.0952
260

265

306.0772

278.0807

262.0856
270

275

280

285

290

295

300

305

310

315

320

325

330

+ESI Product Ion (rt: 12.818 min) Frag=175.0V CID@19.4 (336.1302[z=1] -> **)

335

340

336.1232

Roots

1.4
1.2
1
0.8

321.0980

0.6
0.4

292.0961

0.2
0
x10 5
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

260

265

306.0766

278.0826

263.0903
270

275

280

285

290

295

300

305

310

315

320

325

330

+ESI Product Ion (rt: 12.843 min) Frag=175.0V CID@19.4 (336.1193[z=1] -> **)

335

340

336.1224

Stems

321.0981
292.0963
260

265

306.0753

278.0804

262.0818
270

275

280

285

290

295

300

305

310

313.6016
315

320

325

330

335

340

Mass to Charge (m/z)

Figure 5. MS/MS of berberineStandard berberine chloride yielded an [M + H]+ peak at a (m/z) value of 336.1256. The MS/MS
spectrum of standard berberine analyzed by ESI-MS/MS yielded 306.0783 [M–OCH2]+,
292.0992 [M–CO-CH3-H]+, and 278.0825 [M–CH2O-CO]+ respectively. This result indicatesthat the ESI-MS/MS were adequate for the confirmation of the presence of alkaloid berberine(Son et al., 2014) in leaves, stems and roots of B. frutescens.

15

CONCLUSION
Although G. physocarpus possesses medicinal properties, natural products derived fromthis plant lack potential for protein kinase inhibition activity as evidenced in our study. Further
studies on the protein kinase inhibition activity of B. frutescens and its alkaloids are currently
underway. B. frutescens could exhibit these properties by inhibiting protein kinases or via othermechanisms. TLC, LC-MS, and MS/MS all confirmed the presence of berberine andchelerythrine, in crude extracts derived from B. frutescens.
In future studies, the percentage that each alkaloid contributes to the protein kinase
inhibition properties of B. frutescens will be quantified. The other alkaloids and non-alkaloids
that were shown by TLC analysis can be investigated. The seeds of B. frutescens could not be
assessed during this study because of a paucity of materials. Even though crude extracts derived
from G. physocarpus did not show protein kinase inhibition activity against Streptomyces 85E,
compounds derived from this plant could display biological activity against other organisms. The
medicinal properties of phytochemicals derived from G. physocarpus will be investigated.

ACKNOWLEDGMENTS
This project was supported by grants from the Daniel K. Inouye College of Pharmacy, Universityof Hawai‘i at Hilo, Hilo. We wish to thank the Big Island Invasive Species Committee (BIISC)
for helping us collect samples.

16

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