Journal of Ethnopharmacology 162 (2015) 163–167
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Efficacy of the saponin component of Impatiens capensis Meerb.in preventing urushiol-induced contact dermatitis Vicki A. Motz a,n, Christopher P. Bowers b, Alexander R. Kneubehl a,1, Elizabeth C. Lendrum a,1, Linda M. Young a, David H. Kinder c a
Department of Biological and Allied Health Sciences, Ohio Northern University 525 S Main St, Ada OH 45801, USA Department of Chemistry and Biochemistry, Ohio Northern University 525 S Main St, Ada OH 45801, USA c Raabe College of Pharmacy, Ohio Northern University 525 S Main St, Ada OH 45801, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 October 2014 Received in revised form 7 December 2014 Accepted 16 December 2014 Available online 24 December 2014
Ethnopharmacological relevance: Many different tribes of American Indians used jewelweed, Impatiens capensis Meerb, as a plant mash to reduce development of poison ivy dermatitis. Saponins are a natural soapy constituent found within plants. A 2012 study suggested that saponins may be present in jewelweed which could be responsible for its efficacy in preventing rash development following contact with Toxicodendron radicans (L.) Kuntze (poison ivy). This study validated this hypothesis and demonstrated additional biological activity of the jewelweed saponin containing extract. Materials and methods: Fresh I. capensis leaves were extracted with methanol and further partitioned between ethyl acetate and water, with a final separation between water and n-butanol, to obtain a saponin containing extract. The presence of saponins in the extract was demonstrated by the observation of foaming and using a vanillin colorimetric assay for total saponins. Efficacy of the saponin containing extracts in rash reduction was tested by brushing poison ivy (PI) onto the forearms of volunteers (N ¼23) in six locations and treating these PI exposed areas with distilled water (control), saponin containing extracts, fresh plant mashes, and soaps made with and without plant extracts. Saponin containing extracts were further tested for biological activity against both gram negative and gram positive bacteria and against cancer cell lines A-375, HT-29, and MCF-7. Additionally, because saponins have been shown to have a stimulatory effect in cardiac muscle 2 ml saponin extract was applied superficially to black worms, Lumbriculus variegatus (N¼5). Results, and conclusions: Both saponin containing extracts and all soaps tested were effective in reducing poison ivy dermatitis; thus, saponin content correlates with PI rash prevention. No apparent antibiosis was observed against any bacteria tested; however, dose response cytotoxicity was documented against MCF-7 breast cancer cells and cytostatic activity was seen against the HT-29 colon cancer cell lines. Lumbriculus variegatus exhibited a 138% increase in heart rate over baseline rate five minutes post treatment implying a possible positive chronotropic effect. & 2014 Elsevier Ireland Ltd. All rights reserved.
Key words: 2.536: saponins 2.644: Traditional medicine Northern America Poison ivy Dermatitis Urushiol Pharmacognosy
1. Introduction The Potawatomi (Smith, 1923), Chippewa (Gilmore, 1933), Meskwaki (Smith, 1928), and Omaha (Gilmore, 1919) used jewelweed, Impatiens capensis Meerb. (see Fig. 1), a member of the Balsaminaceae family, as a plant mash to reduce development of poison ivy (PI)
n
Corresponding author. Tel.: þ 419 772 2063; fax: þ419 772 2330. E-mail addresses: [email protected] (V.A. Motz), [email protected] (C.P. Bowers), [email protected] (A.R. Kneubehl), [email protected] (E.C. Lendrum), [email protected] (L.M. Young), [email protected] (D.H. Kinder). 1 Undergraduate researcher. http://dx.doi.org/10.1016/j.jep.2014.12.024 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
dermatitis. Although this ethnobotanical usage has been validated (Motz et al., 2012), the active agent responsible for rash reduction has not been determined. Lawsone, a naphthoquinone found in the closely related species, Impatiens balsamina, was proposed as the active agent (Duke, 1999); however, it was not effective in rash reduction (Motz et al., 2012). Many plants contain saponins which act as natural soaps. Although there is no evidence that the Native Americans used soaps to treat poison ivy, there is evidence to support the use of the yucca plant by southwestern Native Americans as “soap” (Vestal, 1952). All soaps tested for efficacy in preventing poison ivy rash development (either hand formulated or purchased commercially) were effective (Stibich et al., 2000; Motz et al., 2012). This study examined whether saponins
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V.A. Motz et al. / Journal of Ethnopharmacology 162 (2015) 163–167
Fig. 1. Impatiens capensis.
found in the jewelweed, could be the active agents in rash reduction acting in a similar fashion to the soap preparations. Saponins are high molecular weight steroid or triterpenoid plant glycosides which have been credited with forming a portion of the plant's defense system, increasing anti-fungal (Osbourn, 1996) and general anti-microbial (Ohana et al., 1998) activity. They derive their name from the Latin, sapo, meaning soap, and have detergent properties. Many studies have examined their effects in animal models. Some actions appear to be due to their ability to increase membrane permeability by forming pores in plasma membranes (Francis et al., 2002) rendering them hemolytic (Oda et al., 2000); and alteration of calcium permeability in myocyte sarcolemma may explain their cardiac activity (Yamasaki et al., 1987). Furthermore, activity of saponins from many plants has been shown to alter function of the Naþ /Kþ ATPase pump, producing cardiotonic effects (Polya, 2003). Saponin interference with enzyme activity has been credited with their ability to decrease cancer cell proliferation (Oh and Sung, 2001) and to deactivate viruses (Sindambiwe et al., 1998). Although they have not been previously identified in I. capensis, saponins have been seen in both the aerial parts (Anwer et al., 2013) and seeds (Shoji et al., 1994a, 1994b) of I. balsamina as well as the aerial parts of Impatiens siculifer (Li et al., 2009) and the rhizomes of Impatiens pritzellii (Zhou et al., 2007). The saponin content of plants varies with the specific cultivar as well as the physiological season and abiotic factors specific to conditions under which they were grown; as well as having uneven distribution throughout the organs of a plant (Hostettmann and Marston, 2005; Kang et al., 2013 ). The component of poison ivy, Toxicodendron radicans (L.) Kuntze, associated with contact dermatitis is urushiol oil (Armstrong and Epstein, 1995). The structure and oily nature of poison ivy urushiol was elucidated in 1954 (Sims and Dawson, 1954). The detergent action of soaps on oils has long been understood (Fall, 1927) thus it is likely that the saponin content of the jewelweed is preventing dermatitis by emulsifying the urushiol rendering it less capable of eliciting the allergic response (scaling, vesiculation, fissuring, and weeping (Pray, 2007)).
plant name was checked with www.theplantlist.org (9/15/2014). Leaves were extracted in 50% methanol (300 ml) for one week at room temperature without agitation. The extract was then stirred for 24hours at room temperature, vacuum filtered using a Whatman Grade 1 filter and refiltered using a Whatman Grade GF/F filter. The solvent was removed from the filtrate under reduced pressure to produce a gummy, reddish-brown residue. This residue was then rinsed twice from the round bottom flask with 10 ml of distilled water into a 125 ml separation funnel and extracted following a procedure similar to Li et al., (2009). The extract was partitioned three times by adding 20 ml of ethyl acetate each time preserving the aqueous layer. To the aqueous layer, 20 ml n-butanol was added to partition three times, each time preserving the n-butanol layer. The n-butanol partitions were added together and were allowed to air dry at room temperature for 24hours. A 2x concentration was made by adding water by weight to equal the mass of the dried residue plus half the mass of original plant material resulting in a final concentration of 1 gram of extract/2 grams of plant material. The plant strength concentration was made by diluting an aliquot of the 2x solution 1:1 with water resulting in a final concentration of 1 gram of extract/ gram of plant material. Presence of saponins was confirmed both by observation of a persistent foam and by using a vanillin colorimetric assay as described by Hiai et al., (1976). The dried residue was analyzed as a KBr pellet with a Nicolet 6700 FT-IR. 2.2. Evaluation of efficacy against urushiol induced rash development To establish whether these saponin containing extracts of I. capensis decreased urushiol induced rash development, an Institutional Review Board approved protocol was followed. The anterior forearm surfaces of 23 volunteer subjects aged 18–65 years were examined and determined to be free of prior inflammation. Forearms were then washed with tap water and patted dry with a cotton towel. Three 2 cm X 2 cm squares were drawn on each arm with a permanent marker. Poison ivy, Rhus toxicodendron L., plants were identified and collected by Dr. V. Motz, the plant name was checked with www.theplantlist.org (9/15/2014), and a voucher specimen (VAM-01-001) was deposited in the herbarium at the Ohio Northern University (Natural History) Museum. Fresh poison ivy leaves were attached to an applicator stick and crushed on a metal grater to ensure exposure to urushiol. The marked areas of the forearms were exposed to PI by drawing the ivy covered swab five times in the same direction within each square. After two minutes each area was wiped with distilled water to remove any remaining poison ivy. According to a previously assigned, randomized sequence, the six areas were then treated with (1) distilled water as a control, (2) fresh I. capensis mash, (3) a saponin containing extract of I. capensis, (4) a double strength concentrate of the same infusion, (5) Grandma's Pure and Natural Lye soap© made by Remwood Products, Tulsa, OK and (6) Grandma's Poison Ivy and Oak Bar©, an identical soap prepared with a jewelweed infusion. Each test substance was applied with standard cotton-tipped applicators which had been saturated with the treatment using five one-way strokes across the application area perpendicular to the application of PI. After five minutes, all squares were rinsed with distilled water and the areas were bandaged with gauze squares left in place for 48 hours. Rash development was tracked for 18 days and scored on a scale of 0–14 (Motz et al., 2012) (see Table 1).
2. Methods 2.3. Additional physiological effects of saponin containing extracts 2.1. Preparation of plant extracts Fresh leaves of late season I. capensis (50.0 g) were identified and collected by Dr. Vicki Motz, in September of 2013 in Marysville, OH, and a voucher specimen (VAM-01-002) was deposited in the herbarium at the Ohio Northern University (Natural History) Museum; the
Antimicrobial activity was evaluated by triplicate Kirby-Bauer assays using 100 μl of 1 gm of plant material/gm of saponin containing extract (equivalent to the extract of 0.1 gm of plant) against Staphylococcus epidermidis (ATCC-12228), Bacillus subtilis (Wards 85V0228), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25925)
V.A. Motz et al. / Journal of Ethnopharmacology 162 (2015) 163–167
Table 1 Scoring scale for determining degree of response to PI exposure. Score
Description
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
No reaction Redness 1-3 papules 4-8 papules 4 8 papules but not continuous Continuous covering 0–10% of area Continuous covering 11–20% of area Continuous covering 21–30%, of area Continuous covering 31–40%, of area Continuous covering 41–50% of area Continuous covering 51–60% of area Continuous covering 61–70% of area Continuous covering 71–80% of area Continuous covering 81–90% of area Continuous covering 91–100% of area
and Candida albicans (hospital control culture). Mueller-Hinton plates were incubated at 37 1C with 5% CO2 for 24hours and zones of inhibition were recorded. Anticancer in vitro activity was examined in MCF-7 breast cancer cells (ATCCs Number: HTB-22™), A-375 melanoma (ATCCs Number: CRL-1619™), and HT-29 colon cancer (ATCCs Number: HTB-38D™) cell lines. Cells obtained from ATCC, were grown in DMEM supplemented with 10% FBS and penicillin/streptomycin (all reagents obtained from Mediatechs). For determining in vitro anticancer activity, the appropriate cell line was grown to 80% or higher confluence. Cells were trypsinized, suspended in media, and further diluted such that the cells were plated at a density of 500 cells/ml in a 96 well plate. The cells were incubated for 1 hour, then test solution prepared in DMSO was added in additional media. Test solutions were prepared from the dried extract in biological grade DMSO for concentrations of 100, 80, 64, 48, 34 mg/ml. The control group was DMSO only in the same concentration as that of the treatment groups (VC¼vehicle control). These concentrations were added to the cell culture media to give final concentrations of 100, 80, 64, 48, 34 μg/ml for the dose response experiment. The plates were incubated for 48hours, after which the media was removed and replaced with CellGrows complete media without phenol red containing XTT reagent (Sigma, used according to manufacturer's instructions). After 2–4hours, the plate was read on a Bioteks plate reader at 450 nm. Absorption was converted to % growth inhibition relative to vehicle control after subtracting the blank reading. Cell morphology was observed under magnification. Effects of I. capensis saponin extract on cardiac activity were evaluated in black worms, Lumbriculus variegatus. The resting heart rate of black worms in a parafilm counting chamber containing 100 μl of “pond water” in which the worms were raised (modified from Lesiuk and Drewes, 1999) was recorded five times in seconds/beat. The aqueous extract was added to the bath bringing the concentration in the bath to 0.1 gm extracted plant material/ml of “pond water.” Heart rate was again measured five times. Heart rates were re-recorded after five minutes. Mean heart rates were calculated and experimental rates were expressed as a % of initial rate. Heart rates of untreated control worms were observed initially and after five minutes.
(N¼ 9) and those who had no rash development. Scores were reported as a percent of score in control square (poison ivy with only distilled water rinse). Following ANOVA testing, ad hoc Students t-tests at 95% confidence levels were used to assess significance of difference in rash score between treatments groups. Kirby Bauer testing was performed in triplicate and average zones of inhibition in mm were assessed by ANOVA at 95% confidence. Anti-cancer testing was run in triplicate, absorption was converted to percent growth inhibition relative to vehicle control after subtracting the blank reading. Mean and standard deviation of growth at each concentration was reported and compared by ANOVA at 95% confidence.
3. Results 3.1. Chemical evaluation of extract The method for the extraction of saponins was based upon procedures previously described (Shoji et al., 1994a, 1994b; Li et al., 2009). Saponin presence in the extract was qualitatively assessed by vigorous shaking. A persistent froth was observed indicating that saponins were indeed present in the extract (Onwukaeme et al., 2007). The vanillin colorimetric assay was positive for the presence of saponins with a higher concentration in the 2x sample as expected (see Fig. 2). Major IR peaks observed were at 3395, 2920, 1609, 1384, 1282, 1073 and 610 cm 1 (see Fig. 3). It should be noted that no previous study has shown the presence of saponins in I. capensis. 3.2. Efficacy of rash prevention Thirty seven percent of participants (n¼ 7) developed a severe poison ivy rash ( 410 on a scale of 1–14) (see Fig. 4), 47% developed a mild rash and 16% did not develop any rash. Data were collected for 18 days; and the first day upon which the participant developed their highest score was used in the calculations. This was necessary, as both onset and recovery rate were variable and using rash stage rather than a specific time frame gave the most valid comparator of rash development. The untreated control had an average rash score of 13.43 71.13. Mash treated areas scored 11.00 73.21 on the rash scale which was an 18% reduction in rash severity compared to the untreated control. The saponin containing extracts at plant strength and double strength scored 9.93 75.07 and 8.79 74.69 respectively, representing 26% and 35% rash reduction. There was no difference between the jewelweed infused soap and the straight lye soap with rash scores of 6.71 74.27 and 6.147 2.34 and both soaps averaged a 52% rash reduction. The rashes in areas treated with soaps and the double strength extract were all significantly reduced in severity (p o0.05 by Students' paired 1 tailed t-tests) and were not significantly different from each other (see Fig. 5). Just under half of the participants (n ¼9; 47%) developed a milder rash with the control areas scoring 5.11 71.79 on the rash development scale. In those participants who developed a mild rash, three had at least twice as high a score in the extract squares than in the control squares, indicating sensitivity to extracts. These
2.4. Statistical analysis Rash development scores used for analysis were the first day that a volunteer recorded their personal highest score in the control square. This allowed for comparison of maximum rash development normalizing differences in rash development rate. Mean and standard deviation were calculated for those who developed maximum rash scores above 10 (N¼7), those who had rash scores between 0 and 10
165
Fig. 2. Vanillin colorimetric assay results.
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Fig. 3. FT-IR spectrum of jewelweed extract.
Mild Rash Group
% of Control Rash
100
1x Extract 2x Extract Plant Mash Jewelweed Soap
80
* 60
% of Control Rash
80 60
*
*
40 20 E1
E1
E2
S
3.3. Other biological activity of extracts
*
M
M
reduce rash by a third, to 67% of the control and the soaps to just over half at 52% rash reduction when jewelweed was included and 54% without the jewelweed. The rash reduction of the mash and both soaps were significant (p o 0.05 by Students' paired 1 tailed t-tests) with no difference from each other (see Fig. 6).
Severe Rash Group
W
W
Fig. 6. Comparison of rash severity in those participants who developed a mild rash. Control (W) water samples were scored as 100% and others calculated as % of control. Fresh jewelweed mash (M) and soaps both with jewelweed (JS) and without (S) produced significant rash reduction. Rash reduction was observed but inconsistent in full strength saponin containing extract (E1) and double strength extract (E2) due to sensitivity to the extract.
Fig. 4. Arms of one volunteer, day 11 after inoculation.
0
JS
20
Treatment
Lye Soap
100
*
40
0
Control
*
E2
JS
S
Treatment Fig. 5. Comparison of rash severity in those participants who developed a severe rash (n¼ 7). Control (W) water samples were scored as 100% and others calculated as % of control. Fresh jewelweed mash (M) and a full strength extract (E1) containing the saponin component produced minor decreases in rash development. Double strength extract (E2) and soaps both with jewelweed (JS) and without (S) produced significant rash reduction compared to control.
data were included in the analysis as there was no statistical validity to removing them. As a result, mean extract effectiveness reflected only an 11% rash reduction (to 89% of the control) and the double strength extract reduced rash to 72% of control. Notwithstanding, sensitivity to extracts, jewelweed mash was able to
There was no apparent antibiosis of the saponin containing extract against either gram negative or gram positive bacteria tested, nor was antifungal activity observed. Dose response cytotoxicity was demonstrated in MCF-7 breast cancer cells ranging from a 39.8% reduction in cell growth at low concentrations to no cell growth at high concentrations and cytostatic activity was seen in the HT-29 colon cancer cell lines (see Fig. 7). The MCF-7 cells appeared shrunken with blebs, typical of cells undergoing apoptosis. The A-375 and HT-29 cell lines did not show this morphology. No inhibition of growth was observed in the A-375 melanoma cell line. In experimental worms (N¼5), there was an immediate increase in heart rate to 107.5 of resting rate and a further increase after five minutes to 138% of heart rate, whereas control worms (N¼3) had no increase in heart rate after five minutes. This indicates a possible positive chronotropic effect of the saponin containing extract. 4. Discussion The mash of fresh jewelweed reduced rash by 18% for severe rash cases and a significant 30% reduction, in mild cases, reaffirming the
% Growth relative to vehicle control
V.A. Motz et al. / Journal of Ethnopharmacology 162 (2015) 163–167
Supression of Cancer Cell Growth
140 120 100 80 60
*
40
*
20 0 -20
VC
36
48
64
80
* 100
Extract Concentration µg/ml MCF7
HT-29
A-375
Fig. 7. Dose response cell growth inhibition of the plant strength saponin extract in MCF-7 breast cancer cells and cytostatic activity in HT-29 colon cancer and A-375 melanoma cell lines. VC ¼vehicle control.
efficacy of jewelweed mash in decreasing urushiol induced poison ivy dermatitis. Greatest rash reduction was seen in the soap preparations. Since the soaps with and without the jewelweed extracts were essentially identical, it is presumed that the addition of jewelweed did not act synergistically with the basic soap product, indicating that the detergent action of the soaps was effective in reducing rash development. Since the efficacy of the double strength extract was not significantly different from that of the soaps in severe rash reduction, it is reasonable to presume that, working through detergent action, the saponin in the extract was the causative agent. The failure of the extracts to reduce mild rash development was skewed by three participants developing worse rashes in the extract areas than in the control areas. This was interpreted as sensitivity to the extract. The positive chronotropic effect observed was most likely due to the saponins. This cardiac response is consistent with documented cardiotonic activity of saponins from many plants presumably working on the Naþ/Kþ ATPase pump (Polya, 2003). Polya (2003) also attributed saponin toxicity to cancer cell lines to action on this critical pump, which is consistent with our findings. The lack of antimicrobial activity was surprising. A similar mixed saponin crude extract from Sorghum bicolor had demonstrated antibiotic action on Staphylococcus aureus but not on E. coli or the fungus Candida albicans (Soetan et al., 2006). Saponin antibacterial effects are linked to their detergent action on the cell wall. Acknowledgments The authors would like to thank the volunteers who willingly subjected themselves to poison ivy dermatitis in the name of science, and Remwood Products Co. from Tulsa, OK, who generously provided us with bars of their lye and lye with jewelweed soaps for this experiment. Additionally we wish to express thanks to ONU Office of Academic Affairs for their financial support of this research, and to Dr. Robert Verb, curator the herbarium at the Ohio Northern University (Natural History) Museum, for his gracious assistance in voucher specimen preparation. References Anwer, N., Waqar, M.A., Iqbal, M., Mushtaq, M., Sobia, A., 2013. Phytochemical analysis, free radical scavenging capacity and antimicrobial properties of Impatiens bicolor plant. International Food Research Journal 20 (1), 99–103.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Efficacy of the saponin component of Impatiens capensis Meerb.in preventing urushiol-induced contact dermatitis Vicki A. Motz a,n, Christopher P. Bowers b, Alexander R. Kneubehl a,1, Elizabeth C. Lendrum a,1, Linda M. Young a, David H. Kinder c a
Department of Biological and Allied Health Sciences, Ohio Northern University 525 S Main St, Ada OH 45801, USA Department of Chemistry and Biochemistry, Ohio Northern University 525 S Main St, Ada OH 45801, USA c Raabe College of Pharmacy, Ohio Northern University 525 S Main St, Ada OH 45801, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 October 2014 Received in revised form 7 December 2014 Accepted 16 December 2014 Available online 24 December 2014
Ethnopharmacological relevance: Many different tribes of American Indians used jewelweed, Impatiens capensis Meerb, as a plant mash to reduce development of poison ivy dermatitis. Saponins are a natural soapy constituent found within plants. A 2012 study suggested that saponins may be present in jewelweed which could be responsible for its efficacy in preventing rash development following contact with Toxicodendron radicans (L.) Kuntze (poison ivy). This study validated this hypothesis and demonstrated additional biological activity of the jewelweed saponin containing extract. Materials and methods: Fresh I. capensis leaves were extracted with methanol and further partitioned between ethyl acetate and water, with a final separation between water and n-butanol, to obtain a saponin containing extract. The presence of saponins in the extract was demonstrated by the observation of foaming and using a vanillin colorimetric assay for total saponins. Efficacy of the saponin containing extracts in rash reduction was tested by brushing poison ivy (PI) onto the forearms of volunteers (N ¼23) in six locations and treating these PI exposed areas with distilled water (control), saponin containing extracts, fresh plant mashes, and soaps made with and without plant extracts. Saponin containing extracts were further tested for biological activity against both gram negative and gram positive bacteria and against cancer cell lines A-375, HT-29, and MCF-7. Additionally, because saponins have been shown to have a stimulatory effect in cardiac muscle 2 ml saponin extract was applied superficially to black worms, Lumbriculus variegatus (N¼5). Results, and conclusions: Both saponin containing extracts and all soaps tested were effective in reducing poison ivy dermatitis; thus, saponin content correlates with PI rash prevention. No apparent antibiosis was observed against any bacteria tested; however, dose response cytotoxicity was documented against MCF-7 breast cancer cells and cytostatic activity was seen against the HT-29 colon cancer cell lines. Lumbriculus variegatus exhibited a 138% increase in heart rate over baseline rate five minutes post treatment implying a possible positive chronotropic effect. & 2014 Elsevier Ireland Ltd. All rights reserved.
Key words: 2.536: saponins 2.644: Traditional medicine Northern America Poison ivy Dermatitis Urushiol Pharmacognosy
1. Introduction The Potawatomi (Smith, 1923), Chippewa (Gilmore, 1933), Meskwaki (Smith, 1928), and Omaha (Gilmore, 1919) used jewelweed, Impatiens capensis Meerb. (see Fig. 1), a member of the Balsaminaceae family, as a plant mash to reduce development of poison ivy (PI)
n
Corresponding author. Tel.: þ 419 772 2063; fax: þ419 772 2330. E-mail addresses: [email protected] (V.A. Motz), [email protected] (C.P. Bowers), [email protected] (A.R. Kneubehl), [email protected] (E.C. Lendrum), [email protected] (L.M. Young), [email protected] (D.H. Kinder). 1 Undergraduate researcher. http://dx.doi.org/10.1016/j.jep.2014.12.024 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
dermatitis. Although this ethnobotanical usage has been validated (Motz et al., 2012), the active agent responsible for rash reduction has not been determined. Lawsone, a naphthoquinone found in the closely related species, Impatiens balsamina, was proposed as the active agent (Duke, 1999); however, it was not effective in rash reduction (Motz et al., 2012). Many plants contain saponins which act as natural soaps. Although there is no evidence that the Native Americans used soaps to treat poison ivy, there is evidence to support the use of the yucca plant by southwestern Native Americans as “soap” (Vestal, 1952). All soaps tested for efficacy in preventing poison ivy rash development (either hand formulated or purchased commercially) were effective (Stibich et al., 2000; Motz et al., 2012). This study examined whether saponins
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Fig. 1. Impatiens capensis.
found in the jewelweed, could be the active agents in rash reduction acting in a similar fashion to the soap preparations. Saponins are high molecular weight steroid or triterpenoid plant glycosides which have been credited with forming a portion of the plant's defense system, increasing anti-fungal (Osbourn, 1996) and general anti-microbial (Ohana et al., 1998) activity. They derive their name from the Latin, sapo, meaning soap, and have detergent properties. Many studies have examined their effects in animal models. Some actions appear to be due to their ability to increase membrane permeability by forming pores in plasma membranes (Francis et al., 2002) rendering them hemolytic (Oda et al., 2000); and alteration of calcium permeability in myocyte sarcolemma may explain their cardiac activity (Yamasaki et al., 1987). Furthermore, activity of saponins from many plants has been shown to alter function of the Naþ /Kþ ATPase pump, producing cardiotonic effects (Polya, 2003). Saponin interference with enzyme activity has been credited with their ability to decrease cancer cell proliferation (Oh and Sung, 2001) and to deactivate viruses (Sindambiwe et al., 1998). Although they have not been previously identified in I. capensis, saponins have been seen in both the aerial parts (Anwer et al., 2013) and seeds (Shoji et al., 1994a, 1994b) of I. balsamina as well as the aerial parts of Impatiens siculifer (Li et al., 2009) and the rhizomes of Impatiens pritzellii (Zhou et al., 2007). The saponin content of plants varies with the specific cultivar as well as the physiological season and abiotic factors specific to conditions under which they were grown; as well as having uneven distribution throughout the organs of a plant (Hostettmann and Marston, 2005; Kang et al., 2013 ). The component of poison ivy, Toxicodendron radicans (L.) Kuntze, associated with contact dermatitis is urushiol oil (Armstrong and Epstein, 1995). The structure and oily nature of poison ivy urushiol was elucidated in 1954 (Sims and Dawson, 1954). The detergent action of soaps on oils has long been understood (Fall, 1927) thus it is likely that the saponin content of the jewelweed is preventing dermatitis by emulsifying the urushiol rendering it less capable of eliciting the allergic response (scaling, vesiculation, fissuring, and weeping (Pray, 2007)).
plant name was checked with www.theplantlist.org (9/15/2014). Leaves were extracted in 50% methanol (300 ml) for one week at room temperature without agitation. The extract was then stirred for 24hours at room temperature, vacuum filtered using a Whatman Grade 1 filter and refiltered using a Whatman Grade GF/F filter. The solvent was removed from the filtrate under reduced pressure to produce a gummy, reddish-brown residue. This residue was then rinsed twice from the round bottom flask with 10 ml of distilled water into a 125 ml separation funnel and extracted following a procedure similar to Li et al., (2009). The extract was partitioned three times by adding 20 ml of ethyl acetate each time preserving the aqueous layer. To the aqueous layer, 20 ml n-butanol was added to partition three times, each time preserving the n-butanol layer. The n-butanol partitions were added together and were allowed to air dry at room temperature for 24hours. A 2x concentration was made by adding water by weight to equal the mass of the dried residue plus half the mass of original plant material resulting in a final concentration of 1 gram of extract/2 grams of plant material. The plant strength concentration was made by diluting an aliquot of the 2x solution 1:1 with water resulting in a final concentration of 1 gram of extract/ gram of plant material. Presence of saponins was confirmed both by observation of a persistent foam and by using a vanillin colorimetric assay as described by Hiai et al., (1976). The dried residue was analyzed as a KBr pellet with a Nicolet 6700 FT-IR. 2.2. Evaluation of efficacy against urushiol induced rash development To establish whether these saponin containing extracts of I. capensis decreased urushiol induced rash development, an Institutional Review Board approved protocol was followed. The anterior forearm surfaces of 23 volunteer subjects aged 18–65 years were examined and determined to be free of prior inflammation. Forearms were then washed with tap water and patted dry with a cotton towel. Three 2 cm X 2 cm squares were drawn on each arm with a permanent marker. Poison ivy, Rhus toxicodendron L., plants were identified and collected by Dr. V. Motz, the plant name was checked with www.theplantlist.org (9/15/2014), and a voucher specimen (VAM-01-001) was deposited in the herbarium at the Ohio Northern University (Natural History) Museum. Fresh poison ivy leaves were attached to an applicator stick and crushed on a metal grater to ensure exposure to urushiol. The marked areas of the forearms were exposed to PI by drawing the ivy covered swab five times in the same direction within each square. After two minutes each area was wiped with distilled water to remove any remaining poison ivy. According to a previously assigned, randomized sequence, the six areas were then treated with (1) distilled water as a control, (2) fresh I. capensis mash, (3) a saponin containing extract of I. capensis, (4) a double strength concentrate of the same infusion, (5) Grandma's Pure and Natural Lye soap© made by Remwood Products, Tulsa, OK and (6) Grandma's Poison Ivy and Oak Bar©, an identical soap prepared with a jewelweed infusion. Each test substance was applied with standard cotton-tipped applicators which had been saturated with the treatment using five one-way strokes across the application area perpendicular to the application of PI. After five minutes, all squares were rinsed with distilled water and the areas were bandaged with gauze squares left in place for 48 hours. Rash development was tracked for 18 days and scored on a scale of 0–14 (Motz et al., 2012) (see Table 1).
2. Methods 2.3. Additional physiological effects of saponin containing extracts 2.1. Preparation of plant extracts Fresh leaves of late season I. capensis (50.0 g) were identified and collected by Dr. Vicki Motz, in September of 2013 in Marysville, OH, and a voucher specimen (VAM-01-002) was deposited in the herbarium at the Ohio Northern University (Natural History) Museum; the
Antimicrobial activity was evaluated by triplicate Kirby-Bauer assays using 100 μl of 1 gm of plant material/gm of saponin containing extract (equivalent to the extract of 0.1 gm of plant) against Staphylococcus epidermidis (ATCC-12228), Bacillus subtilis (Wards 85V0228), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25925)
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Table 1 Scoring scale for determining degree of response to PI exposure. Score
Description
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
No reaction Redness 1-3 papules 4-8 papules 4 8 papules but not continuous Continuous covering 0–10% of area Continuous covering 11–20% of area Continuous covering 21–30%, of area Continuous covering 31–40%, of area Continuous covering 41–50% of area Continuous covering 51–60% of area Continuous covering 61–70% of area Continuous covering 71–80% of area Continuous covering 81–90% of area Continuous covering 91–100% of area
and Candida albicans (hospital control culture). Mueller-Hinton plates were incubated at 37 1C with 5% CO2 for 24hours and zones of inhibition were recorded. Anticancer in vitro activity was examined in MCF-7 breast cancer cells (ATCCs Number: HTB-22™), A-375 melanoma (ATCCs Number: CRL-1619™), and HT-29 colon cancer (ATCCs Number: HTB-38D™) cell lines. Cells obtained from ATCC, were grown in DMEM supplemented with 10% FBS and penicillin/streptomycin (all reagents obtained from Mediatechs). For determining in vitro anticancer activity, the appropriate cell line was grown to 80% or higher confluence. Cells were trypsinized, suspended in media, and further diluted such that the cells were plated at a density of 500 cells/ml in a 96 well plate. The cells were incubated for 1 hour, then test solution prepared in DMSO was added in additional media. Test solutions were prepared from the dried extract in biological grade DMSO for concentrations of 100, 80, 64, 48, 34 mg/ml. The control group was DMSO only in the same concentration as that of the treatment groups (VC¼vehicle control). These concentrations were added to the cell culture media to give final concentrations of 100, 80, 64, 48, 34 μg/ml for the dose response experiment. The plates were incubated for 48hours, after which the media was removed and replaced with CellGrows complete media without phenol red containing XTT reagent (Sigma, used according to manufacturer's instructions). After 2–4hours, the plate was read on a Bioteks plate reader at 450 nm. Absorption was converted to % growth inhibition relative to vehicle control after subtracting the blank reading. Cell morphology was observed under magnification. Effects of I. capensis saponin extract on cardiac activity were evaluated in black worms, Lumbriculus variegatus. The resting heart rate of black worms in a parafilm counting chamber containing 100 μl of “pond water” in which the worms were raised (modified from Lesiuk and Drewes, 1999) was recorded five times in seconds/beat. The aqueous extract was added to the bath bringing the concentration in the bath to 0.1 gm extracted plant material/ml of “pond water.” Heart rate was again measured five times. Heart rates were re-recorded after five minutes. Mean heart rates were calculated and experimental rates were expressed as a % of initial rate. Heart rates of untreated control worms were observed initially and after five minutes.
(N¼ 9) and those who had no rash development. Scores were reported as a percent of score in control square (poison ivy with only distilled water rinse). Following ANOVA testing, ad hoc Students t-tests at 95% confidence levels were used to assess significance of difference in rash score between treatments groups. Kirby Bauer testing was performed in triplicate and average zones of inhibition in mm were assessed by ANOVA at 95% confidence. Anti-cancer testing was run in triplicate, absorption was converted to percent growth inhibition relative to vehicle control after subtracting the blank reading. Mean and standard deviation of growth at each concentration was reported and compared by ANOVA at 95% confidence.
3. Results 3.1. Chemical evaluation of extract The method for the extraction of saponins was based upon procedures previously described (Shoji et al., 1994a, 1994b; Li et al., 2009). Saponin presence in the extract was qualitatively assessed by vigorous shaking. A persistent froth was observed indicating that saponins were indeed present in the extract (Onwukaeme et al., 2007). The vanillin colorimetric assay was positive for the presence of saponins with a higher concentration in the 2x sample as expected (see Fig. 2). Major IR peaks observed were at 3395, 2920, 1609, 1384, 1282, 1073 and 610 cm 1 (see Fig. 3). It should be noted that no previous study has shown the presence of saponins in I. capensis. 3.2. Efficacy of rash prevention Thirty seven percent of participants (n¼ 7) developed a severe poison ivy rash ( 410 on a scale of 1–14) (see Fig. 4), 47% developed a mild rash and 16% did not develop any rash. Data were collected for 18 days; and the first day upon which the participant developed their highest score was used in the calculations. This was necessary, as both onset and recovery rate were variable and using rash stage rather than a specific time frame gave the most valid comparator of rash development. The untreated control had an average rash score of 13.43 71.13. Mash treated areas scored 11.00 73.21 on the rash scale which was an 18% reduction in rash severity compared to the untreated control. The saponin containing extracts at plant strength and double strength scored 9.93 75.07 and 8.79 74.69 respectively, representing 26% and 35% rash reduction. There was no difference between the jewelweed infused soap and the straight lye soap with rash scores of 6.71 74.27 and 6.147 2.34 and both soaps averaged a 52% rash reduction. The rashes in areas treated with soaps and the double strength extract were all significantly reduced in severity (p o0.05 by Students' paired 1 tailed t-tests) and were not significantly different from each other (see Fig. 5). Just under half of the participants (n ¼9; 47%) developed a milder rash with the control areas scoring 5.11 71.79 on the rash development scale. In those participants who developed a mild rash, three had at least twice as high a score in the extract squares than in the control squares, indicating sensitivity to extracts. These
2.4. Statistical analysis Rash development scores used for analysis were the first day that a volunteer recorded their personal highest score in the control square. This allowed for comparison of maximum rash development normalizing differences in rash development rate. Mean and standard deviation were calculated for those who developed maximum rash scores above 10 (N¼7), those who had rash scores between 0 and 10
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Fig. 2. Vanillin colorimetric assay results.
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Fig. 3. FT-IR spectrum of jewelweed extract.
Mild Rash Group
% of Control Rash
100
1x Extract 2x Extract Plant Mash Jewelweed Soap
80
* 60
% of Control Rash
80 60
*
*
40 20 E1
E1
E2
S
3.3. Other biological activity of extracts
*
M
M
reduce rash by a third, to 67% of the control and the soaps to just over half at 52% rash reduction when jewelweed was included and 54% without the jewelweed. The rash reduction of the mash and both soaps were significant (p o 0.05 by Students' paired 1 tailed t-tests) with no difference from each other (see Fig. 6).
Severe Rash Group
W
W
Fig. 6. Comparison of rash severity in those participants who developed a mild rash. Control (W) water samples were scored as 100% and others calculated as % of control. Fresh jewelweed mash (M) and soaps both with jewelweed (JS) and without (S) produced significant rash reduction. Rash reduction was observed but inconsistent in full strength saponin containing extract (E1) and double strength extract (E2) due to sensitivity to the extract.
Fig. 4. Arms of one volunteer, day 11 after inoculation.
0
JS
20
Treatment
Lye Soap
100
*
40
0
Control
*
E2
JS
S
Treatment Fig. 5. Comparison of rash severity in those participants who developed a severe rash (n¼ 7). Control (W) water samples were scored as 100% and others calculated as % of control. Fresh jewelweed mash (M) and a full strength extract (E1) containing the saponin component produced minor decreases in rash development. Double strength extract (E2) and soaps both with jewelweed (JS) and without (S) produced significant rash reduction compared to control.
data were included in the analysis as there was no statistical validity to removing them. As a result, mean extract effectiveness reflected only an 11% rash reduction (to 89% of the control) and the double strength extract reduced rash to 72% of control. Notwithstanding, sensitivity to extracts, jewelweed mash was able to
There was no apparent antibiosis of the saponin containing extract against either gram negative or gram positive bacteria tested, nor was antifungal activity observed. Dose response cytotoxicity was demonstrated in MCF-7 breast cancer cells ranging from a 39.8% reduction in cell growth at low concentrations to no cell growth at high concentrations and cytostatic activity was seen in the HT-29 colon cancer cell lines (see Fig. 7). The MCF-7 cells appeared shrunken with blebs, typical of cells undergoing apoptosis. The A-375 and HT-29 cell lines did not show this morphology. No inhibition of growth was observed in the A-375 melanoma cell line. In experimental worms (N¼5), there was an immediate increase in heart rate to 107.5 of resting rate and a further increase after five minutes to 138% of heart rate, whereas control worms (N¼3) had no increase in heart rate after five minutes. This indicates a possible positive chronotropic effect of the saponin containing extract. 4. Discussion The mash of fresh jewelweed reduced rash by 18% for severe rash cases and a significant 30% reduction, in mild cases, reaffirming the
% Growth relative to vehicle control
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Supression of Cancer Cell Growth
140 120 100 80 60
*
40
*
20 0 -20
VC
36
48
64
80
* 100
Extract Concentration µg/ml MCF7
HT-29
A-375
Fig. 7. Dose response cell growth inhibition of the plant strength saponin extract in MCF-7 breast cancer cells and cytostatic activity in HT-29 colon cancer and A-375 melanoma cell lines. VC ¼vehicle control.
efficacy of jewelweed mash in decreasing urushiol induced poison ivy dermatitis. Greatest rash reduction was seen in the soap preparations. Since the soaps with and without the jewelweed extracts were essentially identical, it is presumed that the addition of jewelweed did not act synergistically with the basic soap product, indicating that the detergent action of the soaps was effective in reducing rash development. Since the efficacy of the double strength extract was not significantly different from that of the soaps in severe rash reduction, it is reasonable to presume that, working through detergent action, the saponin in the extract was the causative agent. The failure of the extracts to reduce mild rash development was skewed by three participants developing worse rashes in the extract areas than in the control areas. This was interpreted as sensitivity to the extract. The positive chronotropic effect observed was most likely due to the saponins. This cardiac response is consistent with documented cardiotonic activity of saponins from many plants presumably working on the Naþ/Kþ ATPase pump (Polya, 2003). Polya (2003) also attributed saponin toxicity to cancer cell lines to action on this critical pump, which is consistent with our findings. The lack of antimicrobial activity was surprising. A similar mixed saponin crude extract from Sorghum bicolor had demonstrated antibiotic action on Staphylococcus aureus but not on E. coli or the fungus Candida albicans (Soetan et al., 2006). Saponin antibacterial effects are linked to their detergent action on the cell wall. Acknowledgments The authors would like to thank the volunteers who willingly subjected themselves to poison ivy dermatitis in the name of science, and Remwood Products Co. from Tulsa, OK, who generously provided us with bars of their lye and lye with jewelweed soaps for this experiment. Additionally we wish to express thanks to ONU Office of Academic Affairs for their financial support of this research, and to Dr. Robert Verb, curator the herbarium at the Ohio Northern University (Natural History) Museum, for his gracious assistance in voucher specimen preparation. References Anwer, N., Waqar, M.A., Iqbal, M., Mushtaq, M., Sobia, A., 2013. Phytochemical analysis, free radical scavenging capacity and antimicrobial properties of Impatiens bicolor plant. International Food Research Journal 20 (1), 99–103.
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