- Email: [email protected]
The antimicrobial properties of chile peppers (Capsicum species) and their uses in Mayan medicine
~
Journal of
ETHNOPHARMACOIDGY ELSEVIER
Journal of Ethnopharmacology 52 (1996) 61-70
The antimicrobial properties of chile peppers (Capsicum species) and their uses in Mayan medicine R o b e r t H. Cichewicz a, Patrick A. T h o r p e *b aDepartment of Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA bDepartment of Biology, Grand Valley State University, Allendale, MI 49401, USA Received 3 July 1995; revised 23 September 1995; accepted 8 January 1996
Abstract A survey of the Mayan pharmacopoeia revealed that tissues of Capsicum species (Solanaceae) are included in a number of herbal remedies for a variety of ailments of probable microbial origin. Using a filter disk assay, plain and heated aqueous extracts from fresh Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens, and Capsicum pubescens varieties were tested for their antimicrobial effects with fifteen bacterial species and one yeast species. Two pungent compounds found in Capsicum species (capsaicin and dihydrocapsaiein) were also tested for their antimicrobial effects. The plain and heated extracts were found to exhibit varying degrees of inhibition against Bacillus cereus, Bacillus subtilis, Clostridium sporogenes, Clostridium tetani, and Streptococcus pyogenes.
Keywords: Antimicrobial activity; Capsicum; Ethnobotany; Mayan medicine; Solanaeeae
1. Introduction
The indigenous inhabitants of various regions throughout the world rely upon plants as a source of medicinal agents to cure many of their ailments. Scientists observing these practices have facilitated the 'discovery' of a host of economically important drugs, such as the antihypertensive, reserpine, as well as pseudoephedrine, which reduces congestion (Cox and Balick, 1994). Studies of the botanical pharmacopoeia of the indigenous Mayan inhabitants of Mesoamerica have shown that chile peppers (Capsicum species * Corresponding author.
(Solanaceae)) are incorporated into a number of medicinal preparations (Roys, 1931; Alcorn, 1984). In a collection of 437 Mayan therapeutic remedies compiled by Roys (1931), 32 list the tissues of Capsicumspecies as one of the ingredients. In 29 cases, the fruits are reported as being used alone or in conjunction with the leaves, roots, or seeds. Eighteen of these remedies include instructions for boiling or heating the mixture in some manner before administration. These preparations were applied for a variety of ailments including respiratory problems, bowel complaints, earaches, and sores. Alcorn (1984) has also recorded 14 cases in which Huastec Mayan informants reported using fresh and heated Capsicumspecies
0378-8741/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PIL" 0378-8741(96)01384-0
62
R.H. Cichewicz, P.A. Thorpe/ Journal of Ethnopharmacology 52 (1996) 61-70
for medicinal purposes. The illnesses treated inelude infected wounds and fresh burns. A well-studied chemical component of Capsicum species, capsaicin, one of the pungent capsaicinoid compounds found in chile peppers, has already demonstrated a high degree of biological activity affecting the nervous, cardiovascular, and digestive systems (Virus and Gebhart, 1979; Monsereenusorn et al., 1982; Suzuki and lwai, 1984; Cordell and Araujo, 1993; Surh and Lee, 1995). Furthermore, clinical trials have shown that capsaicin may have potential value in the management of painful conditions such as rheumatoid arthritis and cluster headaches by causing the release of a sensory neuron's supply of substance P (a neurotransmitter) until depleted, resulting in insensitivity to painful stimuli (Cordell and Araujo, 1993). Several attempts have been made to test whether Capsicum tissues have a potential value as agents in the treatment of other medical conditions. Many of these efforts have focused on possible antimicrobial effects (Cardoso and Santos, 1948; Gotshall et al., 1949; Harris, 1949; Bushnell et al., 1950; MacDonald and Bishop, 1953; Fitzpatrick, 1954; Frisby et al., 1954; Masilungan et al., 1955; Masilungan et al., 1963; A1-Delaimy and Ali, 1970; Abdou et al., 1972; Mitscher et al., 1972; Chen et al., 1985; Caceres et al., 1991). A variety of assay methods (filter disk, penicup, pour plate,...), extraction procedures (acetone, aqueous, ethanol,...), and tissue types (predominantly common commercial varieties, some inaccurately identified) have been used with contradictory results reported. These tests fail to provide congruent evidence regarding the efficacy of Capsicum tissues' chemotherapeutic properties. The present study was conducted to examine the potential antimicrobial properties of Capsicum species as used by the Mayan inhabitants of Mesoamerica. 2. Materials and methods
2.1. Microorganism strains Strains of Bacillus cereus, Bacillus subtilis, Clostridium sporogenes, Clostridium tetani, Enterobacter aerogenes, Salmonella typhimurium, and Staphylococcus aureus (coagulase positive)
were cultured from Carolina MicroKwik rM Cultures (Carolina Biological Supply Co.). Enterobacter cloacae (ATCC 23355), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13833), Proteus vuigaris (ATCC 13315), Pseudomonas aeruginosa (ATCC 27853), Serratia marcescens (ATCC 8100), Staphylococcus epidermidis (ATCC 12228), Streptococcus pyogenes (ATCC 19615), and Candida albicans (ATCC 14053) were cultured from Difco Bactrol TM Disks (Difco Laboratories). Candida albicans was cultured in Sabouraud medium, Clostridium species in fluid thioglycollate, and all others in brain-heart infusion (BHI) medium. 2.2. Capsicum tissues Tissues from 11 varieties of five species of Capsicum were tested: C. annuum L. ('Bird' fruits, 'Green Bell' fruits, 'Jalapefio' fruits and leaves, 'Red Chile' fruits, and 'Serrano' fruits), C. baccatum L. (USDA PI 439359 fruits, USDA PI 439371 fruits, and USDA PI 439407 fruits), C. chinense Jacq. ('Habanero' fruits), C. frutescens L. ('Tabasc' fruits), and C. pubescens R. & P. (USDA PI 387838 fruits). The C. baccatum, C. frutescens, and C. pubescens were provided by Paul Bosland of New Mexico State University. The C. annuum ('Green Bell', 'Jalapefio', and 'Red Chile') were grown by the authors. The remaining strains were purchased from produce distributors. All Capsicum varieties were identified according to Bosland et al. (1990) and DeWitt and Bosland (1993) and verified by Paul Bosland (personal communications). Voucher specimens are listed in Table 1 and were deposited at the Grand Valley State University Herbarium (Allendale, Michigan, USA). 2.3. Tissue extracts Approximately 100 g of fresh plant tissue was washed and soaked in distilled water before being blotted dry, chopped into small pieces, and ground intermittently in a blender with no solvent for one to two minutes until a homogenous slurry was obtained. The slurry was then strained through a double layer of cheese cloth and the particulate matter discarded. Approximately 35 ml of the filtrate was centrifuged at 4°C for 30 min at 20 000 x g. The supernatant was collected and
R.H. Cichewicz,P.A. Thorpe~Journalof Ethnopharmacology 52 (1996) 61-70 reeentrifuged under the same conditions. The final supernatant was placed on ice until tested. Combined C. annuum tissue extract ('Jalapefio' fruits and leaves) was prepared by combining equal amounts (by weight) of the two tissue types which were blended together and then processed in the same manner as the other extracts. Cooked extract was made by decanting 20 ml of the centrifuged sample into a large test tube which was placed into a boiling water bath. The sample was swirled every 5 rain and placed immediately on ice after 20 rain of heating. 2.4. Filter disk assay Petri plates (10 cm diameter) were filled with a 20 ml base of sterile BHI agar or Sabouraud agar (for C. albicans). Four ml agar overlays cooled to 48°C (Sabouraud for C. albicans, thioglycollate for C. tetani, and BHI for all other strains) were inoculated with 100 #1 of overnight culture of the test organisms (400 #1 for C. tetanO and poured over the agar base. Trypticase soy agar II TM plates with 5% sheep blood (Beeton Dickson and Co.) were inoculated with 100 #1 of overnight S. pyogenes culture and spread mechanically over the surface of the plate. Three 13 mm diameter filter disks (Schleicher and Schuell no. 740-E) were soaked in the extract, gently tapped to remove excess liquid, and positioned on an inoculated plate. A fourth disk soaked in distilled water was also placed on the plate as a negative control. All plates remained upright at room temperature for at least 30 min before being inverted and incubated at 37°C for 15 h. Plates containing Clostridium species were incubated in a sealed BBL GasPak 100 TM canister with a carbon dioxide-enriched, anaerobic atmosphere. All plates were observed for zones of discoloration, inhibition, and/or stimulation of microbial growth, and the diameter of these zones was measured in mls. 2.5. Capsaicinoids assay Pour-plate and spread-plate cultures were prepared as indicated above for B. cereus, B. subtilis, C. aibicans, C. sporogenes, E. coli, S. typhimurium, S. aureus, and S. pyogenes. Ten mg dihydrocapsaicin, capsaicin (98% purity), and cap-
63
saicin (60% purity) (Sigma Chemical Co.) were each dissolved in 1 ml of methyl alcohol. Four sterile filter disks were then placed on the inoculated plates and 25 #1 of the capsaicinoids or methyl alcohol (control) were pipetted on the disks. Each chemical was tested in triplicate with the indicated bacterial strains. 2.6. Garlic assay . In order to evaluate the qualitative response of this assay method, a known botanically derived antimicrobial substance was selected as a positive control. AIlium sativum L. (garlic bulb) was chosen for this purpose as it has been demonstrated to produce a strong inhibitory effect with several bacterial species (Adetumbi and Lau, 1983; Fenwick and Hanley, 1985; Farbman et al., 1993). Allium sativum, purchased from a produce distributor, was peeled and chopped into small pieces, then combined with distilled water (2 ml water per 3 g A. sativum) before being blended and processed in the same manner as the Capsicum tissues. All microbial species were tested in triplicate with .4. sativum. 3. R ~
The combinations of tissue extracts and microorganisms which were tested are presented in Table 1. A number of effects involving the microorganisms were visually observed as circular zones surrounding the test extract-soaked disks, including complete inhibition (the absence of all microbial growth), partial inhibition (reduced microbial growth), stimulation (increased microbial density), an inner zone of partial inhibition with an outer region of stimulation, and blood plate discoloration. All bacterial and yeast strains were completely inhibited by the A. sativum extract (Table 2). The capsaicinoids showed no activity. The Capsicum tissue extracts exhibited a variety of effects with the test organisms, including complete inhibition, partial inhibition, stimulation, and partial inhibition with an outer zone of stimulation. Tests with C albicans and Capsicum extracts demonstrated a slight stimulatory effect in many cases (Table 3). Bacillus cereus and B. subtilis
Tabasco (GVSC 3010) USDA Pl 387838 (GVSC 3011 )
(GVSC 3OO9)
USDA PI 439371 (gvsc 3007) USDA Pl (GVSC 3008) Habanero
(ovsc 3oo6)
f
f,c f,c f
nt c f,c f,c f
f
f
f,c
f,c f
f
f
f
f
f
f
f
f,c
f
f
f,c f
f
f
f
f f f
f
f,c
f,c
f,c
f,c
f,c f,c
f,c
f,c
f,c
f f,c f,c
f
f,c
f,c
f,c
f,c
f,c f,c
f,c
f,c
f,c
f f,c f,c
nt
nt
f
nt
nt
f nt
f
nt
f
f f f
E. aer
nt
nt
f
nt
nt
f nt
f
nt
f
f f f
E. clo
f
f
f,c
f
f
f,c f
f,c
f
f,c
f f f
E. col
nt
nt
f
nt
nt
f,c nt
f
nt
f
f f f
K. pne
nt
nt
f,c
nt
nt
f nt
f
nt
f
f f f
P. vul
f
f
f
f
f
f,c f
f
f
f
f f f
P. aer
nt
nt
f
nt
nt
f nt
f
f
f
f f f
S. typ
nt
nt
f
nt
nt
f nt
f
nt
f
f f f
S. mar
f
f
f,c
f
f
f,c f
f,c
f
f,c
f f f
S. aur
f
f
f
f
f
f f
f
nt
f
ff f f
S. epi
f
f
f,c
f
f
f,c f
f
f
f
f,c f
S. p y o
aB. cer, B. cereus; B. sub, B. subtilis; C. alb, C. albicans; C. spo, C. sporogenes; C. tet, C. tetani E. aer, E. aerogenes; E. clo, E. cloacae; E. col, E. coli; K. pne, K. pneumonia; P. vul, P. vulgaris; P. aer, P. aeruginosa; S. typ, S. typhimurium; S. mar, S. marcescens; S. aur, S. aureus; S. epi, S. epidermidis; S. pyo, S. pyogenes. bf, fresh; Cc, cooked; dnt, not tested.
C. pubescens
C frutescens
C chinense
C baccatum
f,c
f,c
f
f f,c f
f,c b
fa
C. tet
q"
-~ o~
~
~"
'~
~"
¢,
.~ .~
Garlic (GVSC 3000) Bird {GVSC 3001) Green Bell (GVSC 3003) Jalapeno leaves (GVSC 3003) Jalapeno leaves and fruit (GVSC 3003) Red chile (GVSC 3OO4) Serrano (GVSC 3005) USDA Pl 439359
C annuum
B. sub
C. spo
A. sativum
B. cer
C. alb
"~
Variety and voucher no.
Species
Microorganisms"
.~
Table 1 Microbial strains and plant tissue extracts tested for antimicrobial activity
R.H. Cichewicz, P.A. Thorpe/Journal of Ethnopharmacology 52 (1996) 61-70 Table 2 Zones of complete inhibition produced by Allium sativum extract Microorganism
Mean zone diameter 495% confidence interval
(mm) B. cereus B. subtilis C. albicans C. sporogenes C. tetani E. aerogenes E. cloacae E. coli K. pneumonia P. vulgaris P. aeruginosa S. typhimurium S. marcescens S. aureus S. epidermidis S. pyogenes
38.4 40.1 41.2 44.7 39.0 20.6 23.1 24.4 23.2 28.8
4- 2.6 4- 1.9 ± 0.8 4- 3.8 ± 2.5 4- 0.8 ± 1.0 4- 1.0 4- 1.9 4- 1.2
18.2 23.7 22.3 30.7 33.3 29.8
444444-
1.3 2.6 1.9 ~.6 1.9 3.6
showed increased growth in the presence of several tissue extracts; however, the cooked and uncooked C. annuum ('Bird' and 'Jalapefio' leaves) partially inhibited both Bacillus test strains. Similarly, uncooked C. annuum ('Red Chile') partially inhibited B. cereus. In most cases of inhibition, an outer zone of stimulation was apparent (Table 3). Clostridium sporogenes and C. tetani were completely or partially inhibited by most cooked and uncooked Capsicum tissues (Table 4). Cooking the Capsicum extract altered its activity by increasing or decreasing the diameter of the zone of inhibition or altering the inhibitory effect from complete to partial inhibition, partial to complete inhibition, inhibition to no inhibition, or no inhibition to inhibition. In 11 cases, cooking the Capsicum extract reduced the inhibitory effect from complete to partial or no effect with C. sporogenes and in 4 eases the same effect was noted for C. tetani. Furthermore, cooking the extract increased the diameter of the inhibitory zone with C. sporogenes in 4 cases, while the same effect was noted in 10 cases with C. tetani. Streptococcus pyogenes showed signs of inhibition with fresh C. annuum ('Bird', 'Jalapefio'
65
leaves, and 'Red Chile') and cooked C. annuum ('Bird') (Table 5). All tissue extracts produced some degree of a brown to green zone surrounding the filter disk, while the remainder of the plate showed E-hemolytic activity. The discolored zone exhibited by A. sativum could be produced on an uninoculated blood agar plate, while this effect from Capsicum extracts could only be produced in the presence of the S. pyogenes bacterium. Observations made after an additional 24 h showed that the brown to green discolored zone returned to its original red color, while the remainder of the inoculated plate was clear. Microscopic examination of this red zone revealed the presence of red blood cells in the agar which were absent in the clear regions.
4. Discussion and conclusions Capsicum species are small perennial herbs native to tropical South America. The majority of researchers believe that this genus is comprised of more than twenty species, five of which (C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens) are the result of domestication (Bosland, 1994; McLeod et al., 1982). In the past, C. annuum varieties are believed to have been the predominant domesticated chile pepper in Mesoamerica; however, in more recent times C. chinense, C. frutescens, and C. pubescens have also been found throughout this region (Bosland, 1994; Eshbaugh, 1976). These and other Capsicum species can presently be found in scores of applications throughout the world; however, the plant's fruits are most noted as a food item and medicinal agent. One such marked example of the substantial role which Capsicum species play can be seen in Mesoamerica's Mayan culture. Early European observers noted the omnipresent nature of chiles in the Mayan diet, reporting that nothing was eaten without them (Coe, 1994). Today, chiles are found in a wide array of shapes, sizes, colors, and levels of pungency in their food and drink. While typically regarded as a spice, the substantial role which chiles occupy in this culture's diet may have important nutritional consequences for these people. Chemical analysis has demonstrated that Capsicum fruits contain relatively high concentrations of several essential
Serrano U S D A PI U S D A PI U S D A PI Habanero Tabasco U S D A PI
fruit R e d chile
387838
439359 439371 439407
(-) 13.9 + 24.3 + 23.7 + 29.7 + 28.7 + 28.0 + 22.6 + 27.7 + 27.3
20.1 16.3 29.0 20.1
b(_), partial inhibition; c+, stimulation; dnt, n o t tested.
1.5 0.9 0.9 1.7
4. 0.6 4. 0.9 ± 1.0 4. 1.4 4. 2.9 4. 2.5 4. 2.5 4. 2.9 ± 1.4
4. 4. 4. 4.
23.0 14.0 29.0 22.7 26.0
4. 4. 4. 4. 4.
1.2 0.6 2.7 1.4 2.5
+ + + + + + +
24.4 26.7 23.3 28.0 24.3 26.0 27.7
± i 4. ± ± 4. ±
1.0 1.4 1.4 0 1.6 0 1.4
+ 24.4 4. 0.8
+ (-) + + +
(-) 16.9 4- 0.7 + 24.7 4. 1.2
Fresh
Fresh
aB. cer, B. cereus; B. sub, B. subtilis; C.alb, C. albicans
C. chinense C. frutescens C. pubescens
C. baccatum
+c (-) + + nt d
G r e e n Bell J a l a p e n o Leaves
J a l a p e n o fruit J a l a p e ~ o leaves and
(.)b 17.9 4. 1.1
Bird
C. annuum
B. sub
R cer
Microorganism a/treatment
Variety
Species
+ 22.0 4. 0 n o effect n o effect no effect + 21.7 4. 0.6 n o effect n o effect
+ 22.3 4- 1.4
+ 20.0 4. 1.9 n o effect
+ 17.0 4. 0.9 n o effect
+ 17.8 4- 0.8
Fresh
C. aib
M e a n zone d i a m e t e r 4- 95% c o n f i d e n c e interval (nun)
+ 22.3 4- 2.9 nt nt nt + 25.0 4. 2.5 nt nt
+ 28.3 ± 1.3
nt (-) 17.3 ± 1.4 + 33.0 ± 2.5 nt nt
(-) 17.8 4. 3.4
Cooked
B. cer
+ 25.7 4. 2.9 nt nt nt + 26.3 4. 1.4 nt nt
+ 24.0 4- 0
nt (-) 19.0 4. 2.5 + 30.7 4. 2.9 nt nt
(-) 17.0 ± 2.4 + 25.0 4. 2.3
Cooked
B. sub
Table 3 Zones of partial i n h i b i t i o n a n d / o r s t i m u l a t i o n for Bacillus cereus, Bacillus subtilis, a n d Candida albicans p r o d u c e d b y Capsicum e x t r a c t s
C. alb
+ 20.7 ± 2.9 nt nt nt + 18.3 4. 1.4 nt nt
nt
nt nt
nt nt
nt
Cooked
I
ga
e~
R.H. Cichewiez, P.A. Thorpe~Journal of Ethnopharmacology 52 (1996) 61-70
67
Table 4 Zones o f complete or partial inhibition for Clostridium sporogenes and CIostridium tetani produced by Capsicum extract Species
Variety
M e a n zone diameter ± 95% confidence interval (mm) Microorganisma/treatment
C. annuum
Bird
C. chinense C. frutescens C. pubescens
C. tet
C. spo
C. tet
Fresh
Cooked
Cooked
_ b 15.3 ± 0.3
Green Bell
C. baecatum
C. spo Fresh
(_)c 13.8 ± 0.2
Jalapeno leaves Jalapet~o fruit Jalapet~o leaves and fruit Red chile Serrano U S D A PI 439359 U S D A PI 439371 U S D A PI 439407 Habanero Tabasco U S D A PI 387838
(-)
16.8 ± 14.9 ± 17.0 ± 17.0 ± 15.1 ± 16.6 ± 15.4 ± 16.3 ± 15.6 ± 14.9 ± 15.7 ±
0.5 0.4 0 0.2 0.3 0.8 0.5 0.5 0.4 0.4 0.7
- 14.8 ± 0.3
no effect no (-) (-)
17.1 ± effect 16.2 ± 16.5 ± 14.3 ± 15.0 ± 15.5 ± 16.0 ± 14.0 ± 14.0 ± 19.7 ±
1.0 0.3 0.6 0.4 0.9 0.6 0.7 0.5 0 0.4
( - ) 19.5 ± 1.4 ( - ) 15.3 ± 0.4 ( - ) 18.4 ± ( - ) 14.6 ± ( - ) 18.3 ± no effect ( - ) 15.9 ± no effect no effect no effect no effect no effect nt d
1.3 0.4 0.9 0.8
- 15.8 ± 0.9 - 16.3 ± 0.3 - 19.9 ± 0.8 - 16.5 ± 0.6 - 20.7 ± 0.5 no effect - 16.8 ± 0.3 ( - ) 20.0 ± 0 ( - ) 17.7 ± 1.4 ( - ) 18.7 ± 1.4 - 16.0 ± 0 ( - ) 19.3 ± 1.4 nt
aC. spo, C. sporogenes; C. tet, C. tetani b_, complete inhibition; c(_), partial inhibition; dnt, not tested.
Table 5 Zones of complete inhibition a n d / o r discoloration for Streptococcus pyogenes produced by Capsicum extracts Species
Variety
Mean zone diameter ± 95% confidence interval (mm) Microorganisma/treatment
C annmon
Bird Green Bell Jalapet~o leaves Jalapet~o fruit Jalapeffo leaves and fruit Red chile
C. baccatum
C. chinense C. frutescens C. pubescens
Serrano U S D A PI U S D A PI U S D A PI Habanero Tabasco U S D A PI
439359 439371 439407
387838
S. pyo
S. pyo
Fresh
Cooked
_ b 15.0 ± d e 25.0 ± d 22.0 ± - 15.3 ad 22.0 ± d 26.5 ± d 21.8 ± - 16.3 ± d 31.5 ± d 22.2 ± d 31.3 ± d 28.7 ± d 34.7 ± d 29.3 -v d 20.3 ± d 29.7 ±
0 0 2.5 1.4 0 1.8 1.0 0.7 4.9 3.3 3.8 2.9 2.9 1.5 1.4 !.4
aS. pyo, S. pyogenes; b , complete inhibition; Cd, discoloration; ant, not tested.
- 15.0 ± 0 d 25.0 ± 0 nt d nt nt nt nt d 24.3 ± 1.4 nt nt nt d 31.0 ± 2.5 nt nt
68
R.H. Cichewicz, P.A. Thorpe / Journal o f Ethnopharmacology 52 (1996) 61 - 70
nutrients, including vitamin C (up to Iour to six times more than an orange) (Bosland, 1994; Combs, 1992) and provitamins A, E, P, B1 (thiamine), B2 (riboflavin), and B3 (niacin) (Bosland, 1994). Capsicum tissues have also had an important role in the botanical pharmacopoeia of the Mayan people. As demonstrated by Roys (1931) and Alcorn (1984), Capsicum tissues have been used to treat a variety of ailments, many of which could have been caused or complicated by microbial infection. In this experiment, the inhibitory effects produced by Capsicum species against B. cereus, B. subtilis, C. sporogenes, C. tetani, and S. pyogenes suggests that these tissues may have played a medicinal role in the healing practices of the Mayan people. Bacillus species are common microbes found in most natural environments including soil, water, and plant and animal tissues. While most Bacillus species are regarded as having little pathogenic potential, both B. cereus and B. subtilis have been known to act as a primary invader or secondary infectious agent in a number of diseases and have been implicated in some cases of food poisoning (Turnbull and Kramer, 1991). Some varieties of C. annuum in the food and medicine of the Maya may have helped to combat these microbes. Clostridium species are ubiquitous anaerobic organisms found as vegetative cells or spores in soil, sewage, aquatic sediments, intestines of animals, and decaying plant and animal matter (Allen and Baron, 1991). Members of this genus are known to cause a number of deadly diseases, including C. botulinum (botulism), C. perfringens (gas gangrene), and C. tetani (tetanus) (Hatheway, 1990). The observed inhibitory effect against Clostridium species indicates the genus Capsicum may have played a role in the prevention of or recovery from Clostridium-related diseases in Mayan society. Streptococcus pyogenes is a common pathogenic bacteria responsible for a variety of cutaneous and systemic infections (Bisno, 1995). The observed inhibitory and blood plate discoloring effects with some Capsicum varieties and S. pyogenes may indicate an important role for these tissues in Mayan medicine as an inhibitory agent and source of anti-
/~-hemolytic compound(s). Streptococcus species are known to cause a variety of hemolytic effects, including a-hemolysis and B-hemolysis (Bisno, 1995; Facklam and Washington, 1991). Alphahemolysis gives rise to the formation of a brown to green zone of discoloration on a blood agar plate due to the formation of a hemoglobin reductant. Beta-hemolysis results in the rupturing of the red blood cells, causing the plate to fade from red to clear. Beta-hemolysis is facilitated by two distinct enzymes: streptolysin O, which is oxygen sensitive, and streptolysin S, which is not. These enzymes are toxic to a variety of cells and cell organelles ineluding erythrocytes, platelets, and cardiac tissues (Bisno, 1995). The presence of intact red blood cells surrounding the Capsicum extract-soaked disks in the presence of S. pyogenes under aerobic conditions may be due to inhibition of the streptolysin S enzyme. Variation between the observed effects with the Capsicum extracts and the microorganisms might be due to a number of factors. Capsicum species may contain an assortment of chemicals which were responsible for the inhibitory, stimulatory, and anti-hemolytic effects noted in this assay. These chemicals may also be present in dissimilar concentrations in different Capsicum varieties at different stages of maturity (Balbaa et al., 1968). The crude extracts used in this test may allow for synergistic or antagonistic interactions between Capsicum's numerous chemical constituents creating or prohibiting biological activity which may or may not have been apparent with a purified chemical. In addition, cooking the extract will further serve to alter the extract's chemical properties, which may explain why, in several instances, both the fresh and heated extracts displayed dissimilar activity. Moreover, the Capsicum varieties selected for this assay may vary from those incorporated into the Mayan pharmacopoeia. It is not possible to make a direct correlation between the observed activity of the Capsicum extracts in vitro and the actual effects when applied in vivo for the diseases observed by the Maya. One analogous example is that while dried Capsicum tissues have been shown in vitro to inhibit C. botulinum growth (Huhtanen, 1980),
R. H. Cichewicz, P. A. Thorpe/Journal of Ethnopharmacology 52 (1996) 61-70
numerous cases exist in which people have succumbed to botulinal neurotoxins following ingestion of improperly canned chile peppers (Center for Disease Control, 1979). Therefore, it is important that the Capsicum species which have demonstrated growth-inhibiting activity in this assay should be further studied to evaluate the significance of these tissues' clinical role in the Mayan medical system. Additional research is also necessary to investigate the potential antihemolytic activity of Capsicum extracts with S.
pyogenes. While it is uncertain why Capsicumspecies have been incorporated into the Mayan pharmacopoeia, it is possible that the reason(s) are based upon these tissues' potential therapeutic properties as antimicrobial and anti-hemolytic agents. Other research has suggested the possibility of some alternative potentially beneficial medicinal roles and effects of Capsicum species, including capsaicin's strong analgesic properties (Cordell and Araujo 1993), capsaicin-induced alterations in the pH and epithelial cell properties of the digestive tract, preventing microbial infection (Tellez et al., 1993), and possible anticancer effects (Bosland, 1994; Surh and Lee 1995). The questions raised by this research related to the use of Capsicumspecies in the Mayan culture are open for extensive study. Chiles not only function as important spices, but also serve as nutritional and medicinal agents. It is certain that, through ethnobotanical observations, Capsicum species harbor many economically significant benefits awaiting 'discovery'.
Acknowledgments The authors would like to thank Dean P. Douglas Kindschi and the members of the Grand Valley State University Science and Math Student Undergraduate Research Program Committee for their support and financial assistance. The Grand Valley State University library staff are most appreciated for their efforts in obtaining the necessary research materials and references. We are grateful to Dr. Paul Bosland of New Mexico State University for his contribution of peppers and verification of the tested Capsicum varieties.
69
References Abdou, I.A., Abou-Zcid, A.A., EI-Sherbeeny, M.R. and AbouEI-Gheat, Z.H. (1972) Antimicrobial activities of Allium sativum, Allium cepa, Raphanus sativus, Capsicum frutescens, Eruca sativa, Allium kurrat, on bacteria. Qualitas Plantarum et Materiae Vegetabiles 1, 29-35. Adetumbi, M.A. and Lau, B.H.S. (1983) Allium sativum (garlic) - - A natural antibiotic. Medical Hypotheses 12, 227-237. AI-Delaimy, K.S., and All, S.H. (1970) Antibacterial action of vegetable extracts on the growth of pathogenic bacteria. Journal of Science and Food Agriculture 21, 110-112. Alcorn, J.B. (1984) Huastec Mayan Ethnobotany. University of Texas Press, Austin, pp. 578-580. Allen, S.D. and Baron, E.J. (1991) Clostridium. In: A. Balows, W.J. Hausler Jr., K.L. Herrmann, H.D. Isenberg and H.J. Shadomy (Eds.), Manual of Clinical Microbiology. 5th Ed. American Society for Microbiology, Washington, D.C., pp. 505-521. Balbaa, S.I., Karawya, M.S. and Girgis, A.N. (1968) The capsaicin content of Capsicum fruits at different stages of maturity. Lloydia 31,272-274. Bisno, A.L. (1995) Streptococcus pyogenes. In: G.L. Mandell, J.E. Bennett and R. Dolin (Eds.), Principles and Practice of Infectious Disea.ses, 4th Ed., vol. 2. Churchill Livingstone, New York, pp. 1786-1799. Bosland, P.W. (1994) Chiles: History, cultivation, and uses. In G. Charalambous (Ed.), Spices, Herbs and Edible Fungi, Elsevier Science, New York, pp. 347-366. Bosland, P.W., Bailey, A.L. and Iglesias-Olivas, J. (1990) Capsicum Pepper Varieties and Classification. Circular 530, Cooperative Extension Service, College of Agriculture and Home Economics, New Mexico State University, New Mexico, pp. 1-12. Bushnell, O.A., Fukuda, M. and Makinodan, T. (1950) The antibacterial properties of some plants found in Hawaii. Pacific Science 4, 167-183. Caceres, A., Alvarez, A.V., Ovando, A.E. and Samayoa, B.E. (1991) Plants used in Guatemala for the treatment of respiratory diseases. 1. Screening of 68 plants against grampositive bacteria. Journal of Ethnopharmacology 31, 193-208. Cardoso, H.T. and Santos, M.D.L. (1948) Estudos sobre a presenca de antibioticos nos vegetais. Brasil-Medico 62, 67-70. Center for Disease Control (1979) Botulism in the United States, 1899-1977. Handbook for Epidemiologists, Clinicians, and Laboratory Workers. Public Health Service, Atlanta, Georgia, pp. 25, 36-41. Chen, H., Chang, M. and Chang, T. (1985) Antibacterial properties of some spice plants before and after heat treatment. Chinese Journal of Microbiology and Immunology 18, 190-195. Coe, S.D. (1994) America's First Cuisines. University of Texas Press, Austine, p. 62. Combs, G.F., Jr. (1992) The Vitamins: Fundamental Aspects in Nutrition and Health. Academic Press, New York, p. 225.
70
R.H. Cichewicz, P.A. Thorpe/Journal of Ethnopharmacology 52 (1996) 61-70
Cordell, G.A. and Araujo, O.E. (1993) Capsaicin: Identification, nomenclature, and pharmacotherapy. The Annals of Pharmacotherapy 27, 330-336. Cox, P.A. and Balick, M.J. (1994) The ethnobotanical approach to drug discovery. Scientific American 271, 82-87. DeWitt, D. and Bosland, P.W. (1993) The Pepper Garden. Ten Speed Press, Berkeley pp. 1-69. Eshbaugh, W.H. (1976) XII. Genetic and biochemical systematic studies of chili peppers (Capsicum-Solanaceae). Bulletin of the Torrey Botanical Club 102, 396-403. Facklam, R.R. and Washington, J.A., II (1991) Streptococcus and related catalasc-negative gram-positive cocci. In: A. Balows, W.J. Hausler Jr., K.L. Herrmann, H.D. Isenberg and H.J. Shadomy (Eds.), Manual of Clinical Microbiology, 5th Ed. American Society for Microbiology, Washington, D.C., pp. 238-257. Farbman, K.S., Barnett, E.D., Bolduc, G.R. and Klein, J.O. (1993) Antibacterial activity of garlic and onions: A historical perspective. The Pediatric Infectious Disease Journal 12, 613-614. Fenwick, G.R. and Hanley, A.B. (1985) The genus Allium - Part 3. CRC Critical Reviews in Food Science and Nutrition 23, 1-73. Fitzpatrick, F.K. (1954) Plant substances active against Mycobacterium tuberculosis. Antibiotics and Chemotherapy 4, 528-536. Frisbey, A., Gottshall, R.Y., Jennings, J.C. and Lucas, E.H. (1954) The occurrence of antibacterial substances in seed plants with special reference to Mycobacterium tuberculosis. Michigan Agricultural Experiment Station Quarterly Bulletin 36, 477-488. Gottshall, R.Y., Lucas, E.H., Lickfeldt, A. and Roberts, J.M. (1949) The occurrence of antibacterial substances active against Mycobacterium tuberculosis in seed plants. Journal of Clinical Investigation 28, 920-923. Harris, H.A. (1949) Antibacterial activity of seedling extracts of cultivated plants. Bulletin of the Torrey Botanical Club 76, 244-254. Hatheway, C.L. (1990) Toxigenic clostridia. Clinical Microbiology Reviews 3, 66-98. Huhtanen, C.N. (1980) Inhibition of CIostridium botulinum by spice extracts and aliphatic alcohols. Journal of Food Protection 43, 195-196, 200. MacDonald, R.E. and Bishop, C.J. (1953) A further survey of
plants for antibacterial substances. Canadian Journal of Botany 31, 123-131. McLeod, M.J., Guttman, S.I. and Eshbaugh, W.H. (1982) Early evolution of chile peppers (Capsicum). Economic Botany 36, 361-368. Masilungan, V.A., Kalaw, C.V. and Masilungan, F.C. (1963) Screening of plants from the Makiling area for antimicrobial substances. The Philippine Journal of Science 92, 421-430. Masilungan, V.A., Maranon, J. and Valencia, V.V. (1955) Some common Philippine medicinal plants containing antibacterial substances. Journal of the Philippine Pharmaceutical Association 42, 72-79. Mitscher, L.A., Leu, R., Eathala, M.S., Wu, W., Beal, J.L. and White, R. (1972) Antirnicrobial agents from higher plants. I. Introduction, rationale, and methodology. Lloydia 35, 157-166. Munscrecnusorn, Y., Kongsamut, S. and Pezalla, P. (1982) Capsaicin: A literature survey. CRC Critical Reviews in Toxicology 10, 321-339. Roys, R.L. (1931) The Ethno-Botany of the Maya. Reprinted 1976, Institute for the Study of Human Issues, Philadelphia, pp. 2, 7, 9, 12, 13, 14, 15, 17, 31, 32, 34, 35, 38, 49, 57, 58, 61, 63, 65, 67, 90, 93, 95, 106, 129, 150, 153, 184, 189, 195. Surh, Y.J. and Lee, S.S. (1995) Capsaicin, a double-edged sword: Toxicity, metabolism, and chemopreventive potential. Life Sciences 56, 1845-1855. Suzuki, T. and lwai, K. (1984) Constituents of red pepper species: Chemistry, biochemistry, pharmacology, and food science of the pungent principle of Capsicum species. In: A. Brossi (Ed.), The Alkaloids: Chemistry and Pharmacology. Academic Press, New York, pp. 227-299. Tellez, G.I., Jaeger, L., Dean, C.E., Corrier, D.E., DeLoach, J.R., Williams, J.D. and Hargis, B.M. (1993) Effect of prolonged administration of dietary capsaicin on Salmonella enteritidis infection in leghorn chicks. Avian Diseases 37, 143-148. Turnbuli, P.C.B. and Kramer, J.M. (1991) Bacillus. In: A. Balows, W.J. Hausler Jr., K.L. Herrmann, H.D. Isenberg, and H.J. Shadomy (Eds.), Manual of Clinical Microbiology. 5th Ed. American Society for Microbiology, Washington, D.C., pp. 296-303. Virus, R.M. and Gebhart, G.F. (1979) Pharmacologic actions of capsaicin: Apparent involvement of substance P and serotonin. Life Sciences 25, 1273-1284.
Journal of
ETHNOPHARMACOIDGY ELSEVIER
Journal of Ethnopharmacology 52 (1996) 61-70
The antimicrobial properties of chile peppers (Capsicum species) and their uses in Mayan medicine R o b e r t H. Cichewicz a, Patrick A. T h o r p e *b aDepartment of Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA bDepartment of Biology, Grand Valley State University, Allendale, MI 49401, USA Received 3 July 1995; revised 23 September 1995; accepted 8 January 1996
Abstract A survey of the Mayan pharmacopoeia revealed that tissues of Capsicum species (Solanaceae) are included in a number of herbal remedies for a variety of ailments of probable microbial origin. Using a filter disk assay, plain and heated aqueous extracts from fresh Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens, and Capsicum pubescens varieties were tested for their antimicrobial effects with fifteen bacterial species and one yeast species. Two pungent compounds found in Capsicum species (capsaicin and dihydrocapsaiein) were also tested for their antimicrobial effects. The plain and heated extracts were found to exhibit varying degrees of inhibition against Bacillus cereus, Bacillus subtilis, Clostridium sporogenes, Clostridium tetani, and Streptococcus pyogenes.
Keywords: Antimicrobial activity; Capsicum; Ethnobotany; Mayan medicine; Solanaeeae
1. Introduction
The indigenous inhabitants of various regions throughout the world rely upon plants as a source of medicinal agents to cure many of their ailments. Scientists observing these practices have facilitated the 'discovery' of a host of economically important drugs, such as the antihypertensive, reserpine, as well as pseudoephedrine, which reduces congestion (Cox and Balick, 1994). Studies of the botanical pharmacopoeia of the indigenous Mayan inhabitants of Mesoamerica have shown that chile peppers (Capsicum species * Corresponding author.
(Solanaceae)) are incorporated into a number of medicinal preparations (Roys, 1931; Alcorn, 1984). In a collection of 437 Mayan therapeutic remedies compiled by Roys (1931), 32 list the tissues of Capsicumspecies as one of the ingredients. In 29 cases, the fruits are reported as being used alone or in conjunction with the leaves, roots, or seeds. Eighteen of these remedies include instructions for boiling or heating the mixture in some manner before administration. These preparations were applied for a variety of ailments including respiratory problems, bowel complaints, earaches, and sores. Alcorn (1984) has also recorded 14 cases in which Huastec Mayan informants reported using fresh and heated Capsicumspecies
0378-8741/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PIL" 0378-8741(96)01384-0
62
R.H. Cichewicz, P.A. Thorpe/ Journal of Ethnopharmacology 52 (1996) 61-70
for medicinal purposes. The illnesses treated inelude infected wounds and fresh burns. A well-studied chemical component of Capsicum species, capsaicin, one of the pungent capsaicinoid compounds found in chile peppers, has already demonstrated a high degree of biological activity affecting the nervous, cardiovascular, and digestive systems (Virus and Gebhart, 1979; Monsereenusorn et al., 1982; Suzuki and lwai, 1984; Cordell and Araujo, 1993; Surh and Lee, 1995). Furthermore, clinical trials have shown that capsaicin may have potential value in the management of painful conditions such as rheumatoid arthritis and cluster headaches by causing the release of a sensory neuron's supply of substance P (a neurotransmitter) until depleted, resulting in insensitivity to painful stimuli (Cordell and Araujo, 1993). Several attempts have been made to test whether Capsicum tissues have a potential value as agents in the treatment of other medical conditions. Many of these efforts have focused on possible antimicrobial effects (Cardoso and Santos, 1948; Gotshall et al., 1949; Harris, 1949; Bushnell et al., 1950; MacDonald and Bishop, 1953; Fitzpatrick, 1954; Frisby et al., 1954; Masilungan et al., 1955; Masilungan et al., 1963; A1-Delaimy and Ali, 1970; Abdou et al., 1972; Mitscher et al., 1972; Chen et al., 1985; Caceres et al., 1991). A variety of assay methods (filter disk, penicup, pour plate,...), extraction procedures (acetone, aqueous, ethanol,...), and tissue types (predominantly common commercial varieties, some inaccurately identified) have been used with contradictory results reported. These tests fail to provide congruent evidence regarding the efficacy of Capsicum tissues' chemotherapeutic properties. The present study was conducted to examine the potential antimicrobial properties of Capsicum species as used by the Mayan inhabitants of Mesoamerica. 2. Materials and methods
2.1. Microorganism strains Strains of Bacillus cereus, Bacillus subtilis, Clostridium sporogenes, Clostridium tetani, Enterobacter aerogenes, Salmonella typhimurium, and Staphylococcus aureus (coagulase positive)
were cultured from Carolina MicroKwik rM Cultures (Carolina Biological Supply Co.). Enterobacter cloacae (ATCC 23355), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13833), Proteus vuigaris (ATCC 13315), Pseudomonas aeruginosa (ATCC 27853), Serratia marcescens (ATCC 8100), Staphylococcus epidermidis (ATCC 12228), Streptococcus pyogenes (ATCC 19615), and Candida albicans (ATCC 14053) were cultured from Difco Bactrol TM Disks (Difco Laboratories). Candida albicans was cultured in Sabouraud medium, Clostridium species in fluid thioglycollate, and all others in brain-heart infusion (BHI) medium. 2.2. Capsicum tissues Tissues from 11 varieties of five species of Capsicum were tested: C. annuum L. ('Bird' fruits, 'Green Bell' fruits, 'Jalapefio' fruits and leaves, 'Red Chile' fruits, and 'Serrano' fruits), C. baccatum L. (USDA PI 439359 fruits, USDA PI 439371 fruits, and USDA PI 439407 fruits), C. chinense Jacq. ('Habanero' fruits), C. frutescens L. ('Tabasc' fruits), and C. pubescens R. & P. (USDA PI 387838 fruits). The C. baccatum, C. frutescens, and C. pubescens were provided by Paul Bosland of New Mexico State University. The C. annuum ('Green Bell', 'Jalapefio', and 'Red Chile') were grown by the authors. The remaining strains were purchased from produce distributors. All Capsicum varieties were identified according to Bosland et al. (1990) and DeWitt and Bosland (1993) and verified by Paul Bosland (personal communications). Voucher specimens are listed in Table 1 and were deposited at the Grand Valley State University Herbarium (Allendale, Michigan, USA). 2.3. Tissue extracts Approximately 100 g of fresh plant tissue was washed and soaked in distilled water before being blotted dry, chopped into small pieces, and ground intermittently in a blender with no solvent for one to two minutes until a homogenous slurry was obtained. The slurry was then strained through a double layer of cheese cloth and the particulate matter discarded. Approximately 35 ml of the filtrate was centrifuged at 4°C for 30 min at 20 000 x g. The supernatant was collected and
R.H. Cichewicz,P.A. Thorpe~Journalof Ethnopharmacology 52 (1996) 61-70 reeentrifuged under the same conditions. The final supernatant was placed on ice until tested. Combined C. annuum tissue extract ('Jalapefio' fruits and leaves) was prepared by combining equal amounts (by weight) of the two tissue types which were blended together and then processed in the same manner as the other extracts. Cooked extract was made by decanting 20 ml of the centrifuged sample into a large test tube which was placed into a boiling water bath. The sample was swirled every 5 rain and placed immediately on ice after 20 rain of heating. 2.4. Filter disk assay Petri plates (10 cm diameter) were filled with a 20 ml base of sterile BHI agar or Sabouraud agar (for C. albicans). Four ml agar overlays cooled to 48°C (Sabouraud for C. albicans, thioglycollate for C. tetani, and BHI for all other strains) were inoculated with 100 #1 of overnight culture of the test organisms (400 #1 for C. tetanO and poured over the agar base. Trypticase soy agar II TM plates with 5% sheep blood (Beeton Dickson and Co.) were inoculated with 100 #1 of overnight S. pyogenes culture and spread mechanically over the surface of the plate. Three 13 mm diameter filter disks (Schleicher and Schuell no. 740-E) were soaked in the extract, gently tapped to remove excess liquid, and positioned on an inoculated plate. A fourth disk soaked in distilled water was also placed on the plate as a negative control. All plates remained upright at room temperature for at least 30 min before being inverted and incubated at 37°C for 15 h. Plates containing Clostridium species were incubated in a sealed BBL GasPak 100 TM canister with a carbon dioxide-enriched, anaerobic atmosphere. All plates were observed for zones of discoloration, inhibition, and/or stimulation of microbial growth, and the diameter of these zones was measured in mls. 2.5. Capsaicinoids assay Pour-plate and spread-plate cultures were prepared as indicated above for B. cereus, B. subtilis, C. aibicans, C. sporogenes, E. coli, S. typhimurium, S. aureus, and S. pyogenes. Ten mg dihydrocapsaicin, capsaicin (98% purity), and cap-
63
saicin (60% purity) (Sigma Chemical Co.) were each dissolved in 1 ml of methyl alcohol. Four sterile filter disks were then placed on the inoculated plates and 25 #1 of the capsaicinoids or methyl alcohol (control) were pipetted on the disks. Each chemical was tested in triplicate with the indicated bacterial strains. 2.6. Garlic assay . In order to evaluate the qualitative response of this assay method, a known botanically derived antimicrobial substance was selected as a positive control. AIlium sativum L. (garlic bulb) was chosen for this purpose as it has been demonstrated to produce a strong inhibitory effect with several bacterial species (Adetumbi and Lau, 1983; Fenwick and Hanley, 1985; Farbman et al., 1993). Allium sativum, purchased from a produce distributor, was peeled and chopped into small pieces, then combined with distilled water (2 ml water per 3 g A. sativum) before being blended and processed in the same manner as the Capsicum tissues. All microbial species were tested in triplicate with .4. sativum. 3. R ~
The combinations of tissue extracts and microorganisms which were tested are presented in Table 1. A number of effects involving the microorganisms were visually observed as circular zones surrounding the test extract-soaked disks, including complete inhibition (the absence of all microbial growth), partial inhibition (reduced microbial growth), stimulation (increased microbial density), an inner zone of partial inhibition with an outer region of stimulation, and blood plate discoloration. All bacterial and yeast strains were completely inhibited by the A. sativum extract (Table 2). The capsaicinoids showed no activity. The Capsicum tissue extracts exhibited a variety of effects with the test organisms, including complete inhibition, partial inhibition, stimulation, and partial inhibition with an outer zone of stimulation. Tests with C albicans and Capsicum extracts demonstrated a slight stimulatory effect in many cases (Table 3). Bacillus cereus and B. subtilis
Tabasco (GVSC 3010) USDA Pl 387838 (GVSC 3011 )
(GVSC 3OO9)
USDA PI 439371 (gvsc 3007) USDA Pl (GVSC 3008) Habanero
(ovsc 3oo6)
f
f,c f,c f
nt c f,c f,c f
f
f
f,c
f,c f
f
f
f
f
f
f
f
f,c
f
f
f,c f
f
f
f
f f f
f
f,c
f,c
f,c
f,c
f,c f,c
f,c
f,c
f,c
f f,c f,c
f
f,c
f,c
f,c
f,c
f,c f,c
f,c
f,c
f,c
f f,c f,c
nt
nt
f
nt
nt
f nt
f
nt
f
f f f
E. aer
nt
nt
f
nt
nt
f nt
f
nt
f
f f f
E. clo
f
f
f,c
f
f
f,c f
f,c
f
f,c
f f f
E. col
nt
nt
f
nt
nt
f,c nt
f
nt
f
f f f
K. pne
nt
nt
f,c
nt
nt
f nt
f
nt
f
f f f
P. vul
f
f
f
f
f
f,c f
f
f
f
f f f
P. aer
nt
nt
f
nt
nt
f nt
f
f
f
f f f
S. typ
nt
nt
f
nt
nt
f nt
f
nt
f
f f f
S. mar
f
f
f,c
f
f
f,c f
f,c
f
f,c
f f f
S. aur
f
f
f
f
f
f f
f
nt
f
ff f f
S. epi
f
f
f,c
f
f
f,c f
f
f
f
f,c f
S. p y o
aB. cer, B. cereus; B. sub, B. subtilis; C. alb, C. albicans; C. spo, C. sporogenes; C. tet, C. tetani E. aer, E. aerogenes; E. clo, E. cloacae; E. col, E. coli; K. pne, K. pneumonia; P. vul, P. vulgaris; P. aer, P. aeruginosa; S. typ, S. typhimurium; S. mar, S. marcescens; S. aur, S. aureus; S. epi, S. epidermidis; S. pyo, S. pyogenes. bf, fresh; Cc, cooked; dnt, not tested.
C. pubescens
C frutescens
C chinense
C baccatum
f,c
f,c
f
f f,c f
f,c b
fa
C. tet
q"
-~ o~
~
~"
'~
~"
¢,
.~ .~
Garlic (GVSC 3000) Bird {GVSC 3001) Green Bell (GVSC 3003) Jalapeno leaves (GVSC 3003) Jalapeno leaves and fruit (GVSC 3003) Red chile (GVSC 3OO4) Serrano (GVSC 3005) USDA Pl 439359
C annuum
B. sub
C. spo
A. sativum
B. cer
C. alb
"~
Variety and voucher no.
Species
Microorganisms"
.~
Table 1 Microbial strains and plant tissue extracts tested for antimicrobial activity
R.H. Cichewicz, P.A. Thorpe/Journal of Ethnopharmacology 52 (1996) 61-70 Table 2 Zones of complete inhibition produced by Allium sativum extract Microorganism
Mean zone diameter 495% confidence interval
(mm) B. cereus B. subtilis C. albicans C. sporogenes C. tetani E. aerogenes E. cloacae E. coli K. pneumonia P. vulgaris P. aeruginosa S. typhimurium S. marcescens S. aureus S. epidermidis S. pyogenes
38.4 40.1 41.2 44.7 39.0 20.6 23.1 24.4 23.2 28.8
4- 2.6 4- 1.9 ± 0.8 4- 3.8 ± 2.5 4- 0.8 ± 1.0 4- 1.0 4- 1.9 4- 1.2
18.2 23.7 22.3 30.7 33.3 29.8
444444-
1.3 2.6 1.9 ~.6 1.9 3.6
showed increased growth in the presence of several tissue extracts; however, the cooked and uncooked C. annuum ('Bird' and 'Jalapefio' leaves) partially inhibited both Bacillus test strains. Similarly, uncooked C. annuum ('Red Chile') partially inhibited B. cereus. In most cases of inhibition, an outer zone of stimulation was apparent (Table 3). Clostridium sporogenes and C. tetani were completely or partially inhibited by most cooked and uncooked Capsicum tissues (Table 4). Cooking the Capsicum extract altered its activity by increasing or decreasing the diameter of the zone of inhibition or altering the inhibitory effect from complete to partial inhibition, partial to complete inhibition, inhibition to no inhibition, or no inhibition to inhibition. In 11 cases, cooking the Capsicum extract reduced the inhibitory effect from complete to partial or no effect with C. sporogenes and in 4 eases the same effect was noted for C. tetani. Furthermore, cooking the extract increased the diameter of the inhibitory zone with C. sporogenes in 4 cases, while the same effect was noted in 10 cases with C. tetani. Streptococcus pyogenes showed signs of inhibition with fresh C. annuum ('Bird', 'Jalapefio'
65
leaves, and 'Red Chile') and cooked C. annuum ('Bird') (Table 5). All tissue extracts produced some degree of a brown to green zone surrounding the filter disk, while the remainder of the plate showed E-hemolytic activity. The discolored zone exhibited by A. sativum could be produced on an uninoculated blood agar plate, while this effect from Capsicum extracts could only be produced in the presence of the S. pyogenes bacterium. Observations made after an additional 24 h showed that the brown to green discolored zone returned to its original red color, while the remainder of the inoculated plate was clear. Microscopic examination of this red zone revealed the presence of red blood cells in the agar which were absent in the clear regions.
4. Discussion and conclusions Capsicum species are small perennial herbs native to tropical South America. The majority of researchers believe that this genus is comprised of more than twenty species, five of which (C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens) are the result of domestication (Bosland, 1994; McLeod et al., 1982). In the past, C. annuum varieties are believed to have been the predominant domesticated chile pepper in Mesoamerica; however, in more recent times C. chinense, C. frutescens, and C. pubescens have also been found throughout this region (Bosland, 1994; Eshbaugh, 1976). These and other Capsicum species can presently be found in scores of applications throughout the world; however, the plant's fruits are most noted as a food item and medicinal agent. One such marked example of the substantial role which Capsicum species play can be seen in Mesoamerica's Mayan culture. Early European observers noted the omnipresent nature of chiles in the Mayan diet, reporting that nothing was eaten without them (Coe, 1994). Today, chiles are found in a wide array of shapes, sizes, colors, and levels of pungency in their food and drink. While typically regarded as a spice, the substantial role which chiles occupy in this culture's diet may have important nutritional consequences for these people. Chemical analysis has demonstrated that Capsicum fruits contain relatively high concentrations of several essential
Serrano U S D A PI U S D A PI U S D A PI Habanero Tabasco U S D A PI
fruit R e d chile
387838
439359 439371 439407
(-) 13.9 + 24.3 + 23.7 + 29.7 + 28.7 + 28.0 + 22.6 + 27.7 + 27.3
20.1 16.3 29.0 20.1
b(_), partial inhibition; c+, stimulation; dnt, n o t tested.
1.5 0.9 0.9 1.7
4. 0.6 4. 0.9 ± 1.0 4. 1.4 4. 2.9 4. 2.5 4. 2.5 4. 2.9 ± 1.4
4. 4. 4. 4.
23.0 14.0 29.0 22.7 26.0
4. 4. 4. 4. 4.
1.2 0.6 2.7 1.4 2.5
+ + + + + + +
24.4 26.7 23.3 28.0 24.3 26.0 27.7
± i 4. ± ± 4. ±
1.0 1.4 1.4 0 1.6 0 1.4
+ 24.4 4. 0.8
+ (-) + + +
(-) 16.9 4- 0.7 + 24.7 4. 1.2
Fresh
Fresh
aB. cer, B. cereus; B. sub, B. subtilis; C.alb, C. albicans
C. chinense C. frutescens C. pubescens
C. baccatum
+c (-) + + nt d
G r e e n Bell J a l a p e n o Leaves
J a l a p e n o fruit J a l a p e ~ o leaves and
(.)b 17.9 4. 1.1
Bird
C. annuum
B. sub
R cer
Microorganism a/treatment
Variety
Species
+ 22.0 4. 0 n o effect n o effect no effect + 21.7 4. 0.6 n o effect n o effect
+ 22.3 4- 1.4
+ 20.0 4. 1.9 n o effect
+ 17.0 4. 0.9 n o effect
+ 17.8 4- 0.8
Fresh
C. aib
M e a n zone d i a m e t e r 4- 95% c o n f i d e n c e interval (nun)
+ 22.3 4- 2.9 nt nt nt + 25.0 4. 2.5 nt nt
+ 28.3 ± 1.3
nt (-) 17.3 ± 1.4 + 33.0 ± 2.5 nt nt
(-) 17.8 4. 3.4
Cooked
B. cer
+ 25.7 4. 2.9 nt nt nt + 26.3 4. 1.4 nt nt
+ 24.0 4- 0
nt (-) 19.0 4. 2.5 + 30.7 4. 2.9 nt nt
(-) 17.0 ± 2.4 + 25.0 4. 2.3
Cooked
B. sub
Table 3 Zones of partial i n h i b i t i o n a n d / o r s t i m u l a t i o n for Bacillus cereus, Bacillus subtilis, a n d Candida albicans p r o d u c e d b y Capsicum e x t r a c t s
C. alb
+ 20.7 ± 2.9 nt nt nt + 18.3 4. 1.4 nt nt
nt
nt nt
nt nt
nt
Cooked
I
ga
e~
R.H. Cichewiez, P.A. Thorpe~Journal of Ethnopharmacology 52 (1996) 61-70
67
Table 4 Zones o f complete or partial inhibition for Clostridium sporogenes and CIostridium tetani produced by Capsicum extract Species
Variety
M e a n zone diameter ± 95% confidence interval (mm) Microorganisma/treatment
C. annuum
Bird
C. chinense C. frutescens C. pubescens
C. tet
C. spo
C. tet
Fresh
Cooked
Cooked
_ b 15.3 ± 0.3
Green Bell
C. baecatum
C. spo Fresh
(_)c 13.8 ± 0.2
Jalapeno leaves Jalapet~o fruit Jalapet~o leaves and fruit Red chile Serrano U S D A PI 439359 U S D A PI 439371 U S D A PI 439407 Habanero Tabasco U S D A PI 387838
(-)
16.8 ± 14.9 ± 17.0 ± 17.0 ± 15.1 ± 16.6 ± 15.4 ± 16.3 ± 15.6 ± 14.9 ± 15.7 ±
0.5 0.4 0 0.2 0.3 0.8 0.5 0.5 0.4 0.4 0.7
- 14.8 ± 0.3
no effect no (-) (-)
17.1 ± effect 16.2 ± 16.5 ± 14.3 ± 15.0 ± 15.5 ± 16.0 ± 14.0 ± 14.0 ± 19.7 ±
1.0 0.3 0.6 0.4 0.9 0.6 0.7 0.5 0 0.4
( - ) 19.5 ± 1.4 ( - ) 15.3 ± 0.4 ( - ) 18.4 ± ( - ) 14.6 ± ( - ) 18.3 ± no effect ( - ) 15.9 ± no effect no effect no effect no effect no effect nt d
1.3 0.4 0.9 0.8
- 15.8 ± 0.9 - 16.3 ± 0.3 - 19.9 ± 0.8 - 16.5 ± 0.6 - 20.7 ± 0.5 no effect - 16.8 ± 0.3 ( - ) 20.0 ± 0 ( - ) 17.7 ± 1.4 ( - ) 18.7 ± 1.4 - 16.0 ± 0 ( - ) 19.3 ± 1.4 nt
aC. spo, C. sporogenes; C. tet, C. tetani b_, complete inhibition; c(_), partial inhibition; dnt, not tested.
Table 5 Zones of complete inhibition a n d / o r discoloration for Streptococcus pyogenes produced by Capsicum extracts Species
Variety
Mean zone diameter ± 95% confidence interval (mm) Microorganisma/treatment
C annmon
Bird Green Bell Jalapet~o leaves Jalapet~o fruit Jalapeffo leaves and fruit Red chile
C. baccatum
C. chinense C. frutescens C. pubescens
Serrano U S D A PI U S D A PI U S D A PI Habanero Tabasco U S D A PI
439359 439371 439407
387838
S. pyo
S. pyo
Fresh
Cooked
_ b 15.0 ± d e 25.0 ± d 22.0 ± - 15.3 ad 22.0 ± d 26.5 ± d 21.8 ± - 16.3 ± d 31.5 ± d 22.2 ± d 31.3 ± d 28.7 ± d 34.7 ± d 29.3 -v d 20.3 ± d 29.7 ±
0 0 2.5 1.4 0 1.8 1.0 0.7 4.9 3.3 3.8 2.9 2.9 1.5 1.4 !.4
aS. pyo, S. pyogenes; b , complete inhibition; Cd, discoloration; ant, not tested.
- 15.0 ± 0 d 25.0 ± 0 nt d nt nt nt nt d 24.3 ± 1.4 nt nt nt d 31.0 ± 2.5 nt nt
68
R.H. Cichewicz, P.A. Thorpe / Journal o f Ethnopharmacology 52 (1996) 61 - 70
nutrients, including vitamin C (up to Iour to six times more than an orange) (Bosland, 1994; Combs, 1992) and provitamins A, E, P, B1 (thiamine), B2 (riboflavin), and B3 (niacin) (Bosland, 1994). Capsicum tissues have also had an important role in the botanical pharmacopoeia of the Mayan people. As demonstrated by Roys (1931) and Alcorn (1984), Capsicum tissues have been used to treat a variety of ailments, many of which could have been caused or complicated by microbial infection. In this experiment, the inhibitory effects produced by Capsicum species against B. cereus, B. subtilis, C. sporogenes, C. tetani, and S. pyogenes suggests that these tissues may have played a medicinal role in the healing practices of the Mayan people. Bacillus species are common microbes found in most natural environments including soil, water, and plant and animal tissues. While most Bacillus species are regarded as having little pathogenic potential, both B. cereus and B. subtilis have been known to act as a primary invader or secondary infectious agent in a number of diseases and have been implicated in some cases of food poisoning (Turnbull and Kramer, 1991). Some varieties of C. annuum in the food and medicine of the Maya may have helped to combat these microbes. Clostridium species are ubiquitous anaerobic organisms found as vegetative cells or spores in soil, sewage, aquatic sediments, intestines of animals, and decaying plant and animal matter (Allen and Baron, 1991). Members of this genus are known to cause a number of deadly diseases, including C. botulinum (botulism), C. perfringens (gas gangrene), and C. tetani (tetanus) (Hatheway, 1990). The observed inhibitory effect against Clostridium species indicates the genus Capsicum may have played a role in the prevention of or recovery from Clostridium-related diseases in Mayan society. Streptococcus pyogenes is a common pathogenic bacteria responsible for a variety of cutaneous and systemic infections (Bisno, 1995). The observed inhibitory and blood plate discoloring effects with some Capsicum varieties and S. pyogenes may indicate an important role for these tissues in Mayan medicine as an inhibitory agent and source of anti-
/~-hemolytic compound(s). Streptococcus species are known to cause a variety of hemolytic effects, including a-hemolysis and B-hemolysis (Bisno, 1995; Facklam and Washington, 1991). Alphahemolysis gives rise to the formation of a brown to green zone of discoloration on a blood agar plate due to the formation of a hemoglobin reductant. Beta-hemolysis results in the rupturing of the red blood cells, causing the plate to fade from red to clear. Beta-hemolysis is facilitated by two distinct enzymes: streptolysin O, which is oxygen sensitive, and streptolysin S, which is not. These enzymes are toxic to a variety of cells and cell organelles ineluding erythrocytes, platelets, and cardiac tissues (Bisno, 1995). The presence of intact red blood cells surrounding the Capsicum extract-soaked disks in the presence of S. pyogenes under aerobic conditions may be due to inhibition of the streptolysin S enzyme. Variation between the observed effects with the Capsicum extracts and the microorganisms might be due to a number of factors. Capsicum species may contain an assortment of chemicals which were responsible for the inhibitory, stimulatory, and anti-hemolytic effects noted in this assay. These chemicals may also be present in dissimilar concentrations in different Capsicum varieties at different stages of maturity (Balbaa et al., 1968). The crude extracts used in this test may allow for synergistic or antagonistic interactions between Capsicum's numerous chemical constituents creating or prohibiting biological activity which may or may not have been apparent with a purified chemical. In addition, cooking the extract will further serve to alter the extract's chemical properties, which may explain why, in several instances, both the fresh and heated extracts displayed dissimilar activity. Moreover, the Capsicum varieties selected for this assay may vary from those incorporated into the Mayan pharmacopoeia. It is not possible to make a direct correlation between the observed activity of the Capsicum extracts in vitro and the actual effects when applied in vivo for the diseases observed by the Maya. One analogous example is that while dried Capsicum tissues have been shown in vitro to inhibit C. botulinum growth (Huhtanen, 1980),
R. H. Cichewicz, P. A. Thorpe/Journal of Ethnopharmacology 52 (1996) 61-70
numerous cases exist in which people have succumbed to botulinal neurotoxins following ingestion of improperly canned chile peppers (Center for Disease Control, 1979). Therefore, it is important that the Capsicum species which have demonstrated growth-inhibiting activity in this assay should be further studied to evaluate the significance of these tissues' clinical role in the Mayan medical system. Additional research is also necessary to investigate the potential antihemolytic activity of Capsicum extracts with S.
pyogenes. While it is uncertain why Capsicumspecies have been incorporated into the Mayan pharmacopoeia, it is possible that the reason(s) are based upon these tissues' potential therapeutic properties as antimicrobial and anti-hemolytic agents. Other research has suggested the possibility of some alternative potentially beneficial medicinal roles and effects of Capsicum species, including capsaicin's strong analgesic properties (Cordell and Araujo 1993), capsaicin-induced alterations in the pH and epithelial cell properties of the digestive tract, preventing microbial infection (Tellez et al., 1993), and possible anticancer effects (Bosland, 1994; Surh and Lee 1995). The questions raised by this research related to the use of Capsicumspecies in the Mayan culture are open for extensive study. Chiles not only function as important spices, but also serve as nutritional and medicinal agents. It is certain that, through ethnobotanical observations, Capsicum species harbor many economically significant benefits awaiting 'discovery'.
Acknowledgments The authors would like to thank Dean P. Douglas Kindschi and the members of the Grand Valley State University Science and Math Student Undergraduate Research Program Committee for their support and financial assistance. The Grand Valley State University library staff are most appreciated for their efforts in obtaining the necessary research materials and references. We are grateful to Dr. Paul Bosland of New Mexico State University for his contribution of peppers and verification of the tested Capsicum varieties.
69
References Abdou, I.A., Abou-Zcid, A.A., EI-Sherbeeny, M.R. and AbouEI-Gheat, Z.H. (1972) Antimicrobial activities of Allium sativum, Allium cepa, Raphanus sativus, Capsicum frutescens, Eruca sativa, Allium kurrat, on bacteria. Qualitas Plantarum et Materiae Vegetabiles 1, 29-35. Adetumbi, M.A. and Lau, B.H.S. (1983) Allium sativum (garlic) - - A natural antibiotic. Medical Hypotheses 12, 227-237. AI-Delaimy, K.S., and All, S.H. (1970) Antibacterial action of vegetable extracts on the growth of pathogenic bacteria. Journal of Science and Food Agriculture 21, 110-112. Alcorn, J.B. (1984) Huastec Mayan Ethnobotany. University of Texas Press, Austin, pp. 578-580. Allen, S.D. and Baron, E.J. (1991) Clostridium. In: A. Balows, W.J. Hausler Jr., K.L. Herrmann, H.D. Isenberg and H.J. Shadomy (Eds.), Manual of Clinical Microbiology. 5th Ed. American Society for Microbiology, Washington, D.C., pp. 505-521. Balbaa, S.I., Karawya, M.S. and Girgis, A.N. (1968) The capsaicin content of Capsicum fruits at different stages of maturity. Lloydia 31,272-274. Bisno, A.L. (1995) Streptococcus pyogenes. In: G.L. Mandell, J.E. Bennett and R. Dolin (Eds.), Principles and Practice of Infectious Disea.ses, 4th Ed., vol. 2. Churchill Livingstone, New York, pp. 1786-1799. Bosland, P.W. (1994) Chiles: History, cultivation, and uses. In G. Charalambous (Ed.), Spices, Herbs and Edible Fungi, Elsevier Science, New York, pp. 347-366. Bosland, P.W., Bailey, A.L. and Iglesias-Olivas, J. (1990) Capsicum Pepper Varieties and Classification. Circular 530, Cooperative Extension Service, College of Agriculture and Home Economics, New Mexico State University, New Mexico, pp. 1-12. Bushnell, O.A., Fukuda, M. and Makinodan, T. (1950) The antibacterial properties of some plants found in Hawaii. Pacific Science 4, 167-183. Caceres, A., Alvarez, A.V., Ovando, A.E. and Samayoa, B.E. (1991) Plants used in Guatemala for the treatment of respiratory diseases. 1. Screening of 68 plants against grampositive bacteria. Journal of Ethnopharmacology 31, 193-208. Cardoso, H.T. and Santos, M.D.L. (1948) Estudos sobre a presenca de antibioticos nos vegetais. Brasil-Medico 62, 67-70. Center for Disease Control (1979) Botulism in the United States, 1899-1977. Handbook for Epidemiologists, Clinicians, and Laboratory Workers. Public Health Service, Atlanta, Georgia, pp. 25, 36-41. Chen, H., Chang, M. and Chang, T. (1985) Antibacterial properties of some spice plants before and after heat treatment. Chinese Journal of Microbiology and Immunology 18, 190-195. Coe, S.D. (1994) America's First Cuisines. University of Texas Press, Austine, p. 62. Combs, G.F., Jr. (1992) The Vitamins: Fundamental Aspects in Nutrition and Health. Academic Press, New York, p. 225.
70
R.H. Cichewicz, P.A. Thorpe/Journal of Ethnopharmacology 52 (1996) 61-70
Cordell, G.A. and Araujo, O.E. (1993) Capsaicin: Identification, nomenclature, and pharmacotherapy. The Annals of Pharmacotherapy 27, 330-336. Cox, P.A. and Balick, M.J. (1994) The ethnobotanical approach to drug discovery. Scientific American 271, 82-87. DeWitt, D. and Bosland, P.W. (1993) The Pepper Garden. Ten Speed Press, Berkeley pp. 1-69. Eshbaugh, W.H. (1976) XII. Genetic and biochemical systematic studies of chili peppers (Capsicum-Solanaceae). Bulletin of the Torrey Botanical Club 102, 396-403. Facklam, R.R. and Washington, J.A., II (1991) Streptococcus and related catalasc-negative gram-positive cocci. In: A. Balows, W.J. Hausler Jr., K.L. Herrmann, H.D. Isenberg and H.J. Shadomy (Eds.), Manual of Clinical Microbiology, 5th Ed. American Society for Microbiology, Washington, D.C., pp. 238-257. Farbman, K.S., Barnett, E.D., Bolduc, G.R. and Klein, J.O. (1993) Antibacterial activity of garlic and onions: A historical perspective. The Pediatric Infectious Disease Journal 12, 613-614. Fenwick, G.R. and Hanley, A.B. (1985) The genus Allium - Part 3. CRC Critical Reviews in Food Science and Nutrition 23, 1-73. Fitzpatrick, F.K. (1954) Plant substances active against Mycobacterium tuberculosis. Antibiotics and Chemotherapy 4, 528-536. Frisbey, A., Gottshall, R.Y., Jennings, J.C. and Lucas, E.H. (1954) The occurrence of antibacterial substances in seed plants with special reference to Mycobacterium tuberculosis. Michigan Agricultural Experiment Station Quarterly Bulletin 36, 477-488. Gottshall, R.Y., Lucas, E.H., Lickfeldt, A. and Roberts, J.M. (1949) The occurrence of antibacterial substances active against Mycobacterium tuberculosis in seed plants. Journal of Clinical Investigation 28, 920-923. Harris, H.A. (1949) Antibacterial activity of seedling extracts of cultivated plants. Bulletin of the Torrey Botanical Club 76, 244-254. Hatheway, C.L. (1990) Toxigenic clostridia. Clinical Microbiology Reviews 3, 66-98. Huhtanen, C.N. (1980) Inhibition of CIostridium botulinum by spice extracts and aliphatic alcohols. Journal of Food Protection 43, 195-196, 200. MacDonald, R.E. and Bishop, C.J. (1953) A further survey of
plants for antibacterial substances. Canadian Journal of Botany 31, 123-131. McLeod, M.J., Guttman, S.I. and Eshbaugh, W.H. (1982) Early evolution of chile peppers (Capsicum). Economic Botany 36, 361-368. Masilungan, V.A., Kalaw, C.V. and Masilungan, F.C. (1963) Screening of plants from the Makiling area for antimicrobial substances. The Philippine Journal of Science 92, 421-430. Masilungan, V.A., Maranon, J. and Valencia, V.V. (1955) Some common Philippine medicinal plants containing antibacterial substances. Journal of the Philippine Pharmaceutical Association 42, 72-79. Mitscher, L.A., Leu, R., Eathala, M.S., Wu, W., Beal, J.L. and White, R. (1972) Antirnicrobial agents from higher plants. I. Introduction, rationale, and methodology. Lloydia 35, 157-166. Munscrecnusorn, Y., Kongsamut, S. and Pezalla, P. (1982) Capsaicin: A literature survey. CRC Critical Reviews in Toxicology 10, 321-339. Roys, R.L. (1931) The Ethno-Botany of the Maya. Reprinted 1976, Institute for the Study of Human Issues, Philadelphia, pp. 2, 7, 9, 12, 13, 14, 15, 17, 31, 32, 34, 35, 38, 49, 57, 58, 61, 63, 65, 67, 90, 93, 95, 106, 129, 150, 153, 184, 189, 195. Surh, Y.J. and Lee, S.S. (1995) Capsaicin, a double-edged sword: Toxicity, metabolism, and chemopreventive potential. Life Sciences 56, 1845-1855. Suzuki, T. and lwai, K. (1984) Constituents of red pepper species: Chemistry, biochemistry, pharmacology, and food science of the pungent principle of Capsicum species. In: A. Brossi (Ed.), The Alkaloids: Chemistry and Pharmacology. Academic Press, New York, pp. 227-299. Tellez, G.I., Jaeger, L., Dean, C.E., Corrier, D.E., DeLoach, J.R., Williams, J.D. and Hargis, B.M. (1993) Effect of prolonged administration of dietary capsaicin on Salmonella enteritidis infection in leghorn chicks. Avian Diseases 37, 143-148. Turnbuli, P.C.B. and Kramer, J.M. (1991) Bacillus. In: A. Balows, W.J. Hausler Jr., K.L. Herrmann, H.D. Isenberg, and H.J. Shadomy (Eds.), Manual of Clinical Microbiology. 5th Ed. American Society for Microbiology, Washington, D.C., pp. 296-303. Virus, R.M. and Gebhart, G.F. (1979) Pharmacologic actions of capsaicin: Apparent involvement of substance P and serotonin. Life Sciences 25, 1273-1284.