Carcinogenicity of Camellia sinensis (Tea) and Some Tannin-Containing Folk Medicinal Herbs Administered Subcutaneously in Rats 1 , 2, 3 Govind J. Kapadia,4 B. D. Paul,4 E. B. Chung,5 B. Ghosh,S and S. N. Pradhan s , 7 ABSTRACT-In an attempt to correlate the high incidence of esophageal carcinoma in natives of certain places with their habit of using herbaceous folk medicines, we performed bioassays of several plant extracts and the fractions prepared from them. Fourteen extracts and fractions from 6 plants were injected sc into NIH Black rats. The tannin fractions from Quercus fa/cata pagodaefo/ia, Diospyros virginian a, and Camellia sinensis were very aotive and produced tumors at the injection site in 66% or more of the treated animals. Tannin fractions from 3 other plants and total aqueous extracts from 5 of 6 tested plants were also tumorigenic in rats. The induced tumors were malignant fibrous histiocytomas similar, if not identical, to those encountered in humans. The experiment indicated a possibility of induction of tumor in man by the tested plant materials.-J Natl Cancer Inst
57: 207-209, 1976.
Tea infusion is consumed as a beverage by many people around the world. Recent epidemiologic surveys (1-4) have associated a higher observed incidence of carcinomas with the consumption of tea. Aqueous tea extract and the steam-volatile phenolic fraction from the ether extract of tea, painted on Swiss mice initially treated with the carcinogenic hydrocarbon benzo[a]pyrene, have been shown by Kaiser and Bartone (5) and Kaiser (6) to promote cancer growth. "In addition, several herbal folk remedies are used in the low country of South Carolina, one of the few areas of the United States where the localized incidence of esophageal cancer has attracted considerable scientific attention. Morton (7) attributed the high incidence of esophageal carcinoma in this region in part to the usage of the herbal folk remedies. In earlier bioassay and chemical investigations of plants for their potential carcinogenicity (8), we reported that the T AE and TF of Krameria ixina, K. triandra, and Acacia villosa given sc to NIH Black rats produced malignant fibrous histiocytomas. The TFF had little carcinogenicity. In this study (9), we investigated the carcinogenicity of the T AE and TF from Camellia sinensis (tea) and 5 folk medicinal herbs indigenous to the United States (10) by injecting them sc into NIH Black rats. MATERIALS AND METHODS
Plant materials.-The following plant materials were used: C. sinensis (tea) grown in northeast India and processed to yield the black tea commonly used for preparing the beverage; Myrica cerifera (southern bayberry and sweet myrtle/wax myrtle) bark and Liquidambar styraciflua (sweet gum) leaves obtained from Johns Island, South Carolina; Quercus falcata pagodaefolia (cherry bark oak) inner bark obtained from Southeast Forest Service, Huger, South Carolina; Diospyros virginiana (persimmon) unripe fruits obtained from Brookgreen Gardens, Murrells Inlet, South Carolina, and VOL. 57, NO.
I, JULY 1976
207
Limonium nashii (marsh rosemary) roots procured from Isle of Palms, South Carolina. The plant materials, except tea, were powdered in a Wiley mill and extracted and fractionated into TF and TFF, as reported in (8). For D. virginiana fruits, the tannin-caffeine complex was separated into a methanolsoluble complex, whereas the methanol-insoluble tannin complex was dissolved in a minimum amount of acetone; we removed the caffeine by adding water and continuously extracting the aqueous layer with chloroform. The percentage yield of the various fractions is recorded in table 1. Bioassay.-NIH Black rats were used when 1-2 months old. A dose of a plant material was injected sc once a week alternately in either flank of each of 15 males and 15 females; 30 controls were similarly inoculated with saline. When a tumor had grown to a sufficient size, the animal was killed; tumor tissue and certain organs (i.e., regional lymph nodes, lungs, liver and spleen, and kidneys) were collected. Such tissues. with or without tumor, were also collected when an animal died or was killed for any pathologic reason, and when the experiment was terminated arbitrarily at 78 weeks. All tissues were examined grossly for tumor metastasis. All other bioassay procedures, including animal housing and selection of doses, were identical with those of previous experiments (8). Tumor incidence in the groups treated with individual plant materials was compared with that in the saline-treated controls, and the significance of their difference was estimated by chisquare test. ABBREVIATIONS USED: T AE=total aqueous extract; TF=tannin-containing fraction; TFF=tannin-free fraction; TF-I =tannin fraction (caffeine complex, methanol-soluble); TF-2=tannin fraction (caffeine complex, acetone-soluble). Received November 17, 1975; accepted January 2,1976. Supported by Public Health Service contract NOI CP33266 from the Division of Cancer Cause and Prevention, National Cancer Institute (NCI). 3 Presented in part at the Second Joint Meeting of the American Society of Pharmacognosy and the Gesellschaft fUr Arzneipflanzenforschung, July 31, 1975, at the University of Connecticut, Storrs, Conn. 4 Department of Biomedicinal Chemistry, College of Pharmacy and Pharmacal Sciences, Howard University, Washington, D.C. 20059. 5 Department of Pathology, College of Medicine, Howard U niversity. 6 Department of Pharmacology, College of Medicine, Howard U niversity. 7 We are grateful to Dr. Julia F. Morton (University of Miami) for her suggestions concerning selection of plant materials and her cooperation in collecting and providing them; to Mr. G. L. Tarbox (Brookgreen Gardens, Murrells Inlet, S.C.) for providing unripe fruits of Diospyros virginiana; to Dr. B.K. Chowdhury (Howard University) for isolating myricitrin from the tannin-free fraction of D. virginiana; and to Dr. Elizabeth K. Weisburger (NCI) and Dr. K. L. Khanna (Howard University) for helpful discussions. 1
2
J NATL CANCER INST
208
KAPADIA ET AL. TABLE
Plant materials
I.-Development of tumor after sc injection of plant materials in NIH Black rats Percent yield a
Dose, mg
Number of rats with tumor; mean No. of weekly injections (range)b Male
C. sinensis (Assam tea, leaf) M. cerifera (Wax myrtle, bark)
L. nashii (Marsh Rosemary, root) Q. falcata pagodaefolia (Cherry bark oak, bark) L. styraciflua (Sweet gum, leaf) D. virginiana (Persimmon,unripe fruit)
TAE: 26.8 TF: 2.3 (8.58) TAE: 11.2 TF: 3.9 (34.82) TFF: 3.8 TAE: 15.0 TF: 2.3 (20.12) TAE: 4.2 TF: 1.2 (28.57) TAE: 15.0 TF: 2.3 (15.33) TAE: 38.3 TF-l: 3.7 (9.66) TF-2: 14.4 (37.60)
12 8 3 2
10 5 2 8 4 10 5 8 8 6
Saline
1; 69 10 d; 58 2; 68 5 e ; 69 7 e ; 69 3; 33 9 / ; 66 15 d ; 47 15 d ; 45 15 d ; 64 15 d; 56
o
(45-77) (59-76) (59-77) (33-78) (26-47) (52-78) (33-59) (33-68) (45-73) (39-71)
13 d ; 57 (37-77)
o o
Female 1; 11d; 1; 3; 3; 2; 4;
70 68 (45-77) 75 62 (56-68) 65 (59-71) 70 (69-71) 61 (48-72) 15 d ; 49 (32-62) 13 d ; 46 (31-73) 5 e ; 70 (63-75) 11d; 61 (45-78) 0 8 / ; 63 (48-77)
0 0
Number dead or killed for other reasons C Male
Female 1 (f)
2 2 1 1
(a,f) (b) (d) (a)
4 (a,c,e,f) 2 (a,c)
1 (c,f) 2 (a,f) 2 (a,d) 1 (d) 2 (a,f) 1 (c) 2 (d) 1(~
: Weight offracti?n/l00 g plant material. Numbers in parentheses indicate yield ofTF, TF-l, or TF-2 as percent of TAE. E~ch dose. was gIven weekly to 15 ~ale ~nd 15 female. rats (by sc injection in alternate flanks) until tumor development was detected. DIed or kIlled because of: a-lung mfectIon; b-paralysIs; c-abdominal abscess; d-ear infection' e-enlarged teeth' f-accidental or unknown causes. ' , d P
57: 207-209, 1976.
Tea infusion is consumed as a beverage by many people around the world. Recent epidemiologic surveys (1-4) have associated a higher observed incidence of carcinomas with the consumption of tea. Aqueous tea extract and the steam-volatile phenolic fraction from the ether extract of tea, painted on Swiss mice initially treated with the carcinogenic hydrocarbon benzo[a]pyrene, have been shown by Kaiser and Bartone (5) and Kaiser (6) to promote cancer growth. "In addition, several herbal folk remedies are used in the low country of South Carolina, one of the few areas of the United States where the localized incidence of esophageal cancer has attracted considerable scientific attention. Morton (7) attributed the high incidence of esophageal carcinoma in this region in part to the usage of the herbal folk remedies. In earlier bioassay and chemical investigations of plants for their potential carcinogenicity (8), we reported that the T AE and TF of Krameria ixina, K. triandra, and Acacia villosa given sc to NIH Black rats produced malignant fibrous histiocytomas. The TFF had little carcinogenicity. In this study (9), we investigated the carcinogenicity of the T AE and TF from Camellia sinensis (tea) and 5 folk medicinal herbs indigenous to the United States (10) by injecting them sc into NIH Black rats. MATERIALS AND METHODS
Plant materials.-The following plant materials were used: C. sinensis (tea) grown in northeast India and processed to yield the black tea commonly used for preparing the beverage; Myrica cerifera (southern bayberry and sweet myrtle/wax myrtle) bark and Liquidambar styraciflua (sweet gum) leaves obtained from Johns Island, South Carolina; Quercus falcata pagodaefolia (cherry bark oak) inner bark obtained from Southeast Forest Service, Huger, South Carolina; Diospyros virginiana (persimmon) unripe fruits obtained from Brookgreen Gardens, Murrells Inlet, South Carolina, and VOL. 57, NO.
I, JULY 1976
207
Limonium nashii (marsh rosemary) roots procured from Isle of Palms, South Carolina. The plant materials, except tea, were powdered in a Wiley mill and extracted and fractionated into TF and TFF, as reported in (8). For D. virginiana fruits, the tannin-caffeine complex was separated into a methanolsoluble complex, whereas the methanol-insoluble tannin complex was dissolved in a minimum amount of acetone; we removed the caffeine by adding water and continuously extracting the aqueous layer with chloroform. The percentage yield of the various fractions is recorded in table 1. Bioassay.-NIH Black rats were used when 1-2 months old. A dose of a plant material was injected sc once a week alternately in either flank of each of 15 males and 15 females; 30 controls were similarly inoculated with saline. When a tumor had grown to a sufficient size, the animal was killed; tumor tissue and certain organs (i.e., regional lymph nodes, lungs, liver and spleen, and kidneys) were collected. Such tissues. with or without tumor, were also collected when an animal died or was killed for any pathologic reason, and when the experiment was terminated arbitrarily at 78 weeks. All tissues were examined grossly for tumor metastasis. All other bioassay procedures, including animal housing and selection of doses, were identical with those of previous experiments (8). Tumor incidence in the groups treated with individual plant materials was compared with that in the saline-treated controls, and the significance of their difference was estimated by chisquare test. ABBREVIATIONS USED: T AE=total aqueous extract; TF=tannin-containing fraction; TFF=tannin-free fraction; TF-I =tannin fraction (caffeine complex, methanol-soluble); TF-2=tannin fraction (caffeine complex, acetone-soluble). Received November 17, 1975; accepted January 2,1976. Supported by Public Health Service contract NOI CP33266 from the Division of Cancer Cause and Prevention, National Cancer Institute (NCI). 3 Presented in part at the Second Joint Meeting of the American Society of Pharmacognosy and the Gesellschaft fUr Arzneipflanzenforschung, July 31, 1975, at the University of Connecticut, Storrs, Conn. 4 Department of Biomedicinal Chemistry, College of Pharmacy and Pharmacal Sciences, Howard University, Washington, D.C. 20059. 5 Department of Pathology, College of Medicine, Howard U niversity. 6 Department of Pharmacology, College of Medicine, Howard U niversity. 7 We are grateful to Dr. Julia F. Morton (University of Miami) for her suggestions concerning selection of plant materials and her cooperation in collecting and providing them; to Mr. G. L. Tarbox (Brookgreen Gardens, Murrells Inlet, S.C.) for providing unripe fruits of Diospyros virginiana; to Dr. B.K. Chowdhury (Howard University) for isolating myricitrin from the tannin-free fraction of D. virginiana; and to Dr. Elizabeth K. Weisburger (NCI) and Dr. K. L. Khanna (Howard University) for helpful discussions. 1
2
J NATL CANCER INST
208
KAPADIA ET AL. TABLE
Plant materials
I.-Development of tumor after sc injection of plant materials in NIH Black rats Percent yield a
Dose, mg
Number of rats with tumor; mean No. of weekly injections (range)b Male
C. sinensis (Assam tea, leaf) M. cerifera (Wax myrtle, bark)
L. nashii (Marsh Rosemary, root) Q. falcata pagodaefolia (Cherry bark oak, bark) L. styraciflua (Sweet gum, leaf) D. virginiana (Persimmon,unripe fruit)
TAE: 26.8 TF: 2.3 (8.58) TAE: 11.2 TF: 3.9 (34.82) TFF: 3.8 TAE: 15.0 TF: 2.3 (20.12) TAE: 4.2 TF: 1.2 (28.57) TAE: 15.0 TF: 2.3 (15.33) TAE: 38.3 TF-l: 3.7 (9.66) TF-2: 14.4 (37.60)
12 8 3 2
10 5 2 8 4 10 5 8 8 6
Saline
1; 69 10 d; 58 2; 68 5 e ; 69 7 e ; 69 3; 33 9 / ; 66 15 d ; 47 15 d ; 45 15 d ; 64 15 d; 56
o
(45-77) (59-76) (59-77) (33-78) (26-47) (52-78) (33-59) (33-68) (45-73) (39-71)
13 d ; 57 (37-77)
o o
Female 1; 11d; 1; 3; 3; 2; 4;
70 68 (45-77) 75 62 (56-68) 65 (59-71) 70 (69-71) 61 (48-72) 15 d ; 49 (32-62) 13 d ; 46 (31-73) 5 e ; 70 (63-75) 11d; 61 (45-78) 0 8 / ; 63 (48-77)
0 0
Number dead or killed for other reasons C Male
Female 1 (f)
2 2 1 1
(a,f) (b) (d) (a)
4 (a,c,e,f) 2 (a,c)
1 (c,f) 2 (a,f) 2 (a,d) 1 (d) 2 (a,f) 1 (c) 2 (d) 1(~
: Weight offracti?n/l00 g plant material. Numbers in parentheses indicate yield ofTF, TF-l, or TF-2 as percent of TAE. E~ch dose. was gIven weekly to 15 ~ale ~nd 15 female. rats (by sc injection in alternate flanks) until tumor development was detected. DIed or kIlled because of: a-lung mfectIon; b-paralysIs; c-abdominal abscess; d-ear infection' e-enlarged teeth' f-accidental or unknown causes. ' , d P
