Antimutagenic activity of a freeze-dried aqueous
extract of Uncaria tomentosa: A randomized double-blind
study of smokers and non-smokers*

*Preliminary findings of this study were presented at the Machu Picchu Symposium on Medicinal Plants (Urubamba, Peru, 9 May 1996). Correspondence may be addressed to the first author.
^aINCAFLORA
^bLaboratorio de Genética, Clínica Ricardo Palma, Av. Javier Prado Este 1066, Lima 27, Perú.
^cUniversidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima 25, Perú.
^dInstituto Nacional de Medicina Tradicional, Ministerio de Salud. Av. Salaverry s/n, Lima 11, Perú.

Smoker (n=12) and non-smoker (n=12) volunteers randomly assigned to treatments took daily 0 (placebo), 90, or 270 mg of a freeze-dried aqueous extract of Uncaria tomentosa. Their urine's mutagenic activity was assessed under a double-blind design with the Salmonella/ mammalian microsome test (Ames et al., 1975) using S. typhimurium TA 98 tester strain with metabolic activation. Posttests took place at days 17 and 31 of treatment on average. Among the smokers, complete disappearance of mutagenic activity reached its peak at posttest 1 and depended on treatment dosis (r_pb = -.66, p = .01). With placebo effects statistically removed, degree of mutagenic activity decreased linearly as a function of treatment dosis at posttest 2 (r = -.50, p = .05). The urine of all the non-smokers under U. tomentosa, but not of all those under placebo, remained nonmutagenic at posttests 1 and 2. These findings are consistent with those of Rizzi et al. (1993) and lend empirical support to the hypothesis that the plant extract inhibits the mutagenic activity of procarcinogens and other mutagens of cigarette smoke.

1. Introduction

Asháninka indians of the Peruvian rain forest use an aqueous extract of the root or stem bark of "cat's claw" [Uncaria tomentosa (Willd.) (Rubiaceae)] to treat cancer, arthritis, and other diseases (Jones, 1996). Some of the therapeutic properties of the plant can be attributed to flavonoids; the earliest chemical studies identified catechin tannins (including cis-epicatechin) and procyanidins in the bark (Montenegro et al., 1976). Research on the biological activities of U. tomentosa has focused on other compounds. Wagner et al. (1985) isolated oxindole alkaloids that showed a pronounced enhancement of phagocitosis. Cerri et al. (1988) isolated quinovic acid glycosides that exhibit antiviral and anti-inflammatory activities (Aquino et al., 1989, 1991).

Published research findings relevant to the antitumoral use of the plant are scarce. Stuppner et al. (1993) reported selective antiproliferative effects of five oxindole alkaloids of U. tomentosa on leukemic cell lines HL 60 and U-937. Rizzi et al. (1993), using Ames' Salmonella/mammalian microsome test, reported that several extracts and fractions of the plant exhibited a protective action against mutagenesis induced by 8-methoxy-psoralen plus UVA irradiation in S. typhimurium TA 102. Additionally, using tester strains TA 98 and TA 100, they found (i) a reduction of the high initial level of mutagenic activity in the urine of a smoker who took a decoction of U. tomentosa during 15 days and (ii) no effects on the low initial level of mutagenicity observed in the urine of a non-smoker.

In the in vivo test, Rizzi et al. (1993) did not use a placebo for comparison, only had one case per cell, and performed the laboratory observations with knowledge of the treatment given to each subject. The present study, conducted in Peru, verified the antimutagenic action of U. tomentosa using a randomized double-blind design.

2. Materials and methods

2.1. Freeze-dried extract

Tablets of U. tomentosa approved for commercial use by Peru's Ministry of Health and available over the counter since 1994 were used.

Each tablet contained 90 mg of a freeze-dried extract stemming from 3,000 mg of virgin stem bark. The bark was harvested by Piro indians in the central Peruvian rain forest (between the towns of Sepahua on the Urubamba river and Atalaya on the Ucayali river, at about 550 meters above sea level) and left to dry at room temperature during 4-6 weeks. The extract was obtained by the manufacturers following a version of the Asháninka formula consisting in boiling the bark in water at 85^o C during 1h 30 min. Then it was processed at the manufacturers' freeze-drying plant and the resulting powder was converted into tablets at a pharmaceutical laboratory. The excipient of the tablets (70 mg) included corn starch (27.55 mg), lactose (25.47 mg), shellac (13.82 mg), rincinus oil (2.75 mg), and carnauba wax (0.70 mg). Routine quality control included microbiological and chromatographic analyses. The latter verified alkaloid content (about 0.7% of the freeze-dried powder).

Two studies on mice were conducted by the Pharmacology Department of Universidad Peruana Cayetano Heredia simultaneously with this study. One showed that the tablets, diluted in water and taken orally, had no lethal toxicity even at a maximal dosis of 18 g/kg during 72 hours of observation (Tasayco & Castro de la Mata, 1996). The other verified the strong anti-inflammatory activity of both the freeze-dried powder and tablets and confirmed their alkaloid content (Castro de la Mata, 1995).

Placebo tablets were made of excipient (160 mg).

2.2. Subjects

Healthy male and female volunteers aged 18-65 were recruited. According to medical examinations, they were free of major diseases and had normal offsprings. They did not use antioxidant vitamins or psychoactive drugs other than tobacco or alcohol, were not exposed to industrial mutagens, and showed normal kidney and liver functions in blood and urine tests. The smokers had smoked 5 to 40 cigarettes daily over the past 5 years. The non-smokers had smoked 0 cigarettes in the past 5 years and were not passive smokers. Smokers with nonmutagenic urines and non-smokers with mutagenic urines were to be discarded.

Handling of the subjects was individualized and consistent with the Helsinki Declaration. They signed a document of informed consent that described the study aims and procedures in detail, including random assignment and exact treatment doses. Then they were submitted to the clinical check up. If they qualified as healthy donors, were asked to return to the laboratory to submit a urine sample in late afternoon (pretest) and receive the treatments. Posttests were scheduled for days 15 and 30 of treatment, also in late afternoon. A project coordinator made reminder calls to the subjects one day prior and the same day of their appointments.

2.3 Double-blind design

Subjects were processed in cohorts of three within each group (smokers, non-smokers). Within each triad, they were randomly assigned to the treatment conditions, one per cell, and received a ration of 30-tablet aluminum blisters showing the brand name MANAXX, which they knew was a brand of U. tomentosa. Whereas the blisters and tablets were identical in appearance across experimental conditions, the former varied in number and the latter in content. Subjects assigned to the 90 mg and 270 mg conditions received respectively two and four blisters with U. tomentosa tablets and the instruction to take one (those under 90 mg) or three tablets (those under 270 mg) daily with breakfast. Subjects assigned to the 0 mg condition received tablets of placebo; some were given two blisters and the instruction to take one tablet daily with breakfast and some were given four blisters and instructed to take three tablets daily. The subjects ignored their treatment condition throughout the study.

The first author of the study controlled the treatment codes and deposited them in a notary public prior to receiving any laboratory report. The second author performed Ames test at her genetics laboratory only having information on the subject's code, and the third author determined creatinine at his clinical laboratory with similar information; that is, they were blind to the treatments as they performed their tasks.

2.4 Preparation of urine samples

Amberlite XAD-2 resin (60-150 mesh) and dimethyl sulphoxide, spectrometric grade (DMSO) were purchased from Sigma (St. Louis, MO, USA). Methanol and acetone were analytical grade. The resin was prepared as suggested by Yamasaki and Ames (1977); 25 g was washed five times with 150 ml of acetone, five times with 150 ml of methanol and five times with 150 ml of Milli-Q water. The washed resin was stored at 8^o C for no more than 1 week. Urine samples were collected in 250-ml plastic bottles at the clinical laboratory. Part of the urine was sent immediately to the genetics laboratory for implementation of Ames test. The other part was used to determine creatinine concentrations by the Jaffé reaction.

Upon arrival in the genetics laboratory, the urine samples were frozen and stored at -15^o C until they were processed. The extraction and concentration of urinary mutagens were performed adapting the method of Yamasaki and Ames (1977). Urine samples were thawed at room temperature, filtered through Whatman paper no. 1 and added to 7 x 100 mm glass columns containing 0.90 g of washed resin/100 ml urine. Luer three-way stopcocks regulated the flow rate at 1-2 ml/min. The resin was then washed with 10 ml of Milli-Q water, mutagens were eluted with 10 ml of acetone, and the eluate was fractioned into 20 ml and 100 ml doses. The eluates were evaporated to dryness at 60^o C under a Teflon iron. The organic residue was re-suspended in DMSO (400 ml/100 ml urine) for each study sample. Two controls per day were also included by replacing a urine sample with 9 ml of Milli-Q water for the determination of spontaneous revertants and the other with 9 ml of Daunomycin for the determination of mutagen-induced revertants.

2.5 Preparation of liver homogenates (S-9 mix)

Ames test has been adapted to detect potential human carcinogens by adding homogenates of rat liver directly to the petri plates, thus incorporating essential aspects of mammalian metabolism into the in vitro test. Polychlorinated biphenyl mixture (Aroclor 1254), glucose-6-phosphate (G6P), and nicotinamide adenine dinucleotide phosphate, sodium salt (NADP) were purchased from Sigma (St. Louis, MO, USA). Induction of rat liver enzymes for carcinogen activation was made at Universidad Nacional Agraria La Molina following Ames et al.'s (1975) suggestions. Male rats of about 200 g each received a single i.p. injection of Aroclor 1254 (diluted in corn oil to a concentration of 200 mg/ml) at a dosage of 500 mg/kg 5 days before sacrifice. Livers of about 10-15 g each were obtained and processed at 0-4^o C using cold, sterile solutions and glassware. Livers were placed in a beaker containing 3 vol. of 0.15 M KCL (3 ml/g wet liver), minced with sterile scissors, and homogenized in an Equatherm stirring hot plate from Curting Matheson Scientific, Inc. The homogenate was centrifuged for 10 min at 9000 g (9000 rev./min in a Fisher Scientific Microcentrifuger) and the supernatant (S-9 fraction) was decanted and saved. 1 ml of S-9 fraction contained microsomes from about 250 mg of wet liver. The fresh S-9 fractions were sterilized with Millipore 0.22 mm filters, distributed in 2-ml portions, frozen in dry-ice and stored at -80^o C. A fresh S-9 mix was prepared each day. It contained per ml: S-9 (0.04-0.1 ml), MgCl₂ (8 mmoles), KCl (33 mmoles), G6P (5 mmoles), NADP (4 mmoles), and sodium phosphate, pH 7.4 (100 mmoles).

2.6 Ames test

In Ames test, S. typhimurium tester strains are reverted from a histidine requirement back to prototrophy by mutagens from cigarette smoke and other sources. S. typhimurium tester strain TA 98, kindly provided by Dr. B. N. Ames, was selected for this study since it displays the highest sensitivity to cigarette smokers' urine concentrates (Yamasaki and Ames, 1977; Putzrath et al., 1981; Recio et al., 1982). The tester strain was tested for sodium deoxycholate and crystal violet sensitivities, UVA light sensitivity and ampicillin resistance. Ames' test was implemented according to procedures described by Maron and Ames (1983) and De Méo et al. (1988). Tester bacteria were grown at 37^o C overnight in Oxoid Nutrient broth no. 2. After this incubation period, the following ingredients were added to 12 x 75-mm sterile polystyrene tubes on ice: 0.1 ml of S-9 mix, a dosis of urine concentrate (or control compound) not exceeding 10 ml, and 0.1 ml of the overnight culture. This mixture was incubated for 60 min at 37^o C with rapid shaking. After the contact period, the tubes were placed on ice, taken out one at a time, and 2 ml of molten top agar, 0.2 ml of L-histidine and 0.2 ml of Biotin were added; then the combined mixture was poured onto petri plates (100 x 15 mm style, Falcon Plastics) containing 30 ml minimal-glucose agar medium. The seeded plates were incubated for 48 h at 37^o in the dark. Following the incubation period, the number of revertants observed on each plate were counted.

3. Results

There were six cells in the two-factor design of smoking status (smokers, non-smokers) by treatments (0, 90, and 270 mg of U. tomentosa). The useful cases of this study were the first four per cell that completed the one-month treatment and had complete data on all the dependent variables at each measurement phase (pretest, posttest 1, posttest 2). Data from eight cases were discarded since they failed to satisfy this requirement (1 discontinued treatment due to the presence of blood in the urine sample, 3 offered scarce or contaminated urine, and 4 deserted owing to travel obligations, schedule problems, or family prohibition to continue in the study).

Few subjects were able to donate urine samples for posttests 1 and 2 exactly at the 15th and 30th days of treatment, since the clinical laboratory was closed on Saturdays and Sundays and this, plus menstruation and travel, forced early or delayed appointments. On average over the 12 smokers, 12 non-smokers, and 24 subjects, effective days of treatment were respectively 17.23, 16.50, and 16.88 until posttest 1 and 32.57, 30.25, and 31.42 until posttest 2.

To test the hypothesis of this study concerning the antimutagenic action of U. tomentosa, we defined mutagenic activity by (i) a number of observed revertants > 0 under each urine concentrate and (ii) a greater number of revertants under 100 ml than under 20 ml concentrates.

3.1 Complete inhibition of mutagenicity

In the smoker group, all cases showed mutagenic activity in the pretest. Under treatment, four reduced the number of observed revertants to 0, and this occurred equally under the 20 ml and 100 ml concentrates. This was an unexpected result, since (i) Rizzi et al. (1993) reported numbers of revertants > 0 for the smoker and the non-smoker of their in vivo study before and after treatment and (ii) in our own study Milli-Q water generated spontaneous revertants in all cases.

The distribution of cases with 0 revertants across treatment groups was unequal. Whereas all the subjects under placebo maintained counts > 0 in the posttests, only 75% of those under 90 mg of U. tomentosa and 25% of those under 270 mg did so (Figure 2). With the change in condition (pretest count > 0, posttest count = 0) coded -1 and the lack of change coded 0, we obtained a significant point-biserial correlation between this nominal variable and the treatment dosis (0, 90, or 270 mg) either at posttest 1 or posttest 2 (r_pb= -.66, p = .01, one-tailed, n = 12). Thus, the rigorous data of this study concerning complete inhibition of mutagenicity were consistent with the hypothesis that U. tomentosa inhibits mutagenic activity.

3.2 Degree of inhibition

The nominal data in Figure 2 suggests that the effects were similar under 17 or 33 days of treatment. Yet, an analysis of the degree of mutagenic inhibition suggests a different conclusion. Table 1 offers the average number of observed revertants per treatment group and urine concentrate at each measurement phase. Figure 3 depicts the average change in number

of revertants from pretest to each posttest. The Pearson correlation between change score and treatment dosis over the 12 cases provides a synthetic quantitative index of treatment effects. Under the 20 ml urine concentrate, the correlation for posttest 2 came very close to signific-ance (r = -.47, p = .06) but that for posttest 1 did not (r = -.30, p = .17). Similarly, under the 100 ml concentrate, the correlation for posttest 2 attained significance (r = -.54, p = .03) but that for posttest 1 did not (r = -.37, p = .11). Thus, the data on degree of mutagenic inhibition showed greater effects of U. tomentosa after 33 than after 17 days of treatment.

3.3 Placebo effect

A placebo effect is evidenced in the diminishing numbers of revertants observed from pretest to posttest 1 and then to posttest 2 in the group under 0 mg of U. tomentosa (Table 1, Figure 3). Changes in the sensitivity of Ames test cannot explain this trend. As shown in Table 2, the number of spontaneous revertants (in response to Milli-Q water) remained approximately constant and that of mutagen-induced revertants (caused by Daunomycin) actually increased. Changes in the concentration of urine samples can also be discarded since the creatinine concentrations were greater at posttest 2 than at the pretest.

This leaves observer bias as the most likely cause of the placebo effect, for the genetic analyists were blind to treatment dosis but not totally blind to measurement phase and thus might have been prone to count larger numbers of revertants in the pretest than in the posttests. If there was an observer bias associated with measurement phase, the data for cases under U. tomentosa may have been biased, too. Yet, if the bias operated approximately as a constant and affected equally the count for the different urine concentrates, it can be controlled statistically.

Ames et al. (1975) suggested to confirm mutagenic activity demonstrating a dose-response effect for a narrow range of concentrations of the suspected carcinogen, and De Méo et al. (1988) used a regression approach to measure mutagenic activity. We computed for each case at each measurement phase the slope (b coefficient) of the regression of number of revertants per plate on urine concentrate (20 ml, 100 ml) on the assumption that observer bias affected the height of the regression (a coefficient) but not its slope. Indeed, the average change in slope from pretest to posttest 2 emerged virtually free of placebo effect (Figure 4), and the correlation between change in slope and treatment dosis reached significance (r = -.50, p = .05). On the other hand, the placebo effect was not fully removed from the average change in slope from pretest to posttest 1, and the correlation between change in slope and treatment dosis failed to reach significance (r = -.19, p = .28).

3.4 Sensitivity of Ames test and concentration of urine samples

The sensitivity of Ames test and the concentration of urine samples varied over treatment groups (Table 2); however, they cannot provide us with credible alternatives to the hypothesis that the dosis of U. tomentosa caused the complete inhibition of mutagenic activity observed at posttest 1. To facilitate com-parisons between variables with different metric, Figure 5 depicts percent changes computed on the data on Table 2. The left panel of the figure shows nearly identical changes in each variable for the 0 and 270 mg treatment groups. Hence, these var-iables cannot account for the observed correlation between treatment dosis and complete inhibition of mutagenicity observed after 17 days of treatment.

Data on the right panel of Figure 5 are relevant to the observed correlations between treatment dosis and degree of mutagenic inhibition after 33 days of treatment (Figures 3 and 4). The number of spontaneous revertants remained constant for the placebo group (change score = 0) but decreased for the two groups under U. tomentosa, while creatinine concentrations increased more in subjects under 270 mg than in those under 90 mg. A combination of test sensitivity (as indexed by the number of spontaneous revertants) and urine concentration, rather than the dosis of U. tomentosa, could have caused the decreasing mutagenicity observed under 0, 90, and 270 mg. To evaluate this possibility, we transformed each relevant variable to log 10 (Variable + 0.0001) and calculated mutagenic activity controlling for Ames test sensitivity (as indexed by the number of spontaneous revertants) and creatinine as:

MA_T = (Observed revertants_T - Spontaneous revertants_T)/Creatinine_T.

The correlation between treatment dosis and change in MA_T was significant under both the 20 ml (r = -.60, p < .03) and 100 ml concentrates (r = -.63, p < .02). Change in the slope of the regression of MA_Ts on urine concentrates also correlated significantly with treatment dosis (r = -.63, p < .02). Thus, differences in Ames test sensitivity (as indexed by the number of spontaneous revertants) and creatinine concentration can be discarded as alternative causes of the findings concerning posttest 2.

3.5 Donor variables

Differences in personal traits and behaviors were observed between the treatment groups (Table 3). However, none of these variables correlated significantly with change in mutagenic activity from pretest to posttest 1 or posttest 2. Age was distributed in a monotonically ascending way over the three treatment groups, yet the age difference between the donors under 90 mg and 270 mg of U. tomentosa was trivial and could not generate an alternative explanation for the substantive findings of this study. Similar is the case of the effective number of treatment days until posttest 2.

3.5 Non-smokers

The 12 non-smokers of this study exhibited revertant counts = 0 both under 20 ml and 100 ml concentrates at the pretest, and all the cases under U. tomentosa (n = 8) maintained this condition at both posttests. This finding is consistent with that of Rizzi et al. (1993), who in their in vitro assay found that U. tomentosa did not cause mutagenicity and in their in vivo test found that the urine of a non-smoker did not become mutagenic under treatment with the plant extract.

Two cases under placebo, however, exhibited counts > 0 at posttest 1 and this was observed under each concentrate (Case # 15, 21 rev. under 20 ml, 54 rev. under 100 ml. Case #16, 11 rev. under 20 ml, 16 rev. under 100 ml). Changes in sensitivity of Ames test or in urine sample concentration cannot explain this result (Table 4). Neither can the differences between donors' personal traits and behaviors (Table 5). The small number of cases per cell prevents induction of reliable conclusions from this result, but the finding suggests the hypothesis that U. tomentosa may protect non-smokers against temporary mutagenicity.

Table 5 Characteristics of the non-smokers
Treatment group		Percent- age female	Mean age	Effective number of treatment days
				Mean posttest 1	Mean posttest 2
0 mg	Rev=0	50	26.0	17.5	29.5
	Rev>0	50	32.5	16.5	31.5
90 mg		100	38.5	16.5	29.7
270 mg		50	39.5	16.0	30.7

4. Discussion

Apart from our use of a placebo group, a double-blind design, and a number of subjects within each cell, there were other methodological differences between this study and Rizzi et al.'s (1993) in vivo test. (i) Owing to resource and workload limitations, we did not use a TA 100 tester strain, ß-glucuronidase, nor a third, intermediate urine concentrate in Ames test. Ours was not a full replication of Rizzi et al.'s study. (ii) They, in turn, did not report the number of spontaneous revertants, control mutagen-induced revertants, nor creatinine concentrations. (iii) Rizzi et al. may have achieved a more sensitive implementation of Ames test. In our study, the number of observed revertants under 20 ml was very low on average among the smokers (not greater than the number of spontaneous revertants) and it was easy for us to find 12 non-smokers that satisfied the requirement of 0 revertants under the 20 ml and 100 ml urine concentrates in the pretest. (iv) Our treatment lasted one month while theirs only lasted 15 days. (v) Finally, we used a daily dosis of freeze-dried extract obtained from 3,000 mg of U. tomentosa bark, plus a triple daily dosis, while Rizzi et al. used a fresh extract from 6,500 mg, and the procedures to obtain the respective extracts were far from identical.

Nevertheless, the results were essentially similar. Thus, we can confidently state not only that our data confirm the internal validity of Rizzi et al.'s (1993) findings and support their conclusion that U. tomentosa inhibits the mutagenic activity in smokers' urine but also that the findings are quite robust and easy to replicate under varied conditions.

Some doubts remain concerning the relative effectiveness of 17 versus 33 days of treatment. Whereas the data on complete inhibition of mutagenicity suggested that treatment beyond the first two weeks does not cause additional effects, the findings concerning degree of inhibition clearly indicated an advantage for the longer treatment. We give more credit to the latter because they emerged after removal of the placebo effect that we attribute to observer bias. Nonetheless, we recommend caution in the interpretation of the difference between the two periods since it may just represent an artifact of calibration problems in the implementation of Ames test. The sole solid conclusion of the study is that U. tomentosa inhibits urinary mutagenesis in smokers. Use of Ames test in future studies to find the most effective treatment period is clearly justified.

However, we believe that future research must focus more directly on the clinical implications of Rizzi et al.'s (and our own) findings. The antimutagenic activity of U. tomentosa may be due to an antioxidant mechanism which acts by inhibiting oxidative/free radicals mediators to the transformation of the procarcinogens present in cigarette smoke (Rizzi et al., 1993, p. 76). An implication of this hypothesis is that the plant extract can prevent cancers associated with cigarette smoking (mouth, larynge, esophagus, lung, pancreas, kidney, bladder). Future research could verify this potential through specific clinical trials. Long-term studies, lasting 5 to 10 years, may be required to demonstrate that symptom-free subjects under U. tomentosa take longer to develop clinically visible tumors, or develop less tumors, than subjects under placebo or alternative treatments. On a shorter frame, research may test whether treatment with the plant extract reduces the number of precursor cells and inhibits their transformation into malignant cells. Bladder tissue precursor cells are easily detectable in human urine.

This, on the other hand, should not preclude research on the potential of the plant extracts to prevent other cancers. During centuries, the Asháninka indians did not smoke cigarettes, yet they used, and still use, the aqueous extract of "cat's claw" to treat diverse tumors. In fact, unpublished in vitro studies conducted at France's Centre National de la Recherche Scientifique (l'Institut de Chimie des Substances Naturelles) (Villanueva, 1996), at Italy's Naples and Palermo universities (Pizza, 1996), and for Canada's Northeastern Ontario Regional Cancer Research Centre (Cano, 1996) have demonstrated the citostatic action of U. tomentosa extracts on breast cancer cell lines. In vivo, clinical studies must follow.

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