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Appendix J
Prototype Focused Monograph: Review of Liver-Related Risks for Chaparral1

I. DESCRIPTION OF THE INGREDIENT

A. Chaparral as a Dietary Supplement Ingredient

Chaparral is one name for an herbaceous woody shrub that grows in the southwestern region of the United States and the northern region of Mexico. It is also called creosote bush or greasewood. The common Spanish names are hediondilla, which means “little smelly one,” because the bush has a strong odor similar to the smell of creosote (a distillate of coal/ wood tar used as a wood preservative), and gobernadora, which means “governess,” because the bushes can dominate an area by creating an ad-

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This is a focused monograph, prepared for the purpose of illustrating how a safety review of a dietary supplement ingredient might be prepared following the format for focused monographs described in this report. While it was prepared as a prototype using the processes described in the report, it was not conducted under the auspices of the Food and Drug Administration utilizing all the resources available to the agency. Thus some pertinent information not available to the Committee could be of importance in evaluating safety to determine if use of this dietary supplement ingredient would present an unreasonable risk of illness or injury. Also, the development and review of this prototype was conducted by individuals whose backgrounds are in general aspects of evaluating science and whose expertise is not necessarily focused specifically on this dietary ingredient, although significant additional assistance was provided by consultants with relevant expertise. Therefore, this prototype monograph, while extensive, does not represent an authoritative statement regarding the safety of this dietary supplement ingredient.



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Dietary Supplements: A Framework for Evaluating Safety Appendix J Prototype Focused Monograph: Review of Liver-Related Risks for Chaparral1 I. DESCRIPTION OF THE INGREDIENT A. Chaparral as a Dietary Supplement Ingredient Chaparral is one name for an herbaceous woody shrub that grows in the southwestern region of the United States and the northern region of Mexico. It is also called creosote bush or greasewood. The common Spanish names are hediondilla, which means “little smelly one,” because the bush has a strong odor similar to the smell of creosote (a distillate of coal/ wood tar used as a wood preservative), and gobernadora, which means “governess,” because the bushes can dominate an area by creating an ad- 1   This is a focused monograph, prepared for the purpose of illustrating how a safety review of a dietary supplement ingredient might be prepared following the format for focused monographs described in this report. While it was prepared as a prototype using the processes described in the report, it was not conducted under the auspices of the Food and Drug Administration utilizing all the resources available to the agency. Thus some pertinent information not available to the Committee could be of importance in evaluating safety to determine if use of this dietary supplement ingredient would present an unreasonable risk of illness or injury. Also, the development and review of this prototype was conducted by individuals whose backgrounds are in general aspects of evaluating science and whose expertise is not necessarily focused specifically on this dietary ingredient, although significant additional assistance was provided by consultants with relevant expertise. Therefore, this prototype monograph, while extensive, does not represent an authoritative statement regarding the safety of this dietary supplement ingredient.

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Dietary Supplements: A Framework for Evaluating Safety verse environment for the growth of other plants, resulting in a monoculture in some areas (Schultz and Floyd, 1999). One remarkable feature of the chaparral bush is the complex resinous coating on the leaves that serves as a chemical defense against grazing by herbivores and against attack by insects. The chemicals in the resin find their way into the desert soil surrounding the chaparral plant and discourage growth by other plant species, thus effectively reducing competition for water and nutrients (Mabry et al., 1977). Chaparral is formally known as Larrea tridentata (Sessé and Moc. ex DC.) Coville (synonymous with Larrea mexicana Moric.) of Zygophyllaceae (McGuffin et al., 1997). Historically, the dry leaves, green stems, and fine twig tips of chaparral were used for various ailments. Since about 1969, these same plant components have been used as dietary supplements. Various forms have been available: dried plant material for making teas (water extracts), aqueous-alcoholic extracts or tinctures, and tablets or capsules containing ground, dried plant material. During the past 10 years, chaparral products have not been as readily available as in the past; however, each of these forms is currently available in the U.S. marketplace in varying degrees. B. Individual Components Table A contains a list of the components of primary interest in chaparral (i.e., present in leaves, stems, and twigs). Some components of chaparral are common in other plants and are widespread in the human diet. The major components of the resinous coating of chaparral are lignans (Mabry et al. 1977; Sakakibara et al. 1976) which can comprise up to 80 percent of some extracts of chaparral, such as methanol extracts of green leaves or green stems (Hyder, 2001). Lignans are low-molecular-weight plant products made up of phenylpropanoid dimers or trimers. Mature chaparral leaves contain lower amounts of lignans than new leaves (Gisvold and Thaker, 1974). The major lignan in chaparral is nordihydroguaiaretic acid (NDGA) (Downum et al., 1988), which is a derivative of guaiaretic acid and is a catechol having two hydroxyl groups on each of the two phenol rings. NDGA comprises approximately 10 percent of the dry leaf weight, but may be as much as 15 percent in some instances (Obermeyer et al., 1995). NDGA comprises approximately 50 percent of the phenolic resin extracted from the external surface of the leaves (Botkin and Duisberg, 1949; Mabry et al., 1977; Sakakibara et al., 1976). Chaparral also contains guaiaretic acid and other substituted guaiaretic acid derivatives (Table A). Other lignans in chaparral are classified as furanoid lignans and 1 aryl tetralin lignans. The latter are structurally related to podophyllotoxins. Chaparral contains flavonoids as non-water-soluble aglycones, as wa-

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Dietary Supplements: A Framework for Evaluating Safety ter-soluble glycosides, and as sulfated flavonoids (Mabry et al., 1977). Chaparral also contains triterpenes, including sapogenins (Mabry et al., 1977). The aglycone forms of the flavonoids and triterpenes are listed in Table A. Chaparral contains volatile oils, wax esters, sterols, and other hydrocarbons (Mabry et al., 1977; Waller and Gisvold, 1945). Although a number of the known components of chaparral exhibit cytotoxic activity under various conditions, these effects are judged to be weak and require high concentrations of the substance, and thus would extrapolate to the ingestion of large amounts of chaparral in order to exhibit potential toxic activity in humans. Additionally, many of these components are present in the diet from other sources. C. Description of Dietary Supplement Preparations and Amounts Ingested in Ordinary Use Chaparral is sold in several forms, one of which is the dried, broken leaves, green stems, and fine twig tips that can be brewed as a tea (i.e., an aqueous extract). An example of the modern preparation of chaparral tea would be to steep 7 to 8 g of crumbled dried leaves, stems, and twigs in one quart of hot water. In ordinary use as a water extract, chaparral might be consumed in the amount of 1 to 3 cups of chaparral tea per day for a period of 2 to 3 weeks (Micromedex, 2002). Another form of chaparral is a tincture or aqueous alcohol extract. The ordinary use of such an extract might be 20 to 30 drops per day for a period of 2 to 3 weeks (Micromedex, 2002). Chaparral is also available as a dried leaf powder (frequently sold in capsule or tablet form). Typical suggested uses of such capsules or tablets would be one to two 500-mg capsules or tablets per day for 2 to 3 weeks. Chaparral is also available as a component of various botanical mixtures sold as tinctures and as loose leaves, stems, and twigs for teas. Chaparral dried leaf capsules are also available in combination with silymarin (a flavanolignan complex from milk thistle), vitamin C, or other antioxidants. II. INFORMATION RELEVANT TO LIVER CONCERNS A. Human Use Information and Safety Data 1. Historical use Chaparral has been used for many centuries for a variety of medicinal purposes (Heron and Yarnell, 2001). Native populations in the southwestern United States have used chaparral tea for decades without published evidence of toxicity. Most processing of chaparral used in American Indian

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Dietary Supplements: A Framework for Evaluating Safety cultures involved aqueous extracts, such as hot water teas (Heron and Yarnell, 2001). A tea has very little NDGA (a constituent of concern, see below) compared with an alcoholic extract or the powdered dry leaf because NDGA is poorly soluble in water (Obermeyer et al., 1995). Pima Indians used the tea orally as a diuretic, emetic, or expectorant, and topically as an antiseptic or poultice (Mabry et al., 1977). In many American Indian cultures, chaparral tea has been used to mitigate colds, bronchitis, and other breathing problems; for menstrual cramps; and for numerous intestinal problems. It has also been applied topically for painful joints, skin infections, snakebites, burns, and allergies (Mabry et al., 1977; Moerman, 1998). The leaves have been used both as a decoction in a bath or as an external poultice for rheumatism and arthritis, as well as for scratches, wounds, and bruises (Moerman, 1998). There are a few reports of the use of chaparral extracts by southwest native healers in the management of type 2 diabetes (Gowri et al., 2000). In the medical literature there is a paucity of reports involving the ingestion of chaparral capsules or tablets, except for those resulting in adverse effects (described below). 2. Adverse effects The clinical data suggest a pattern of hepatotoxicity. This pattern is discussed in more detail below. One difficulty in evaluating the clinical data on chaparral is that in most of the cases, the chaparral preparation ingested was not described in any detail. Additionally, the product purity and quality were not reported. Clinical trial data: Table B provides a summary of a small clinical trial that was conducted among 59 terminal cancer patients to examine the effect of NDGA and chaparral tea on tumor growth. Thirty-six patients consumed chaparral tea (16–24 oz/d) while 23 patients consumed NDGA (250–3,000 mg/day). Selected blood tests and urinalysis were repeated at 2 to 4 week intervals. An analysis of the 45 patients who were treated for at least 4 weeks suggested that there were no hematological or chemical abnormalities that could be attributed to the treatment. Patients reported minor adverse effects as described in Table B. Of the 59 treated patients, no pattern of hepatotoxicity was reported following consumption of either chaparral tea or NDGA by the terminally ill cancer patients. The reasons why 14 subjects dropped out were not reported. Clinical case reports: Table C-1 summarizes clinical case reports of patients who took chaparral without the added complication of additional ingredients. Careful inspection of Table C-1 reveals 9 cases of well-diag-

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Dietary Supplements: A Framework for Evaluating Safety nosed hepatotoxicity (Cases #1–9) and 6 cases of suspected or probable hepatotoxicity (Cases #10–15). The cases are arranged in order of apparent severity with the most severe case, which required a liver transplant, presented as Case #1. Many of these patients ingested chaparral in capsule or tablet form (Cases #1–3, 5–11). Most of the chaparral products were unidentified as to whether they contained dried plant material or extracts. The listed amount of chaparral ingested ranged from 0.3 to 6 g/day; however, this information was not included in all case reports. The duration of chaparral use (which is not indicated in 2 of the cases) ranged from 20 days to “many years.” It is notable that Case #15 was the only patient known to use chaparral tea: 4 bags daily for 1.5 years. The product used by this patient was examined using microscopic and chromatographic analysis and was correctly identified as Larrea tridentata with no evidence of biochemical or biological contamination (Sheikh et al., 1997). The severity of the liver damage in these case reports does not seem to correlate directly with either the amount of chaparral consumed or the duration of use. There are five cases with documented recovery from liver damage after cessation of chaparral use (Cases #2, 5, 8, 9, 10). There is one case (#8) documenting a return of jaundice following resumption of chaparral ingestion. Table C-2 summarizes the clinical case reports of patients who took chaparral in combination with other supplements or ingredients, primarily other botanicals. The six cases of hepatotoxicity found in Table C-2 are difficult to evaluate because of the confounding factor of possible adverse effects due to these other substances. These cases include well-documented hepatotoxicity (Cases #17–20) but the cause of the liver damage is difficult to interpret. Two cases returned to normal after cessation of chaparral (Cases #18, 22). There are also reports (Cases #25–29 and Series A) of subjects taking an aqueous alcoholic extract (90 percent ethanol) as 8 to 10 percent of a formula with other herbs, ingesting a total of 30 to 240 mL over a period of 40 days to 5 months, with no indication of liver damage according to liver function tests. Adverse event reports to Special Nutrition/Adverse Event Monitoring System (SN/AEMS): Table D presents the available information on cases reported in the SN/AEMS. The 18 reports include 12 cases indicating varying degrees of liver damage. (These 12 cases are included among the patients in Table C-1.) It should be noted that in the SN/AEMS reports there is no indication of whether a causal relationship exists between the adverse event and chaparral ingestion. 3. Interactions There are no known interactions with chaparral.

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Dietary Supplements: A Framework for Evaluating Safety 4. Consequences of unusually large intake or chronic cumulative use There may be adverse effects associated with consumption of excessively large amounts of chaparral (Heron and Yarnell, 2001). This type of overuse is typically related to encapsulated chaparral products. B. Animal Studies Animal studies on chaparral: There were no animal studies identified that showed liver toxicity as the result of chaparral administration. In studies with rats, significant toxic effects were demonstrated following administration of chaparral; however, the nature of the toxicity was not documented (Nakazato et al., 1998; Ulreich et al., 1997). The ethanol:water tincture of chaparral administered to the rats was lethal in the relatively large amounts administered in these studies. In all, there is evidence of considerable toxic effects from four different animal models: using rats (Konno et al., 1987; Nakazato et al., 1998; Ulreich et al., 1997), hamsters (Granados and Cardenas, 1994), chickens (Zamora, 1984), and insects (Mabry et al., 1977) with relatively high exposures to chaparral. In considering all of the animal studies (Table E), the evidence evaluating any aspects of the safety of chaparral in animal studies is minimal. Acute studies on NDGA in animals: The evidence evaluating the safety of NDGA, a major component of chaparral, is more substantial but is still incomplete (Table E). NDGA administered by gavage to rats and mice was reported to have an LD50 of > 4 g/kg body weight. NDGA was somewhat more toxic in guinea pigs, with an LD50 of 0.8 g/kg body weight. Thus, the LD50 is less than 100× a typical human intake. Chronic studies on NDGA in animals: Chronic studies on the safety of NDGA are limited to toxicity studies conducted primarily in small rodents (Table E). Rats fed NDGA at 0.5 percent of their diet exhibited massive hemorrhages and multiple renal cysts in experiments reported only in abstract (Cranston et al., 1947) and reviews (Lehman et al., 1951). Strong evidence has been published that NDGA fed to rats at high doses (1 or 2 percent of the diet) clearly leads to various pathological changes. In various rat models, growth inhibition and structural changes in or near the kidney have been shown to develop within 2 to 6 months (Cranston et al., 1947; Gardner et al., 1986, 1987; Lehman et al., 1951). Renal and mesenteric cysts form within 6 to 12 months of NDGA feeding (Goodman et al., 1970; Grice et al., 1968; Lehman et al., 1951). By 18 months of feeding 1 percent NDGA (Grice et al., 1968) or 6 months of feeding 2 percent NDGA (Evan and Gardner, 1979), the development of renal and mesenteric cysts is profound. The renal cysts contained degenerating tubular cells and the renal

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Dietary Supplements: A Framework for Evaluating Safety damage was predominantly in the proximal convoluted tubules. Biochemical analysis showed no free NDGA in the lymph nodes or kidney extracts; only the orthoquinone metabolite of NDGA could be detected (Grice et al., 1968). In complementary studies, single dose administration of 250 mg of NDGA into the small intestine of rats revealed formation of the orthoquinone metabolite at the region of the ileocecal junction (Grice et al., 1968). Studies using other species and/or other routes of administration verify the toxicity of NDGA (Giri and Hollinger, 1996; Hsu et al., 2001; Madrigal-Bujaidar et al., 1998; Mikuni et al., 1998; Telford et al., 1962). Other observations: Pretreatment of rats with NDGA (50 mg/kg body weight, by gavage) significantly aggravated indomethacin-induced gastric ulcers (Cho and Ogle, 1987). Treatment of rats with NDGA (10 μg/kg body weight, by intravenous administration) worsened ischemia-reperfusion injury to liver (Okboy et al., 1992). C. In Vitro Studies Investigations on the in vitro effects of chaparral and NDGA on a variety of chemical and biological systems are summarized in Tables F-1 and F-2. While a few studies involved extracts of chaparral, most focused on the effects of NDGA, and a few considered the effects of other lignans with structural similarities to NDGA. Some cytochrome P450 oxidations are inhibited by NDGA in vitro (Agarwal et al., 1991; Capdevila et al., 1988). D. Liver-Related Information About Related Substances Studies on taxonomically related substances: Five species of Larrea are recognized: the bifolate species, L. tridentata (native to the southwestern United States and northern Mexico), L. divaricata (native to northwestern Argentina and parts of Peru), and L. cuneifolia (native to Argentina), plus two multifolate species that grow at high altitudes, L. nitida (native to certain parts of South America, especially Argentina) and L. ameghinoi (native to a few parts of South America) (Brinker, 1993–1994). No useful safety data were found on L. cuneifolia, L. nitida, or L. ameghinoi. The toxicity of L. divaricata, a South American species that is taxonomically related to L. tridentata, has been studied to a very limited extent. A water extract of the dried leaves was injected into mice intraperitoneally and the LD50 was found to be 10 g/kg body weight for males and 4 g/kg for females (Anesini et al., 1997).

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Dietary Supplements: A Framework for Evaluating Safety Substances related to the individual components of chaparral: Table G contains a list of substances that were considered as structurally, taxonomically, or functionally related to the components of chaparral (present in leaves, stems, and twigs). Known toxicities of these related substances were considered in evaluating the potential toxicity of chaparral. For comparison, Table A contains a listing of the known components of chaparral with the chemical structures of those that may be relevant to the safety of chaparral. In Table G it should be noted that larreantin is a potential hepatotoxin and is known to be present in the root of L. tridentata (Luo et al., 1988). Several mechanisms were considered whereby it might be possible that chaparral products could contain larreantin. First, chaparral root might be included with the other plant material (leaves, stems, and twigs). Second, under certain environmental conditions, a component of the root of a plant might physiologically be present in the leaves. Third, the presence of trace amounts of larreantin in the leaves, stems, or twigs could have been undetected. Although each of these mechanisms is possible, it seems unlikely that larreantin is present in chaparral preparations in significant amounts. Functionally related substances: It was reported that NDGA is metabolized to an orthoquinone derivative (De Smet, 1993; Grice et al., 1968), which could be further metabolized by conjugation to glutathione. Because hepatic levels of glutathione are often limiting, drugs undergoing glutathione conjugation could interact negatively with the quinone derivative of NDGA by both substances drawing on glutathione reserves in the liver, leading to glutathione depletion (Slattery et al., 1987). Knowledge about chemical structures of chaparral components: As stated above, NDGA is metabolized to an orthoquinone derivative (De Smet, 1993; Grice et al., 1968). Acetaminophen is also a quinone, but one that is understood to be cytotoxic and to cause substantial liver problems. Large doses of acetaminophen cause centrilobular hepatic necrosis (Hojo et al., 2000). The current understanding is that the hepatotoxicity of acetaminophen is due to cytochrome P450-dependent formation of N-acetyl para-(benzo)quinone imine (NAPQI) in the centrilobular region of the liver (Harman et al., 1991; Hojo et al., 2000; Holme et al., 1984). By extrapolation, the site of chaparral toxicity might be expected to reflect the site of metabolism of NDGA to the quinone. NAPQI causes mitochondrial damage, including inhibition of oxidative phosphorylation (Andersson et al., 1990; Fujimura et al., 1995; Moore et al., 1985). Likewise, NDGA causes inhibition of the mitochondrial electron transport chain. Thus there are some similarities between NAPQI toxicity and NDGA toxicity that can be used to hypothesize a mechanism of hepatotoxicity based on the formation of a NDGA quinone. Acetaminophen-induced toxicity is also

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Dietary Supplements: A Framework for Evaluating Safety seen in the kidney, another site of metabolism of the drug to NAPQI. Therefore, one might hypothesize that chaparral ingestion would lead to toxicity in the kidney also if chaparral toxicity is related to NDGA metabolism to a toxic quinone, which is purely theoretical. Indeed, renal toxicity of NDGA is evident in animal studies (Table E). III. OTHER RELEVANT INFORMATION A. Sources Chaparral grows as a wild desert shrub. It is an evergreen bush that grows in arid regions and can reach a height of 9 feet (Brinker, 1993–1994). The identification of the plant used as L. tridentata is very important to the safety of the chaparral product. Misnaming and species identification: There may be some instances of substitution of another plant product for L. tridentata. L. divaricata has been commonly confused with L. tridentata. The two species are very similar in appearance (Brinker, 1993-1994), but originate from distinct locations. The major source of confusion is the misnaming of the two species, even in published reports of clinical or experimental data (Gisvold, 1947; Smart et al., 1970). Contaminants and adulterants: Adulteration has not been reported. Processing issues: As described in the human use information section, most processing of chaparral used in American Indian cultures involved aqueous extracts, such as hot water teas (Heron and Yarnell, 2001). A tea has very little NDGA compared with an alcoholic extract or the powdered dry leaf because NDGA is poorly soluble in water (Obermeyer et al., 1995). The extraction liquid generally used to make a tincture of chaparral has a high percent of ethanol (up to 95 percent, v/v) so that the extract will contain phenolic compounds, such as NDGA and flavonoids. The amount of solvent-extractable natural products does not change considerably regardless of whether fresh or dried leaves are used in processing (Mabry et al., 1977). The solvent used does make a considerable difference in the quantity of individual components. Diethyl ether gives a high yield, as compared with 85 percent aqueous methanol, which also extracts considerable chlorophyll (Mabry et al., 1977). Analytic issues: Analytical methods have been published for the determination of a number of components of chaparral. These methods include gas liquid chromatography, high pressure liquid chromatography, and mass

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Dietary Supplements: A Framework for Evaluating Safety spectrometric analysis of lignans (Gonzalez-Coloma et al., 1988; Obermeyer et al., 1995; Valentine et al., 1984), and the ammonium molybdate spectrophotometric assay for NDGA (Duisberg et al., 1949). Pharmacokinetic analysis of NDGA administered to mice used a method with a limit of detection of 0.5 μg/mL plasma or serum (Lambert et al., 2001). B. Conditions of Use Suggested or Recommended in Labeling or Other Marketing Material Common popular uses: Chaparral has had numerous ethnomedicinal, homeopathic, and folk medicine uses. Homeopathic medicine has used chaparral tea in the treatment of colds, cold sores, coughs, bronchitis, viral infections, urinary tract infections, indigestion, heartburn, abdominal cramps, enteritis, dysentery, parasites, dysmenorrhea, menstrual cramps, premenstrual syndrome, neuritis, and sciatica (Heron and Yarnell, 2001; Mabry et al., 1977). Chaparral has been used as an abortifacient and as a means to increase fertility (Heron and Yarnell, 2001). Chaparral products have been described as having a beneficial impact on liver metabolic functions (Heron and Yarnell, 2001). In folk medicine, chaparral has been used for leukemia and many different types of cancers. It has been suggested that chaparral contains immune-stimulating polysaccharides and that NDGA may have some antitumor properties. From conventional medical sources there is anecdotal and in vitro evidence of cytotoxic activity with varying toxicity depending on the concentration of NDGA. Currently chaparral is marketed to consumers for arthritis, rheumatism, and bursitis; as an antioxidant; for immune function; for various cancers, such as melanomas, leukemia, breast cancer, ovarian cancer, and Kaposi’s sarcoma; as a blood and liver cleanser; as a diuretic; for colds and the flu; for herpes family viruses including herpes simplex, herpes zoster, cytomegalovirus, and Epstein-Barr; and for acne and skin disorders. C. Liver-Related Cautions Noted Cautions provided in labeling or other marketing material: A review of chaparral product labels and Internet marketing materials indicates that many (but not all) provide cautions to consumers to seek advice from health care providers before using the product if they have a history of liver or kidney disease or currently have digestive problems and to avoid using if pregnant or nursing. One caution indicated that the chaparral product was not intended for long-term use. Two informational websites that did not sell chaparral products suggested that people consuming chaparral tea should drink 3 cups a day for a maximum of 2 weeks unless under the care

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Dietary Supplements: A Framework for Evaluating Safety of a physician or health practitioner experienced in the use of botanical medicines. At least three informational websites that did not sell products cautioned against the use of chaparral capsules since several adverse event reports were associated with this form. Cautions issued by manufacturing associations: In 1994 the American Herbal Products Association (AHPA) commissioned a review. Four case studies were examined and it was concluded that: … since the patients were ingesting chaparral during the time each developed acute hepatitis, most likely of a hepatocellular nature, it is reasonable to conclude a relationship exists between the ingestion and the disease. However, no clinical data were found in the medical records to indicate that chaparral is inherently a hepatic toxin. Moreover, each patient had a medical history not incompatible with prior liver disease. A fair conclusion is [that] the disease in each patient was the result of an individual idiosyncratic reaction to the drug [botanical product], possibly the result of an autoimmunologic reaction, which given the quantity of chaparral ingested in this country, must be exceedingly rare (AHPA, 2002). Following the Food and Drug Administration (FDA) warning issued in 1992 (see Section IIIE, below), many manufacturers voluntarily removed most products containing this botanical (FDA, 1993). In 1995 AHPA recommended that if member companies chose to sell chaparral, all consumer labeling contain the following informational language: Seek advice from a health care practitioner before use if you have had, or may have had, liver disease. Discontinue use if nausea, fever, fatigue or jaundice (e.g., dark urine, yellow discoloration of the eyes) should occur (APHA, 2002). D. Usage Patterns Prevalence of use in the general population: According to a survey conducted by the Herb Research Foundation from 1973 to 1993, at least 200 tons of chaparral was sold in the U.S. market (Blumenthal, 1993). This would be equivalent to 500 million doses at 500 mg/dose. No current data are available; there has been no recent tracking of sales data. Knowledge of use by particular groups: There are no published surveys in the literature that provide knowledge about the use of chaparral by specific groups. However, anecdotal reports suggest that indigenous American Indian groups in the southwestern United States and Hispanics may use chaparral, primarily as an aqueous extract (tea).

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Dietary Supplements: A Framework for Evaluating Safety Species/Study Design Results and Conclusions Rats (male), fed NDGA (0.5% of the diet), 2 y Growth inhibition was seen with NDGA at 0.5% of the diet after 6 mo Massive cecal hemorrhages with single and multiple cysts in the mesentery near the cecum were seen (Lehman et al., 1951) Rats (male), fed NDGA (0.5% of the diet), 2 y Inflammatory cecal lesions and slight cystic enlargement of paracaecal lymph nodes were seen Growth inhibition at 6 mo of NDGA feeding (Lehman et al., 1951) Rats (female), fed NDGA (1% of the diet), 6 mo Growth inhibition at 6 mo of NDGA feeding (Lehman et al., 1951) Rats, fed NDGA (0.5% of the diet) No effect on food intake, kidneys, liver, or spleen Cranston et al., 1947) Rats, fed NDGA (0.1, 0.5, or 1% of the diet) Some animals developed cysts in mesentery at 0.5% and 1.0% NDGA (Cranston et al., 1947) NOTE: Acute toxicity studies are defined as a single administration or exposure for less than 24 h; chronic toxicity studies are defined as repeated administration or exposure for 1 mo or longer, combining what some authors call subchronic and chronic (Klassen, 1995). i.p. = intraperitoneally, S/D = Sprague-Dawley, iv = intravenously, AUC = area under the curve.

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Dietary Supplements: A Framework for Evaluating Safety TABLE F-1 Chaparral: Summary of In Vitro Studies Substance Study Design Results and Conclusions Inhibition of enzymes Chaparral, methanol extract Enzyme assays using rat (male and female, Sprague-Dawley) liver microsomes incubated with 0.1–100 μg chaparral extract/mL At 10 μg/mL the chaparral extract inhibited glutathione S-transferase At 100 μg/mL the chaparral extract also inhibited aminopyrine N-demethylase (various cytochrome P450 forms), aniline hydroxylase (cytochrome P450 2E1), and UDP-glucuronyl transferase The activity of NADPH-cytochrome c reductase was increased by the larger amounts of the extract (Sapienza et al., 1997)

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Dietary Supplements: A Framework for Evaluating Safety TABLE F-2 Nordihydroguaiaretic Acid (NDGA): Summary of In Vitro Studies Substance Study Design Results and Conclusions Apoptosis NDGA, 25 μM Cells in culture: SW 850 (human pancreatic cancer cell line) C4-I (human cervical cancer cell line) NDGA induced apoptosis within 3 hr reaching maximum in 12–16 hr Seufferlein et al., 2002) NDGA, 10 μM, 4–18 h Cells in culture: FL5.12 cells (mouse hematopoietic/ lymphocytic cell line, lipoxygenase-deficient) NDGA induced apoptosis and greatly increased caspase-3-like activity (Biswal et al., 2000) NDGA, IC50 5 μM Cells in culture: Walker-256 cells (rat epithelial carcinoma cell line, LLC-WRC 256 cells) NDGA induced apoptosis (Tang and Honn, 1997) NDGA, 25 μM Cells in culture: MTLN-3 cells (rat tumor cell line) RBL cells (rat tumor cell line) NDGA induced apoptosis (Tang and Honn, 1997) NDGA, 25 μM Cells in culture: A431 cells (human epithelial carcinoma cell line) HEH cells HL-60 cells (human myeloblastic cell line) U937 cells (human monocytes cell line) NDGA induced apoptosis in some human cell lines NDGA Cells in culture: PC-3 cells (human cell line) 1-IL cells DU145 cells (human cell line) WB35 cells (human cell line) WM983A cells (human cell line) neoT cells (human cell line) MCF-7 cells (human epithelial adenocarcinoma cell line) MCF-10A cells (human mammary epithelial cell line) HT-1080 cells (human epithelial fibrosarcoma cell line) NDGA (25–35 μM) did not induce apoptosis in other (human cell line) human cells (Tang and Honn, 1997) NDGA, 30 μM Cells in culture: LN-18 cells (human malignant glioma cell line) NDGA inhibited mediated by CD95 receptor apoptosis (Wagenknecht et al., 1998)

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Dietary Supplements: A Framework for Evaluating Safety Substance Study Design Results and Conclusions Cytotoxicity NDGA, LC50 200 μM Cells in culture: EMT6 cells (mouse mammary carcinoma cell line) NDGA caused slight cytotoxic activity in EMT6 tumor cells, likely related to depletion of sulfhydryl groups (Shi and Pardini, 1995) NDGA, LD50 9–20 μg/mL Cells in culture: Vero cells (African green monkey kidney epithelial cell line) Hep-2 cells (HeLa epithelial cell line) NDGA had weak cytotoxic activity (Zamora et al., 1992) NDGA, 25–250 μM, ≤ 72 hr Cells in culture: 786A cells (sarcoma cell line), IC50 0.24 mM TA3 cells (mammary cell line), IC50 0.21 mM NDGA had weak cytotoxic activity Addition of NDGA (250 μM) decreased cellular respiration and ATP concentration within 1 hr (Pavani et al., 1994) NDGA In cell suspensions: NDGA had cytotoxic anaerobic glycolysis and respiration NDGA inhibited aerobic and activity (Burk and Woods, 1963) NDGA Cells in culture: WISH cells (human HeLa cell line) NDGA decreased viability (ID50 100 μg/mL) Blalock et al., 1981) NDGA Cells in culture: Mouse L cells NDGA decreased viability (ID50 100 μg/mL) (Blalock et al., 1981) NDGA Cells in culture: Ehrlich ascites cells NDGA sensitized cells to X-ray irradiation (1,000 r) (von Ardenne et al., 1969) These data were published in German. Summary is based on the English abstract NDGA, 150–600 mM Tissue slices: Rat liver slices (male, Fisher 344) During incubation of precision-cut rat liver slices with NDGA, cell viability decreased by several indicators (decreased content of potassium, LDH, and glycogen) Ethanol was also cytotoxic and the total effect was additive (Ulreich et al., 1997)

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Dietary Supplements: A Framework for Evaluating Safety Substance Study Design Results and Conclusions NDGA, 150–600 mM Tissue slices: Rat kidney slices During incubation of precision-cut rat kidney slices with NDGA, cell viability decreased by two indicators (decreased content of potassium and LDH) (Nakazato et al., 1998) NDGA, 150–600 mM Tissue slices: Human liver slices Human kidney slices During incubation of precision-cut human liver or kidney slices with NDGA, cell viability decreased by two indicators (decreased content of potassium and LDH) Cytotoxicity was dose-dependent (Nakazato et al., 1998) NDGA, LD50 150 μM Isolated cells: Rat hepatocytes During a 2-hr incubation with NDGA or 21 different flavonoids and polyphenols, NDGA was one of the most cytotoxic, behind galangin and chrysin (Moridani et al., 2002) Inhibition of cellular processes NDGA, Ki 140 μM Isolated jejunal loops from rats (female Wistar), using luminal perfusion NDGA inhibited intestinal glucose absorption, glucose utilization, and lactate production (Kellett et al., 1993) NDGA, 30 μM Cells in culture: 3T3-4 cells NDGA enhanced glucose transport and metabolism (± insulin) (Reed et al., 1998, 1999). NDGA, 30 μM Isolated rat adipocytes NDGA enhanced glucose transport 2-fold (± 100 pM insulin) (Reed et al., 1998, 1999) NDGA Isolated rat pancreatic islets NDGA inhibited insulin secretion induced by glucose (Yamamoto et al., 1982)

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Dietary Supplements: A Framework for Evaluating Safety Substance Study Design Results and Conclusions NDGA, 100 μM Tissue culture: Isolated mouse pancreatic islets in culture for 1–2 wk NDGA in the culture media reduced insulin secretion induced by glucose (20 mM) even though the total insulin content of islets was equivalent in control and NDGA-treated cultures (Hsu et al., 2001) NDGA, 30 μM Isolated rat adipocytes NDGA inhibited lipolysis in response to isoproterenol or 8-chlorophenyltheo cAMP (Gowri et al., 1998) NDGA, 50 μM Isolated rat adipocytes NDGA reduced lipolytic activity induced by isoproterenol and decreased the phosphorylated form of hormone-sensitive lipase (Gowri et al., 1998) NDGA Cells in culture: SW 850 (human pancreatic cancer cell line) C4-I (human cervical cancer cell line) NDGA inhibited anchorage-dependent proliferation (data not shown) After incubation with NDGA for 8 hr, cells began to detach from tissue culture dish Incubation of cells with NDGA (25 μM) inhibited expression of cyclin D1 (while expression of cyclin E was unchanged) Incubation of cells with NDGA (25 μM) resulted in disruption of the cytoskeleton (actin stress fibers but not the circumferential actin filament network) Incubation of cells with NDGA (25 μM) activated stress-activated MAP kinases (JNK1/2 and p38mapk but not ERK1/2) (Seufferlein et al., 2002)

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Dietary Supplements: A Framework for Evaluating Safety Substance Study Design Results and Conclusions NDGA, 15–30 μM Cells in culture in soft agar: SW 850 (human pancreatic cancer cell line) C4-I (human cervical cancer cell line) NDGA inhibited colony formation in response to 0.5% or 10% fetal bovine serum, thought to represent anchorage-independent growth (Seufferlein et al., 2002) NDGA inhibited anchorage-dependent proliferation (data not shown) (Seufferlein et al., 2002) After incubation with NDGA for 8 hr, cells began to detach from tissue culture dish NDGA, 10 μM Cells in culture: HEK293 cells (human embryonic cell line) Porcine coronary arterial smooth muscle cells) NDGA at 10 μM activates the Ca2+-dependent K+ channel, releasing Ca2+ (Yamamura et al., 2002) NDGA, > 10 μM Cells in culture: HEK293 cells (human embryonic cell line) Porcine coronary arterial smooth muscle cells) NDGA at > 10 μM quickly causes a large increase the intracellular concentration of Ca2+ (Yamamura et al., 2002) NDGA, 5–100 μM Cells in culture: Rat C6 glioma cells NDGA increased the concentration of intracellular Ca2+ (Su et al., 2002) NDGA, 1–100 μM Isolated porcine coronary artery smooth muscle cells (inside-out and outside-in patches) NDGA opens the Ca2+-dependent K+ channel, except in the presence of very low cytosolic Ca2+ concentrations (Nagano et al., 1996) NDGA, 100 μM Bovine heart mitochondria NDGA inhibited mitochondrial electron transport (by inhibition of NADH-coenzyme Q reductase and succinate coenzyme Q reductase) (Pardini et al., 1970) NDGA Beef heart mitochondria NDGA reduced microsomal electron transport by inhibiting succinate cytochrome c reductase (Shi and Pardini, 1995)

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Dietary Supplements: A Framework for Evaluating Safety Substance Study Design Results and Conclusions NDGA, IC50 15 ηmoles/mg mitochondrial Rat liver mitochondria NDGA inhibited mitochondrial electron transport (Bhuvaneswaran and Dakshinamurti, 1972) Inhibition of enzymes NDGA Rat epidermal and hepatic microsomal cytochrome P-450 NDGA inhibited aryl hydrocarbon hydroxylase (CYP 1A and 1B) and inhibited 7-ethoxy-resorufin O-demethylase (CYP 1A) activities (Agarwal et al., 1991) NDGA, 100 μM Cells in culture: Hep-G-2 cells NDGA inhibited cytochrome 1A1 induction (hydrodynamic stress-induced) (Mufti and Shuler, 1996) NDGA Rat liver homogenate (IC50 6 μM) Human liver homogenate Human placenta homogenate NDGA inhibited catechol O-methyl transferase and (Burba and Becking, 1969) NDGA, Ki 125 μM Isolated jejunal loops from rats (female Wistar), luminal perfusion NDGA inhibited (Mg2+/Na+/ K+)-ATPase and (Na+/K+)- ATPase in jejunum (Kellett et al., 1993) NDGA Various enzyme sources NDGA inhibited carboxylesterase (2 μM) and inhibited formyltetrahydrofolate synthetase (ED50 100 μM) (Schegg and Welch, 1984) NDGA, 100 μM Microsomes NDGA inhibited cyclooxygenase (Van der Merwe et al., 1993) NDGA, IC50 1 μM Rat platelets NDGA inhibited platelet cyclooxygenase (Ferrandiz et al., 1990) NDGA, IC50 1–42 μM Intact cells and cell-free preparations NDGA inhibited 5-lipoxygenase activity (peritoneal neutrophils from female Wistar rats, IC50 2–4 μM); inhibited 15-lipoxygenase activity (isolated from soybean, IC50 4 μM); and inhibited

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Dietary Supplements: A Framework for Evaluating Safety Substance Study Design Results and Conclusions     cyclooxygenase activity (peritoneal macrophages from male CD-1 mice, IC50 1–42 μM) (Chang et al., 1984). NDGA, IC50 0.2 μM Soybean lipoxygenase NDGA inhibited lipoxygenase (Whitman et al., 2002) NDGA, IC50 5 μM Human platelet 12-lipoxygenase NDGA inhibited human 12-lipoxygenase (Whitman et al., 2002). NDGA, IC50 0.1 μM Human reticulocyte 15-lipoxygenase NDGA inhibited human 15-lipoxygenase (Whitman et al., 2002) NDGA, IC50 10 μM Cells in culture: Caco-2 (human colon epithelial cell line) NDGA (10 μM) inhibited 15-lipoxygenase activity without inhibiting cyclooxygenase activity (data not shown) (Kamitani et al., 1998) NDGA, IC50 0.3 μM Rabbit erythroid 15-lipoxygenase NDGA inhibited 15-lipoxygenase (Luther et al., 1991) NDGA, IC50 180 μM Sheep vesicular gland prostaglandin H synthase NDGA inhibited prostaglandin H synthase (Luther et al., 1991) NDGA, 10 μM Rat alveolar macrophages and Chinese hamster lung fibroblasts phospholipase NDGA inhibited A2(Robison et al., 1990) NDGA, IC50 11 μM Human aromatase: Placental microsomes Choriocarcinoma cell line JEG-3 al., NDGA inhibited human aromatase (estrogen synthetase) (Adlercreutz et 1993) NDGA, Ki 94 μM Rabbit skeletal muscle enzyme NDGA inhibited phosphofructokinase (Kellett et al., 1993) NDGA, IC50 41 μM Rat liver microsomes NDGA inhibited aryl hydrocarbon hydroxylase (Agarwal et al., 1991) Other NDGA, 10 ηM Cells in culture: Human renal tubular cells (epithelial cells) NDGA increased the incorporation of hydroxyproline, a component of basement membrane (Vedovato et al., 1994)

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Dietary Supplements: A Framework for Evaluating Safety Substance Study Design Results and Conclusions NDGA, 10 μM Chemiluminescence was used to indicate production of oxidative metabolites from polymorphonuclear leukocytes interacting with formylmethionyl-leucyl-phenylalanine. NDGA acted as antioxidant that eliminates extracellular and intracellular production of oxidative metabolites (Dahlgren, 1991) NDGA Cells in culture: Chinese hamster V79 cells NDGA reduced cytotoxicity of H2O2 (Nakayama, 1994) Derivatives of NDGA (also present in chaparral) 3′-O-Methyl NDGA, ID50 41 ηmol/mg mitochondrial protein Beef heart mitochondria 3′-O-Methyl NDGA inhibited mitochondrial electron transport (by inhibition of succinoxidase and NADH-oxidase) (Heiser et al., 1977) Meso-dihydroguiaretic acid Rat liver microsomes Inhibited aminopyrene N-demethylase activity (various CYP forms) (Stetler-Stevenson et al., 1992) Secoisolariciresinol Cells in culture: P-388 (mouse lymphocyte leukemia cell line) IC50 8.3 μg/mL (23 μM) KB-16 (human nasopharyngeal carcinoma cell line) IC50 0.8 μg/mL (2.2 μM) A-549 (human lung adenocarcinoma cell line) IC50 1.4 μg/mL (3.9 μM) HT-29 (human colon adenocarcinoma cell line) IC50 0.6 μg/mL (1.7 μM) Weak cytotoxic activity (Shen et al., 1997) NOTE: LC50 = concentration that is lethal to 50 percent of the organisms exposed, LD50 = dose that is lethal to 50 percent of the organisms exposed, ID50 = dose at which the response has decreased to 50 percent of the original response, LDH = lactate dehydrogenase, CYP = cytochrome P450, ED50 = dose required to produce a specified effect in 50 percent of the test organisms exposed, CD-1 = a strain of mice.

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Dietary Supplements: A Framework for Evaluating Safety TABLE G Chaparral: Related Substances That Might Suggest Risk Study Design Structure Results and Conclusions Lignan: substituted guaiaretic acid derivative Tetra-O-methylnordihydroguaiaretic acid, synthetic (Lambert et al., 2001)   No data suggestive of toxicity are available Naphthoquinone Larreantin Present in the roots of L. tridentate (Luo et al., 1988); not known to be present in the aerial parts of L. tridentata, but a possible component   In vitro study: weak cytotoxic activity Potential hepatotoxin: in general, quinones are reactive species and readily form adducts with cellular macromolecules and thus can cause cellular damage; naphthoquinones are lipophilic and readily react with membrane-bound macromolecules (e.g., membrane bound enzymes such as the cytochrome P450s) as well as cytosolic molecules (e.g., glutathione) Another mechanism by which quinones cause cellular damage is by increasing the oxidative stress of the cell as the quinone/semiquinone pair repeatedly cycle, generating oxygen radicals or other intracellular radicals with each cycle (Jaeschke et al., 2002) NOTE: Only the substances considered to be relevant to the risk of chaparral as a dietary supplement are included in the table. “Functionally related” substances may exhibit an activity that chaparral exhibits, based on in vitro or other data; they are not listed here because they have a similar chemical composition.