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National Toxicology Program. NTP Technical Report on the Toxicity Studies of a Gum Guggul Extract Formulation Administered by Gavage to Sprague Dawley (Hsd:Sprague Dawley® SD®) Rats and B6C3F1/N Mice: Toxicity Report 99 [Internet]. Research Triangle Park (NC): National Toxicology Program; 2020 Jun.
NTP Technical Report on the Toxicity Studies of a Gum Guggul Extract Formulation Administered by Gavage to Sprague Dawley (Hsd:Sprague Dawley® SD®) Rats and B6C3F1/N Mice: Toxicity Report 99 [Internet].
Show detailsChemical and Physical Properties
Commiphora mukul is a flowering plant of the Burseraceae family endemic to the semiarid climates of Pakistan and West India (Figure 1). C. mukul is a hardy gynodioecious tree/shrub highly sought for its oleoresin (gum guggul), which is further processed by solvent extraction to produce gum guggul extract (GGE).1
Figure 1
Commiphora mukul, Botanical Species from Which Gum Guggul Oleoresin Is Harvested
C. mukul trees are 4–6 feet tall with ash-colored bark, free of foliage throughout most of the year, and are slow growing, requiring 5–6 years to attain the maturity necessary for harvesting of oleoresin. The oleo-gum resin is contained in the gum resin ducts within the primary and secondary phloem of larger veins present in the leaves and soft base of the stems.2-4
The crude oleo-gum resin exudate contains gums (32%), oleo-gum resin (38%), essential oils (1%), minerals (20%), organic matter (4%), and unidentified components (5%).1 The constituents of gum guggul include a complex mixture of diterpenoids, triterpenoids, steroids, flavonoids, long-chain aliphatic tetrols, aliphatic esters, ferulates, lignans, carbohydrates, amino acids, and numerous inorganic ions.5 Crude gum guggul oleo-gum resin is fractionated via solvent extraction by ethyl acetate to yield a soluble resin fraction (approximately 45%) and an insoluble gum fraction (approximately 55%). The soluble fraction is referred to as guggulipid and contains purported bioactive molecules, whereas the insoluble fraction contains constituents such as gums, minerals, and other components.5,6 The soluble isolate can be fractionated further by use of pH gradients to yield acidic (4%), basic (1%), and neutral fractions (95%). The neutral fraction is divided into ketonic (12%) and nonketonic (88%) subfractions. The nonketonic, inactive sub-fraction contains fatty alcohols, diterpenes, and lignans, whereas the neutral ketonic sub-fraction contains guggulsterones (35–40%) from which Z- and E-guggulsterones (approximately 10%) and other C21 and C27 sterols and ester molecules are derived.7,8 The phytosteroids Z- and E-guggulsterone are widely considered the principal pharmacological components of gum guggul that impart its hypolipidemic and anti-inflammatory properties (Figure 2).9-13
Figure 2
Z- and E-Guggulsterone Isomers
Z- and E-guggulsterone isomers antagonize the farnesoid X receptor (FXR), which is a key regulator of bile acid signaling and cholesterol metabolism.13,14 Guggulsterones transactivate the bile salt export pump (BSEP); the up-regulation of BSEP expression is dominant over its FXR-mediated antagonism.15 Guggulsterones also act as agonists for the pregnane X receptor (PXR), estrogen receptor-α, and the progesterone receptor.16 GGE contains additional, presumed bioactive, constituents including guggulsterols (I–VI), myrrhanol A, and myrrhanone A, among others, that might contribute to its asserted anti-inflammatory, hypolipidemic, and antitumor effects.5 The guggulsterone content of C. mukul varies by geographic location, with higher concentrations endemic to hyperarid agroclimatic regions.17
Production, Use, and Human Exposure
Gum guggul is harvested from the plant’s soft underbark through the process of tapping, which involves making an incision in the tree bark of sufficient depth and positioning a vessel to collect the resin outflow. Harvest season is typically from February to June and is restricted to trees 5 years of age or older. Once tapped, a fluid exudes from the incision and slowly hardens to form the oleo-gum resin, which has a bitter taste and aromatic odor and is yellow, pale green, or reddish brown. The gum resin is typically collected every 10–12 days following the initial incision, with 700–900 g harvested from a single tree.18 In some cases, enhancer compounds, such as the plant growth regulator ethephon, are applied to trees to increase cumulative gum yield.18 After harvest, the oleoresin is graded for quality according to the extent of adulteration with particulates such as bark, soil, and sand.2 Guggul trees often die because of tapping, with pathogenesis associated with an outgrowth of the microbe Xanthomonas axonopodis pv. commiphorae.19 Commercial harvesting, poor seed set, low germination rates, and slow growth rates have led to a dwindling C. mukul population and placement on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species as “critically endangered.”20 More recently, improved nondestructive harvesting techniques have been developed with parameters regarding cut size, technique, and use of enhancing compounds.21 Gum guggul production, as measured by the forest department of Kachchh, India, showed a decline in harvesting from the 1960s, when up to 30 tons were collected, and 1999, when only 2.42 tons were collected.22
GGE has been used in traditional Ayurvedic medicine for centuries, having been first described in Atharva Veda, circa 2000 BC. Indications of gum guggul described in the Ayurveda include atherosclerosis, hypercholesterolemia, skin disorders, broken bones, rheumatism, and obesity.1,6 Consumption of raw oleoresin is associated with the onset of skin rashes, irregular menstruation, intestinal discomfort, and hepatotoxicity.5 Traditional use often involves combining processed gum guggul with other flora or natural products such as dried fruits or plant components.
Human exposure to gum guggul occurs primarily by ingestion of homeopathic preparations, herbal/dietary supplements, and pharmaceuticals. Dosage forms include powdered oleo-gum resin, petroleum ether extracts, ethyl acetate extracts, or other galenical preparations. According to U.S. Pharmacopeia guidelines, the oleoresin gum should not contain less than 1% Z- and E-guggulsterones, and extracts must contain at least 5% Z- and E-guggulsterones calculated on an anhydrous basis as Z-guggulsterone.23 In addition to consumptive use, gum guggul is used in the production of incense, lacquers, varnishes, ointments, and cosmetics and as a fixative agent in perfume.
Currently, standardized extracts of gum guggul are approved for marketing in India as lipid-lowering drugs and in the United States as dietary supplements. Use of herbal remedies and dietary supplements are becoming increasing popular in Western societies. According to the 2012 National Health Information Survey, an estimated 18% of adults in the United States consumed a dietary supplement that was not vitamin/mineral based.24,25 These products often lack sufficient data regarding quality, safety, or efficacy. Commercial guggulipid preparations are reported to contain 2.5–5% Z- and E-guggulsterones.26 Analysis of six products marketed in the United States contained significantly less guggulsterones than the amount claimed on the label, some of which had no detectable levels.8 As summarized by the World Health Organization (WHO), suggested maximum daily intakes range from 3 to 4.5 g of the oleo-gum resin divided in three doses, or 500 mg of petroleum ether extracts of the resin two or three times a day.27 The Indian Pharmacopeia recommends a maximum concentration of guggulsterones in supplements of 4% to 6% and that guggulipid preparations be taken in an amount equivalent to 25 mg guggulsterones three times a day.28 Although concern has been expressed regarding use of synthetic products standardized for up to 99% guggulsterone content,29 recommended doses are commonly based on doses used in available clinical trials. The composition of GGE formulations can vary between manufacturers and among lot preparations from an individual manufacturer.
Multiple human clinical trials have investigated the purported hypolipidemic effects of GGEs, with inconsistent results. Initial trials conducted in India using GGEs have demonstrated decreased total cholesterol, triglycerides, and low-density lipoproteins (LDL), and increased high-density lipoproteins (HDL) in hyperlipidemic cohorts.30-35 Many of these studies were not randomized, did not have a placebo control, contained a limited number of individuals, and did not control for variations in patient diet or exclude those taking additional medications to lower serum cholesterol or lipid concentrations. A well-designed trial in 61 individuals was conducted in which subjects were given 50 mg of standardized GGE containing 25 mg of guggulsterones twice a day for 24 weeks, in combination with dietary interventions that were low in cholesterol and saturated fat and high in soluble fiber.12 Consumption of the GGE was associated with decreased cholesterol (12%), LDL (12.5%), triglycerides (12%), and total cholesterol/HDL ratio (11.1%). Following a 12-week wash-out period, however, blood lipoprotein concentrations were significantly increased in individuals that had been taking GGE compared to controls. A short-term study of gum guggul efficacy was conducted in a Western population of healthy adults in which standardized GGE at doses of 3,000 mg (75 mg guggulsterones) or 6,000 mg (150 mg guggulsterones) was administered daily for 8 weeks. In contrast to previous beneficial findings, consumption of GGE was associated with increased LDL-cholesterol in low- and high-dose groups of 4% and 5%, respectively; reduced HDL-cholesterol concentrations; and no change in other lipoprotein measurements.36 Stimulation of thyroid function has been reported in animal studies, but not observed in human clinical studies.37
Regulatory Status
Dietary supplements are classified by the U.S. Food and Drug Administration (FDA) as products containing vitamins, minerals, amino acids, other dietary/nutritional substances, or herbs and other botanical species that are intended to supplement the diet.38 Products containing gum guggul oleoresin, extract, or its constituents, are marketed as dietary supplements and are therefore regulated under the Dietary Supplement Health and Education Act of 1994. The FDA must monitor reports of adverse effects associated with dietary supplement use and prove a product is unsafe to restrict its use or remove it from the market. Because products in this class are not regulated as drugs, manufacturers must adhere to 21 U.S.C. 343(r)(6), Section 403(r)(6) of the Federal Food, Drug, and Cosmetic Act and not “claim to diagnose, mitigate, treat, cure, or prevent a specific disease or class of diseases.” Products may also be removed from market if the label is false or misleading, for example, if nutrient content information is false or misleading.
Absorption, Distribution, Metabolism, Excretion, and Toxicokinetics
GGEs are complex mixtures of numerous chemical constituents, which has hindered the ability to fully characterize their pharmacokinetic profiles. Studies of absorption, distribution, metabolism, and excretion (ADME) and toxicokinetic profiles of GGE in animal models appear limited to profiles of Z- and E-guggulsterones. Review of the available literature revealed no adequate pharmacokinetic evaluations of GGE in humans.
Experimental Animals
Comparative pharmacokinetic profiles of Z-guggulsterone have been generated following a single oral administration (50 mg/kg) or intravenous administration (18 mg/kg) in male Sprague Dawley rats.39 Z-guggulsterone displayed partial isomerization to E-guggulsterone in rat serum samples. Absolute bioavailability of Z-guggulsterone following oral administration was 42.9%, suggesting significant first pass metabolism. Cmax (maximum serum concentration) values of 1.07 μg/mL and 0.97 μg/mL, terminal half-life values of 4.48 hours and 3.56 hours, and area under the curve (AUC) values of 5.95 μg/mL and 4.75 μg/mL were estimated for Z- and E-guggulsterones, respectively.39 Further studies have investigated the pharmacokinetic behavior following oral (30 mg/kg) and intravenous (10 mg/mL) administration of a (1:1) racemic mixture of Z- and E-guggulsterones, which better mimics the racemic mixture found in GGEs.40 The pharmacokinetic properties of Z- and E-isomers showed high clearance relative to the hepatic plasma flow rate and short elimination half-lives of 0.63 ± 0.25 hours and 0.74 ± 0.35 hours, respectively. The observed in vivo clearance and low metabolic stability of guggulsterones following incubation with rat hepatic microsomes suggest increased metabolic lability and susceptibility to pre-systemic hepatic elimination. Pharmacokinetic parameters such as AUC, volume of distribution (Vd), clearance, and half-life displayed no evidence of stereoselectivity on co-administration of guggulsterone racemates in rats.40 In contrast, pharmacokinetic parameters displayed stereoselectivity in male New Zealand rabbits following intravenous administration of a 1:1 Z- to E-guggulsterone mixture.41 This suggests distinct pharmacokinetic differences between rats and rabbits.
Humans
The metabolism of GGEs and guggulsterones in humans is not well documented. Yang et al. evaluated the metabolism and regulation of cholesterol signaling by Z-guggulsterone in pooled human hepatic microsomes.42 The authors found that contrary to prior assumptions, Z-guggulsterone and its metabolites did not induce CYP7A1, but significantly increased bile salt excretion pump activity facilitating cholesterol elimination. Metabolism of Z-guggulsterone was associated with increased activity of multiple CYP450 enzymes, with CYP3A4 being the most active.42 Consumption of guggulipid (1 g) decreased the bioavailability of the prescribed medications propranolol and diltiazem in healthy male volunteers.43 This result indicates a potential for adverse botanical-drug interactions that could alter bioavailability, efficacy, or toxicity of prescription drugs when taken concomitantly with products containing GGE.
Toxicity
Experimental Animals
In mice intraperitoneally injected with a standardized GGE, the LD50 (median lethal dose) was 1.6 g/kg (5.12082 mmol/kg). In rats and mice orally administered the same GGE, the LD50 was 1.6 g/kg.44 The observed LD50 of orally administered guggul essential oil in mice was 1.7 g/kg.45
No adverse effects were observed in subchronic and chronic toxicity studies in rats, dogs, or rhesus monkeys administered a standardized GGE containing 2.5–7% Z- and E-guggulsterone (125–500 mg/kg) for 90–180 days.44 No mortality was observed in dogs administered 1 g GGE daily for 3 months.33 Rats administered GGE (250 mg/kg) daily (route unspecified) for 3 months had a mortality rate of 50%, relative to 20% in controls.33
Numerous reports suggest that alteration of thyroid signaling pathways contribute to purported effects on serum lipid and cholesterol concentrations. Increased serum triiodothyronine (T3) and T3/T4 (total thyroxine) ratios were measured in mice administered 0.2 g/kg GGE (approximately 11.5% guggulsterones) orally for 15 days.46 This study also found that GGE significantly decreased hepatic lipid peroxidation. Measurements of thyroid function parameters such as iodine uptake by the thyroid gland, thyroid enzyme activity, thyroid hormone release, and oxygen consumption in various tissue were elevated in albino rats following oral gavage of isolated Z-guggulsterone (0.1 mg/kg).37
Humans
Several mild adverse effects associated with GGE ingestion have been described from post-market surveillance, clinical trials, and several patient case reports. Gastrointestinal distress was among the most common side effects reported, typically described as one or more of the following symptoms: upper gastric fullness, eructation, hiccough, loose stools, and diarrhea. These symptoms have been observed across numerous clinical trials investigating the effects of consuming raw guggul or standardized GGEs.32-34,36 Hypersensitivity skin reactions were noted in a clinical trial, occurring in 15% of participants receiving 50 mg of guggulsterones three times daily and in 3% of participants receiving 25 mg of guggulsterones three times daily.26,36 Symptoms typically presented within 48 hours of beginning the therapy regimen and resolved within 1 week following discontinuation of treatment. Administration of GGE was associated with alteration of hematologic parameters such as inhibition of platelet aggregation and increased fibrinolysis.44,47,48 These biological activities could increase an individual’s risk of bleeding, but such adverse events have not been reported.
Numerous case reports involving gum guggul use were identified in the available literature and the FDA MedWatch database. In one report, a 62-year-old male patient presented with loose stools after consuming 1 g of a standardized GGE three times daily for a week. This patient was also concurrently using a nicotine patch, aspirin, saw palmetto, and lycopene.49 In another case, a 63-year-old woman presented with severe hypertransaminasemia that had occurred following consumption of a red yeast rice extract supplement containing guggulsterols.50 By 2014, 17 cases of adverse effects associated with consumption of gum guggul-containing products had been reported.51 In most cases, subjects had also consumed products that contained additional botanical ingredients associated with adverse outcomes, such as kava or ephedra, or were taking additional medications or dietary supplements in combination with gum guggul-containing products. The multi-ingredient nature of some consumed products or consumption of additional dietary supplements/medications is fundamentally confounding to assignments of causality.23
Developmental and Reproductive Toxicity
Experimental Animals
A limited number of developmental and reproductive toxicity studies were identified in the literature. In a study by Amma et al., administration of a gum guggul emulsion (200 mg/kg) or the acidic GGE fraction (20 mg/kg) to adult female albino rats for 7 days reduced reproductive organ weights (uterus, ovaries, and cervix).52 In the same study, increased concentrations of glycogen and sialic acid were measured in reproductive tissues of gum guggul-exposed rats, suggesting antifertility effects.
Humans
No reports of human developmental and reproductive toxicity associated with gum guggul or guggulsterone consumption were identified in the literature. Gum guggul is used traditionally as an emmenagogue and has been reported in clinical trials to shorten the menstrual cycle and increase menstrual blood flow.27,53 According to the WHO, safety of gum guggul use during pregnancy has not yet been established; therefore, use should be discontinued during pregnancy and lactation.27
Immunotoxicity
Guggulsterone has been used in traditional medicine for its anti-inflammatory effects, and has been reported to inhibit proinflammatory signaling, transcription of nuclear factor-kappa B, and tumor cell proliferation.54 The National Toxicology Program (NTP) performed a 28-day study of a GGE formulation (Lot # G51177/H, approximately 2.45% total guggulsterone content) in female B6C3F1/N mice to evaluate potential effects on the immune system at dose concentrations ranging between 31.25 and 500 mg/kg, using the methods described in Frawley et al.55 At the dose concentrations examined, the GGE formulation had no effect on the antibody formation in the hemolytic plaque assay, sheep red blood cell ELISA, or keyhole limpet hemocyanin ELISA, all indicators of effects on humoral immunity. In addition, no effects were observed in assays used to evaluate cell-mediated and innate immunity. Select data for these studies can be found in Appendix F .
Carcinogenicity
No 2-year carcinogenicity studies of gum guggul, GGEs, or guggulsterones were identified in the available literature.
Genetic Toxicity
Aqueous extracts of the gum guggul oleoresin (40 mg/plate) were not mutagenic in Salmonella/microsome assays using S. typhimurium strains TA98 and TA100. Treatments using a hot aqueous extract of gum guggul oleoresin (40 mg/plate) inhibited aflatoxin B1-mediated mutagenesis in S. typhimurium TA98 and TA100 strains. Intraperitoneal injection of aqueous GGEs at doses 10–40 times purported therapeutic doses produced no chromosomal damage or evidence of micronuclei in the bone marrow of treated TAI mice.56 Numerous reports suggest guggulsterone constituents can impart antimutagenic and antitumorigenic activities.57,58
Study Rationale
GGE was nominated for study by the National Institute of Environmental Health Sciences due to widespread human exposure through increasing dietary supplement use, demonstrated metabolic and hormone-altering effects, and lack of adequate toxicological data. NTP conducted 28-day interim and 3-month studies to evaluate the toxic effects of a GGE formulation administered by oral gavage in male and female Hsd:Sprague Dawley® SD® rats and B6C3F1/N mice. Twenty-eight-day interim studies were included to provide additional information pertaining to the temporality of GGE effects on thyroid hormone homeostasis, serum cholesterol/lipid concentrations, and CYP induction potential, and to detect early-induced histopathological lesions.
- Introduction - NTP Technical Report on the Toxicity Studies of a Gum Guggul Extr...Introduction - NTP Technical Report on the Toxicity Studies of a Gum Guggul Extract Formulation Administered by Gavage to Sprague Dawley (Hsd:Sprague Dawley® SD®) Rats and B6C3F1/N Mice
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