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Synephrine and HCA evaluated for health risks

Phytochemical compounds in sport nutrition: Synephrine and hydroxycitric acid (HCA) as examples for evaluation of possible health risks.

1 Introduction
Nutritional supplements containing phytochemical preparations represent a popular group of products intended for use by professional and amateur sportspeople. The use of such products is based on expectations with respect to promotion of desired effects related to e. g. energy increase, athletic performance, weight loss, muscle growth, hastening of recovery or induction of other physiological or metabolic responses [1].

Highly heterogeneous preparations from many different botanical species are currently being used in sports supplements. For example, so called “pre-workout” supplements which are claimed to stimulate fat-burning metabolism and physical performance when taken shortly before training, often contain multiple phytochemicals/herbals together with natural or synthetic caffeine sources [2].

Many consumers tend to consider such products as risk-free, because they are of the opinion that “natural” equals “safe”.. However, depending on the individual phytochemicals, their doses and the combination of compounds within the supplements, certain phytochemical products may potentially induce unwanted side effects, interfere with medications, or even cause serious health damage. For example, sport supplements containing preparations of Ephedra herb (e.g. Ephedra sinica) were widely used for performance-enhancing by athletes.

However, the safety of food supplements containing Ephedra herbs, their preparations or their alkaloids such as ephedrine and its congeners was questioned by several safety authorities, as cases of severe health damage such as sudden cardiac death or stroke resulting from Ephedra herb use were reported, many of which occurred in young adults using Ephedra herb at the dosages recommended on the product labels [3-5].

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It was considered that caffeine was likely to enhance the cardiovascular and central nervous system effects of ephedrine [6]. As a result, the US Food and Drug Administration (FDA) considered food supplements containing ephedrine alkaloids illegal for marketing [4, 7].

In 2013 the European Food Safety Authority (EFSA) concluded, following the “Guidance on safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements” [8], that Ephedra herb and its preparations containing Ephedra alkaloids used as food supplements were of significant safety concern at the estimated use levels [9].

Unlike drugs, which must be approved by the competent Authorities before they can be marketed, food supplement products do not require pre-market review or approval within the EU. The responsibility for the safety of food supplements in the EU and compliance with food law provisions lies with manufacturers and distributors. In the case of concerns regarding potential public health risks in association with the use of particular products or a type of botanical preparation, the safety assessment is conducted by member states Authorities.

The aim of the present review was to analyse the risks of typical natural compounds used in sports food supplements as well as to discuss the current challenges regarding the risk assessment of such phytochemical compounds. For this purpose, two examples of phytochemicals – synephrine and hydroxycitric acid – were chosen.

The currently available toxicological animal data and relevant human studies on these compounds have been summarized and discussed with regard to possible health risks. To this end, the databases PubMed/Medline and Embase were searched with different strategies to identify relevant publications, the last search update being in November 2016. Numerous search strategies were used, including the phytochemicals of interest (e. g. “synephrine” or “hydroxycitric acid”) in connection with the endpoints of interest e.g. “toxicity”, “adverse effects”, “adverse events” or “safety”. In addition, currently published reviews, evaluations by scientific bodies or health authorities regarding further relevant original studies/publications were checked.

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2 Synephrine
Synephrine, also referred to as p-synephrine, is an alkaloid with adrenergic activity which is naturally found in bitter oranges and other citrus fruits. Synephrine may be used as the active ingredient in sports supplements with the intention to promote weight loss as well as to improve athletic performance and to increase energy. It is usually added to the supplements in form of bitter orange (Citrus aurantium) extract. Synephrine is commonly found in “pre-workout” supplements marketed as having “thermogenic properties”, where it has replaced ephedrine, after the latter was prohibited because of its strong association with serious adverse cardiac and cerebrovascular effects [10]. Synephrine is frequently present in products in combination with caffeine and/or multiple herbal ingredients. Because of its known sympathomimetic properties and especially due to possible adrenergic effects on the cardiovascular system, the use of synephrine in food supplements has raised concern for consumer safety. The toxicologically relevant data for synephrine are summarized below.
2.1 Chemical characteristics, dietary occurrence, exposure, medical use
Synephrine or p-synephrine is chemically known as 1[4-hydroxyphenyl]-2-methyl-aminoethanol (CAS No. 94-07-5, chemical formula C9H13NO2, molar mass 167.21 g/mol). Structurally, synephrine is closely related to ephedrine and to the catecholamines epinephrine and norepinephrine (Fig. 1). The p-synephrine, which is found naturally in citrus fruits and used in supplements in form of bitter orange extracts, should not be confused with m-synephrine (synonyms: phenylephrine, neo-synephrine), which is a sympathomimetic drug used primarily as a decongestant. Synephrine exists in two enantiomeric forms: the l-form or (-)-synephrine, which is found in bitter orange and other citrus fruits, and the d-form or (+)-synephrine, which is usually not found in nature [11]. The biological activity of (-)-synephrine was shown to be roughly twice that of the racemate of (±)-synephrine [11]. Hereinafter, the designations “(-)-synephrine”, “(+)-synephrine“, or “(+)-synephrine” are used, if it is possible to distinguish between the individual enantiomers or the racemate, otherwise the term “synephrine” will be used. In humans, synephrine is found in trace amounts in the adrenal gland and is considered a trace bioamine [10].
Synephrine is present in most Citrus fruits, both in their fruits and juice. The dietary exposure to synephrine occurs primarily via ingestion of citrus fruits and their products (juices, marmalade, etc). The total daily intake of synephrine via conventional food, estimated for the German population under consideration of maximum concentrations of synephrine, amounts to 6.7 mg/day for average consumers and to 25.7 mg/day for high consumers [12]. Estimations for the French population considering the maximum levels in citrus fruits yielded an average synephrine intake of 4.3 mg/day and 17.7 mg/day at the 95th percentile [13].
Synephrine is usually added to food supplements in form of extracts prepared from the fruit rinds of bitter oranges (Citrus aurantium) with 6-10% synephrine content or as purified phytochemical (up to 95% purity) [14, 15]. The different food supplements provide highly variable synephrine daily doses which typically range between 5 and 100 mg.
Synthetic synephrine has been medically used worldwide as a sympathomimetic drug under the names Oxedrine or Sympatol®. It consisted of a racemic mixture of (-)- and (+)-synephrine in the form of tartrate and was used for treatment of hypotensive states at oral doses of 100 to 150 mg three times daily [16]. For the German drug Sympatol®, which is no longer available, hypertension, coronary heart disease, tachycardia and arrhythmia were mentioned as contraindications [17].

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2.2 Kinetics and Metabolism
From the plasma concentration data, it appears that synephrine has a low bioavailability when taken orally. After dosing of ten healthy subjects with 46.9 mg synephrine, the measured Cmax was about

2.85 ng/ml, the Tmax about 75 min and the half-life about 3 h [18]. After ingestion of 21 mg synephrine by adults engaging in moderate physical activity, the measured plasma synephrine levels were below 2 ng/ml [19]. Likewise, a pharmacokinetic study of the pharmaceutical Sympatol® showed that the time to peak plasma concentration for orally taken synephrine was 1 to 2 hours, and the elimination half-life was about 2 hours [20]. Synephrine was shown to be a substrate for monoamine oxidase (MAO) enzymes in rat brain mitochondria preparations, displaying Km and Vmax values of 250 μM and of 32.6 nM/mg protein/30 min, respectively [21].
2.3 Toxicological data from animal studies
Rodent studies involving oral administration of synephrine (whether in form of Citrus aurantium extracts or as purified phytochemical) showed that it has the potential to induce cardiovascular toxicity, ranging from an increase in blood pressure and heart rate to ventricular arrhythmias and death. Published animal studies which have addressed the potential toxicity of synephrine/Citrus aurantium when administrated by gavage are summarized in Table 1.
In mice, acute dosing with Citrus aurantium extract (2.5% synephrine) at doses ranging from 1000 to 5000 mg/kg bw produced a reduction of locomotor activity, whereas administration of purified synephrine at doses of ≥300 mg/kg bw induced a reduction of locomotor activity as well as gasping, salivation, piloerection and exophthalmia [22]. These effects were reversible and resolved within 3-4 hours. The authors suggested that the effects may have been due to adrenergic stimulation [22]. In a subsequent subchronic study by the same group [23] mice were treated daily for 28 days with either a methanolic extract of C. aurantium (7.5% synephrine, 400, 2000, or 4000 mg/kg bw) or purified synephrine (30 or 300 mg/kg bw). No clinical signs of toxicity and no deaths were observed in any of the treatment groups.

In the rat, daily treatment for 15 days with hydroethanolic Citrus aurantium extracts (doses ranging from 2.5 to 20 mg/kg bw and day) standardized to either 4 or 6 % synephrine resulted in a dose- dependent decrease of food consumption and body weight gain, as well as an increased mortality in all treatment groups. Furthermore, rats given the highest extract dosage (20 mg/kg bw per day) developed significant electrocardiogram alterations (ventricular arrhythmias and “enlargement of QRS complex”) from 10 to 15 days of treatment [24].

In a 28-days-gavage study rats were treated daily with two different Citrus aurantium extracts (7.25 or 95.0% synephrine) [25]. Synephrine doses were 10 or 50 mg/kg bw per day from each extract. Additionally, caffeine (25 mg/kg bw per day) was added to these doses, since many food sports supplements also contain caffeine. An increase in blood pressure and heart rate was observed in all synephrine-treated groups, whereas more significant effects were observed with less purified (7.25% synephrine) Citrus aurantium extract, suggesting that other components in the botanical preparation may additionally alter these physiological parameters. The increases in blood pressure and heart rate were more pronounced when caffeine was added [25].

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In a subsequent 28-days-gavage study conducted by the same research group [26] the cardiovascular effects of Citrus aurantium extracts containing synephrine (7.25 or 95.0%) were studied in exercising animals (running on a treadmill for 30 min/day, 3 days/week). Similar to the previous study, the rats were dosed daily with synephrine doses of 10 or 50 mg/kg bw per day from each extract with and without addition of caffeine (25 mg/kg bw). Both tested synephrine doses of both extracts significantly increased systolic and diastolic blood pressure for up to 8 hours after dosing. Addition of caffeine to the extract treatments further increased blood pressure parameters, and these combined effects were significantly different compared to the effects of the caffeine alone. Authors concluded that the combination of synephrine, caffeine and exercise can have significant effects on blood pressure and heart rate [26].

2.5 Human data
Studies on acute effects
Only a few human studies have been published in peer-reviewed journals, in which isolated synephrine administration was assessed for cardiovascular effects. The majority of studies have been conducted with products containing not only synephrine (present as extracts of C. aurantium), but also other ingredients such as caffeine, green tea, ginseng extract, guarana extract, etc. Some of these studies reported that synephrine taken alone as C. aurantium extract [29] or in combination with caffeine [18, 19] acutely increased systolic and diastolic blood pressure and heart rate in otherwise healthy normotensive adults. Other studies reported no such acute effects when comparable synephrine doses were tested in healthy young normotensive individuals [30, 31]. The relevant human studies evaluating acute effects of synephrine ingestion on cardiovascular parameters are briefly summarized in the following:

Ingestion of a single synephrine dose of 54 mg by healthy adult subjects (n=15) led to an increase in systolic blood pressure between 1 and 5 hours after ingestion, and an increase in diastolic pressure between 4 and 5 hours after ingestion compared to the placebo group. The maximum difference in systolic pressure was 7.3 ± 4.6 mmHg, observed 3 hours after ingestion, while the maximum difference in diastolic pressure was 2.6 ± 3.8 mmHg, observed 5 hours after ingestion. Heart rate acceleration was observed 2 to 5 hours after ingestion with a maximum deviation from the control group of 4.2 ± 4.5 beats per minute (bpm), observed 4 hours after ingestion [29]. By contrast, providing a lower dose of synephrine did not seem to significantly affect the cardiovascular parameters. A single dose of 27 mg synephrine (ingested as a standardised 6% extract of C. aurantium) showed no effect on systolic or diastolic blood pressure or on the length of the QT interval in 18 healthy volunteers [32]. Also a single dose of 26 mg synephrine (C. aurantium extract) administered to 22 healthy weight-stable subjects had no effect on blood pressure and heart rate [33].

In another study, conducted in healthy adult subjects (n=10 per group), no significant variations in blood pressure or heart rate were observed up to 75 minutes after ingestion of a single oral dose of 50 mg of synephrine alone or combined with the flavonoids hesperidin and naringin [34]. According to a further study in 18 healthy young adults (proven to be free of cardiovascular disease prior to onset of study), consumption of 49 mg synephrine did not result in any significant changes in electrocardiograms, heart rates, blood pressure, blood chemistries, or blood cell counts at any time point (measurements conducted at baseline, 30, 60, 90 min, 2, 4, 6, and 8 h after ingestion) in either control or synephrine-treated group [30].

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In another study, a combination of a synephrine dose of 5.5 mg (C. aurantium extract) with 239 mg caffeine significantly elevated blood pressure and heart rate in healthy normotensive adults (n=10). In the same study, when the synephrine preparation was ingested alone at a dose of 46.9 mg synephrine, no effect on blood pressure, but a significant increase in heart rate was observed [18]. In a further study by the same group [19], healthy, normal-weight adults (n=10) ingested a single dose of a supplement containing 21 mg synephrine in combination with 304 mg caffeine under resting conditions or 1 h prior to moderately intense exercise. A significant increase in diastolic blood pressure (peak at 3 h) was observed after supplement ingestion vs. placebo. Systolic blood pressure was likewise increased between 2 and 3 h in the supplement group, although the difference was not significant in comparison to placebo. According to another study [35], conducted in 23 healthy subjects, ingestion of a food supplement whose composition included, among others, 13 mg of synephrine and 176 mg caffeine did not result in any quantifiable effect on blood pressure or on heart rate, compared to a placebo group.

In a recently published study, aimed to investigate the metabolic, lipolytic and cardiovascular responses to supplementation with synephrine alone and in combination with caffeine during resistance exercise in 12 young healthy men (20-24 years), no changes in heart rate were reported after ingestion of 100 mg of synephrine alone, but a significant increase was observed when taken together with 100 mg caffeine [36]. The blood pressure changes were not assessed in this study.

Studies involving repeated application
In a double-blinded, placebo-controlled study conducted in healthy subjects (25 per group), 49 mg synephrine per single dose (C. aurantium extract) were given alone or in combination with naringin (approx. 101 mg) and hesperidin (approx. 576 mg) twice daily for 60 days. No clinically significant treatment-related changes in systolic or diastolic blood pressures, blood chemistry or blood cell counts were observed at the end of the study in comparison to baseline measurements [37]. However, acute hemodynamic effects could have been missed in this study, since subjects were evaluated only at baseline, day 30 and day 60. Thus, it is not clear at which time point after the last dosing the
measurements were collected. Moreover, given the large within subject variability regarding heart rate 11 and blood pressure throughout a typical day, it may be difficult to detect changes based on a single time measurement over a several week period. The study authors ́ statement “no adverse events were reported by any of the subjects” is difficult to interpret as the used “quality of life questionnaires” were not designed to detect negative treatment-related symptoms [38].

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Case reports observed in conjunction with use of synephrine-containing supplements.
It is important to mention that a number of published case reports have associated products containing synephrine with various adverse effects, such as myocardial infarction [39, 40], ischemic stroke [41], acute arterial hypertension [42], tachycardia [43], bradycardia [44], syncope and QT prolongation [45], ischemic colitis [46], vasospasm and stroke [47], ventricular fibrillation [48] or apical ballooning cardiomyopathy [49]. Health Canada reported about the collection of a series of cases of serious adverse reactions suspected of being associated with synephrine-containing natural health products [50, 51]. In 2014, the French Agency for Food, Enviromental and Occupational Health (ANSES) compiled about 18 reports of adverse reactions collected from 2009 to 2014, which were mainly related to cardiovascular health and were linked to the consumption of food supplements containing synephrine [52]. In general, the majority of reported cases concern people who took these food supplements in a sports context and/or for weight-loss purposes. Despite the fact that most of the mentioned clinical reports contain insufficient information and causality with respect to synephrine is not easy to prove, since the products in question usually also contained caffeine and/or other herbal preparations, these reports indicate that synephrine in particular in combination with caffeine may potentially cause severe cardiovascular effects.

2.6 Mode of action
The available mechanistic data indicate that effects of synephrine on the cardiovascular system are attributable to adrenergic stimulation. As shown in Fig. 1, synephrine is structurally similar to adrenergic agonists such as adrenaline, noradrenaline and ephedrine. Synephrine is supposed to act at α- and (to a lesser extent) β- adrenergic receptors, resulting in vasoconstriction and increased blood pressure. In vitro studies in human and mammal cells and tissues have reported the activity of synephrine on adrenergic receptors of different classes, though the receptor-binding affinity of synephrine is considered to be within orders of magnitude lower than that of endogenous adrenergic agents as norepinephrine [53].

It has been reported, for example, that synephrine interacts directly with α1-adrenergic receptors and causes contraction of the rat aorta, anococcygeus (a type of smooth muscle in rectum), as well as the guinea pig atria and trachea [54]. Synephrine showed vasoconstrictive effects in rat aorta, and these effects were sensitive to α1-adrenergic receptor antagonists but not to β-adrenergic antagonists [55]. Synephrine was reported to reduce portal pressure and elevate mean arterial pressure in sham- operated and portal hypertensive rats [56].

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Synephrine-containing C. aurantium extracts as well as purified synephrine increased glycogenolysis, glycolysis and perfusion pressure in the isolated perfused rat liver, and these effects were sensitive to several adrenergic antagonists [57]. Synephrine induced hemodynamic and metabolic effects in isolated perfused rat liver, which were strongly inhibited by α-adrenergic antagonists and moderately affected by β-adrenergic antagonists [58]. Moreover, synephrine induced an antidepressant-like activity in mouse models of immobility tests, which have been attributed to stimulation of adrenergic receptors [59]. The claimed effect of synephrine as a weight-loss stimulant is also attributed to adrenergic properties, in this case to the activation of β3-adrenergic receptors and consequent induction of thermogenesis [53].

2.7 Health risks of synephrine: Assessment and discussion
The main health concern about synephrine use in food and sports supplements refers to its potential as a sympathomimetic to induce adrenergic effects on the cardiovascular system. Synephrine displays a proven sympathomimetic activity and was used as an active ingredient in medicinal products. For the German drug Sympatol® which is no longer available on the German market, hypertension, sclerotic vessels, coronary heart disease and tachycardic arrhythmia were mentioned as contraindications [17].
Animal studies involving oral administration clearly showed that supplementation with synephrine (in form of C. aurantium extract or as purified phytochemical) can lead to elevated blood pressure; and that ingestion of synephrine in combination with caffeine can induce considerable cardiovascular effects with additional alteration of heart rate [25, 26].

Some human studies showed that certain synephrine-containing food supplements raised blood pressure in healthy, normotensive adults. Such effects were typically observed between 2 and 4 hours post dosing. Though the results from human studies with oral ingestion of synephrine-containing products were not consistent, it should be taken into account that observed effects of synephrine on the cardiovascular system appear to depend on the dose, time point of measurements and the presence of other additional stimulants, e. g. caffeine. Moreover, for many of the available studies it is unclear to which extent potential bias, due to funding by food supplement industry, may exist. In addition, it should be considered that most of the studies were small and tested generally only healthy and normotensive volunteers. Individuals with underlying cardiovascular diseases, that might be unrecognized, or other conditions as described as contraindications for medical uses of synephrine may be vulnerable subgroups of the population. Another limitation is that human studies investigating the acute effect of synephrine on blood pressure were done at rest, thus effects the stimulant ingredients may have had under strenuous physical exercise conditions (i. e. in states with highly increased heart rate and blood pressure) would not have been seen.

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A role of supplements containing synephrine and caffeine has been implicated in many serious adverse events documented in case reports. Caffeine is a central nervous system stimulant and increases systolic blood pressure through interaction with adenosine receptors [60]. It is also known to enhance the effects of other sympathomimetics such as ephedrine [61]. Though these case reports usually do not demonstrate causation or association, considering the repeated co-occurrences, the nature of the observed adverse reactions together with available evidence from animal studies, they amplify the potential safety concerns regarding the use of synephrine-containing supplements.

In its Scientific Opinion on the application of the Qualified Presumption of Safety approach for the safety assessment of botanicals and botanical preparations [62] EFSA regarded synephrine as a principal substance of concern and concluded that: “…the use of extracts of Citrus aurantium in food supplements would have to be restricted to levels where no significant increase of synephrine exposure compared to historical intake levels with traditional foods is to be expected“.

The German Federal Institute for Risk Assessment (BfR) recently issued a risk assessment of sports and weight loss products containing synephrine and caffeine [12], noting that the available toxicity data were not sufficient to allow the derivation of a safe intake level. Since synephrine is naturally present in the human diet (see Section 2.1), the BfR used the “presumption of safety” approach in accordance with the EFSA-guidance document for the safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements to propose a safe intake level of synephrine from food supplements[8]. The BfR concluded that the quantities of synephrine provided by the food supplements should not exceed 6.7 mg/day, which corresponds to the median value of dietary synephrine intake observed from conventional foods [12].

The French food safety authority ANSES in its assessment of synephrine from 2014 [13] concluded that intake levels of synephrine through food supplements must remain below 20 mg/day and recommended not taking synephrine in combination with caffeine. It also recommended to avoid the use of products containing synephrine during physical exercise and discouraged its use by sensitive individuals (people taking certain medications, pregnant or breastfeeding women, children and adolescents)”.

3 Hydroxycitric Acid (HCA)
Hydroxycitric acid (HCA) is a fruit acid naturally occurring in fruits of the tropical plant Garcinia cambogia. HCA is used as the active ingredient in a variety of food supplements intended for weight loss. In sports supplements HCA or Garcinia cambogia extract is often a component of so called “fat burner” mixtures. The phytochemical is claimed to suppress the appetite, inhibit the synthesis of lipids, or burn fat via thermogenesis.

The use of HCA as a slimming supplement is mainly based on its ability to inhibit the ATP citrate lyase, the enzyme which plays an important role in fatty acid synthesis [63-65]. HCA is generally added to the supplements in form of Garcinia cambogia extracts. However, the composition of such commercially available extracts is often not clearly specified. Health concerns regarding safety of the HCA-containing supplements have been raised, based on results from animal studies which have suggested that high doses of certain Garcinia cambogia extracts or of HCA itself may be toxic to the testis. The relevant toxicological data on HCA as food supplement are summarized below.

3.1 Chemical characteristics, dietary occurrence and exposure
HCA is chemically known as 1,2-dihydroxypropane-1,2,3-tricarboxylic acid (CAS No. 27750-10-3, chemical formula: C6H8O8, molar mass: 208.1 g/mol). Due to the presence of two chiral centers in the molecule HCA may exist in form of four stereoisomers: (-)-HCA (2S, 3S), (+)-HCA (2R, 3R), (-)- allo-HCA (2S, 3R) or (+)-allo-HCA (2R, 3S). Only the (-)-HCA stereoisomer is known to be naturally present in fruit rinds of Garcinia species (G. cambogia, G. indica, G. atroviridis) [63, 64, 66]. Commercially available HCA products are usually prepared by water or methanol extraction from the fruit rind of Garcinia cambogia – a small- or medium-sized tree, native to Southeast Asia. In the dried pericarp of Garcinia cambogia fruits HCA is present at levels of up to 30% by weight. In aqueous solution, the free HCA is unstable and is converted to its more stable lactone form (see Fig. 2). In food supplements the salts of HCA with sodium, calcium-potassium or magnesium are usually used, because this modification increases the stability of the acid and prevents it from being converted into its lactone form, which is thought to be less biologically active [64, 66-68].

In some countries specifications for Garcinia cambogia preparations are published [69]. However, within the worldwide market the specification of HCA preparations/Garcinia cambogia extracts used in individual products is often unclear and in addition, available specifications leave significant portions of HCA- preparations/Garcinia cambogia extracts unaccounted for. Different HCA products are available, which contain varied quantities of HCA (usually 50% to 60%) and are generally marketed as HCA or as a “Garcinica cambogia extract” [63, 70]. Detailed information concerning specifications is available only for individual products, such as for example a calcium-potassium HCA double salt with a HCA content of 60 % (Ca/K-60 % HCA-salt) and, among other specified constituents, 16 % potassium and 11 % calcium, prepared from the Garcinica cambogia fruit rinds [68].

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Garcinia cambogia fruit rind is used in Asian countries for culinary purposes, primarily as a condiment or flavoring agent [70, 71]. There is presently no reliable information available concerning the daily intake of HCA via conventional food in these countries. It can be assumed that consumer exposure to HCA in Germany occurs primarily through its use as a food supplement. The daily dosage of HCA recommended according to product labelling ranges in different commercially available products between 500 and 3000 mg [63].

3.2 Kinetics and Metabolism
From the plasma concentration data, it appears that HCA has a limited bioavailability in humans when taken orally. After ingestion of 2 g of calcium-potassium-HCA salt with a HCA content of 60 % (Ca/K-60 % HCA-salt) (corresponding to 1200 mg HCA) by healthy adult volunteers (n=4) the measured peak plasma HCA concentrations ranged between 4.7 and 8.4 μg/ml (reached 90-120 min after dosing), suggesting a limited efficiency of HCA absorption. According to the author’s estimations, in case of complete absorption the plasma HCA concentrations should have reached approximately 46 μg/ml [72]. Calcium and magnesium salts of HCA are slightly soluble in aqueous media and are supposed to be poorly absorbed in the gastrointestinal tract [73].
3.3 Toxicological data from animal studies
HCA showed relatively low acute toxicity in rodents. The determined oral LD50 of a calcium- potassium-HCA-salt with a HCA-content of 60% HCA (Ca/K-60 % HCA-salt) in rats was higher than 5000 mg/kg, which corresponds to 3000 mg/kg HCA [71]. Several repeated-dose studies (28 days and more) reported that high doses of HCA preparations induced testicular toxicity when administered orally to rats. However, in other animal studies these effects were not observed. Studies which addressed the question of potential testicular toxicity of HCA/Garcinia cambogia preparations are summarized in Table 2.
In a subchronic toxicity study, an extract from Garcinia cambogia containing 41.2 % HCA (63.4% of which was present in the lactone form) was tested in developing male Zucker obese rats [74]. The animals were fed for 92 or 93 days with diets containing different HCA levels of 0, 10, 51, 102 and 154 mmol/kg diets, which corresponded to an average intake of HCA doses of 0, 78, 389, 788 and 1244 mg/kg bw. Each diet group was pair-fed to the highest HCA group (154 mmol/kg diet).

Testicular atrophy and toxicity were observed in animals of the highest and second highest HCA 18 groups (778 and 1244 mg HCA/kg bw and day), whereas no significant treatment-induced effects were detected in other organs. A dose-dependent atrophy and degeneration of germ cells was seen in histopathological examination of the testes in the two highest dose HCA groups. Furthermore, significant plasma hormone changes indicating disturbed spermatogenesis (decrease in levels of Inhibin B, a hormone which is produced exclusively by the testis; increase in the follicle-stimulating hormone- (FSH-) plasma concentrations) were observed in these two HCA groups. No changes were observed in plasma concentrations of testosterone and LH in any of the treatment groups.

Additionally, severe diarrhea was observed in animals fed the highest HCA dose. The authors of this study identified a no-observed-adverse-effect-level (NOAEL) of 51 mmol HCA per kg diet, which corresponded to 389 mg HCA/kg bw and day [74]. A lowest-observed-effect-level (LOAEL) was 102 mmol HCA per kg diet, which was equivalent to 778 mg HCA/kg bw and day, respectively.
Comparable effects on the testes were also observed in another rat strain, when the same HCA- containing product (Garcinia cambogia extract containing 41.2 % HCA) was tested in male Fischer 344 rats [75]. Reduced testis weights, degeneration of the germ cells in testis and impaired spermatogenesis were observed in animals ingesting a Garcinia cambogia extract containing a HCA dose of 102 mmol/kg diet (the lowest dose which induced adverse effects in the previous study [74]) for 4 weeks.

Similar to the previous study [74], significant changes in serum hormone levels related to spermatogenesis (decrease in inhibin B and increase of FSH concentrations) were reported in HCA- treated animals compared to the control group. When the same Garcinia cambogia extract (HCA content 41.2 %) was tested in female rats, no changes in sex hormones (FSH, luteinizing hormone, estradiol and progesterone) and no morphological signs of toxicity in the follicle and corpus luteum were observed after feeding the animals a diet containing 154 mmol HCA/kg diet (a dose which induced marked testicular toxicity in the subchronic toxicity study [74]) for a period of 28 days [76].

Another animal study, which is only available as a conference abstract [77], aimed to clarify whether HCA itself or other ingredients of Garcinia cambogia extract caused testicular toxicity. Fischer 344 rats were fed diets containing 0, 0.13, 0.66 and 3.31 % of HCA-calcium-salt (99.5% purity) or 5% Garcinia extract powder containing 66.2% HCA (dose equivalent to 3.31% HCA-calcium-salt) for 28 days. A decrease in epididymal weights, an increase in cell debris in epididymal ducts, a significant decrease of spermatocytes as well as degenerative changes of Sertoli cells and elongated spermatids were reported in rats fed 3.31 % of HCA-calcium-salt and 5% Garcinia extract powder diets. Since the observed effects were almost identical in both former groups with respect to quality and extent of effects, the authors concluded that the observed testicular toxicity might be attributable to HCA as the principle ingredient of Garcinia cambogia preparations [77].

A more recent study, which is only available as a scientific correspondence [78], reported that no histopathological changes were found in testes of adult male rats after dosing with 212-1063 mg/kg bw/day of HCA lactone (98% purity) or equimolar concentrations of HCA (300-1500 mg/kg bw/day; unknown purity) for 8 weeks. No detailed information is available concerning the study findings.
When the Ca/K-60 % HCA-salt was tested in a 90-day subchronic toxicity study in rats, no signs of testicular toxicity were reported [79, 80]. Sprague-Dawley rats received the test product by gavage at dose levels of 0.2, 2.0, and 5.0% of feed intake, which was approximately equivalent to HCA doses of 60, 600, and 1500 mg/kg and day, respectively [68].

After 90 days of administration a significant reduction of food intake (26% in males and 23% in females receiving the highest HCA dose) as well as body weight (16% in males and 13% in females of the highest HCA dose group) was found in HCA-treated animals. No significant effects on organ weights, hepatic DNA fragmentation and lipid peroxidation, no hematological or biochemical alterations and no significant histopathological changes in the different investigated organs (including kidney, liver, prostate and testes) were reported in HCA-treated animals. Notably, testicular atrophy and aspermatogenesis were seen in one animal, but unfortunately no information was provided as to which group this animal belonged to (i.e. control or HCA group, or affected HCA-dose group, respectively). Overall the study authors concluded that the Ca/K-60 % HCA-salt did not cause any changes in major organs or in hematology, clinical chemistry, and histopathology. [79].

The authors identified a NOAEL for the tested HCA-preparation of 2500 mg Ca/K-60 % HCA-salt/kg bw and day, which corresponds to 1500 mg HCA/kg bw and day [68, 79, 80].
The same HCA product (Ca/K-60 % HCA-salt) was further tested in a two-generation reproduction toxicity study in Sprague-Dawley rats [81]. Male and female animals were fed a diet containing 0, 1000, 3000, or 10000 ppm of the HCA-preparation for a period of 10 weeks prior to mating, during mating, and across two generations. No treatment-related adverse effects on reproductive performance in terms of fertility and mating, gestation, parturition, litter properties, lactation, sexual maturity, and development of offspring were observed during HCA-exposure of male and female rats of the F0 and F1 generations. No treatment-related changes in sperm quality parameters (sperm count and sperm motility), no changes in male fertility indices and no histopathological alterations in testes were observed in male rats of the F0 and F1 generations. The authors identified a NOAEL for the tested Ca/K-60 % HCA-salt of 10000 ppm in feed, which was equivalent to a HCA intake of 610.8 and 914.4 mg/kg bw and day, in male and female rats, respectively [81].

Following this two-generation study, a developmental toxicity study with male and female rats of the F2 generation was performed [82]. Animals received the same dietary exposure levels of HCA as those employed for the two-generation study (0, 1000, 3000 and 10000 ppm of the Ca/K- 60 % HCA- salt) until mating, and the female animals up to the 20 day of pregnancy. No evidence of maternal toxicity and no external, soft tissue or skeletal abnormalities in the fetuses were observed on the 20th day of gestation. At the highest HCA dose group a 13% lowering of maternal body weight gain, a non-significant reduction of corpora lutea numbers, a non-significant increase of early resorption numbers as well as a significant reduction of mean litter size were observed. The authors concluded that the tested HCA product was not found to be teratogenic at any of the dietary dose levels tested and identified a NOAEL of 10000 ppm, equivalent to a Ca/K-60 % HCA-salt dose of 1249 mg/kg bw/day, corresponding to a HCA dose of 744 mg/kg bw/day [82].

3.4 Information on genotoxic effects
No genotoxic activity was observed for the Ca/K-60 % HCA-salt when tested in an Ames bacterial reverse mutation assay at concentrations up to 25000 μg per plate or in a chromosomal aberration test in CHO cells at concentrations up to 125000 μM/ml [68, 83]. In a mouse micronucleus assay, the same HCA preparation (Ca/K-60 % HCA-salt) caused a significant increase in the number of micronucleated polychromatic erythrocytes at the dose of 12500 μmol/kg (i. p. application) [83]. However, there are several methodological questions to the validity of the latter study (the use of DMSO as a solvent in test groups, no clear dose-dependency of observed effect, etc.) which complicates its interpretation [84].
No genotoxic activity was observed for a “Ca-type” Garcinia cambogia extract (65% HCA content) when tested in a chromosomal aberrations test in Chinese hamster lung cells (up to 4000 μg/ml) or in an in vivo micronucleus induction test in male Slc mice (up to 2000 mg/kg) [85]. Taken together, the available data indicate that HCA is unlikely to have a mutagenic potential.

3.5 Human data
Clinical studies
The majority of the human studies with HCA-preparations published so far were conducted with slightly to moderately overweight persons to examine the claimed effects of HCA on weight loss, promotion of appetite suppression and other beneficial effects on body composition (reduction of body fat, improvement of plasma cholesterol or fat oxidation parameters, etc.) and were not designed to detect possible adverse effects of tested HCA products (studies are reviewed elsewhere [63, 86, 87]). Table 3 summarizes human studies involving HCA ingestion that are relevant for the risk assessment and which provide detailed information on observed adverse effects and/or safety-relevant clinical and laboratory parameters. In general, no severe or serious adverse effects were reported in any of these studies, and the observed symptoms were mild, infrequent and included headache, nausea, diarrhea, and other gastrointestinal symptoms (see Table 3).
A HCA dose of 3000 mg/day (in form of Garcinia cambogia extract containing 60% HCA, no further specifications) was administered to 10 healthy male volunteers (26 – 56 years) for 30 days [88]. No treatment-relevant changes were observed in hematological and biochemical blood parameters. No changes in serum testosterone levels were observed in comparison to the initial values and no serious adverse effects were reported. In a further study with the same Garcinia cambogia extract, a HCA dose of 3000 mg/day was ingested by 48 healthy subjects (24 female, 24 male, normal weights) for a period of 12 weeks with [89]. This study included a 2-week “run-in” placebo treatment period.

No treatment-related changes in hematology, blood biochemistry or urinalysis were detected and no serious adverse effects were observed. To address the question of possible effects of the tested HCA- preparation on male testicular function, the levels of serum inhibin B and FSH (markers of spermatogenesis) were investigated in male test subjects. Slight increases of serum inhibin B and FSH levels (in comparison to those at week 0) observed at week 12 were interpreted by the study authors as being of no physiological significance [89]. No significant differences were observed for serum LH and testosterone levels in male as well as for serum FSH, LH, and progesterone in female test subjects. The identical Garcinia cambogia extract was further tested in a randomized, placebo- controlled, double-blind study on 18 overweight volunteers (placebo group n=21) for 12 weeks with subsequent 4 weeks follow-up [90, 91]. No significant alteration of serum testosterone, estrone, and estradiol levels were observed in the treatment group when compared to the placebo group. Similarly, hematology and serum clinical pathology parameters did not reveal any significant adverse effects.

Another randomized, placebo-controlled, double-blind study tested a dose of 1200 mg HCA (given as Garcinia cambogia extract containing 60% HCA, no further specifications) in 29 overweight volunteers (placebo group n=29) over 10 weeks [92]. Analysis of blood biochemistry revealed no treatment-related changes in plasma liver enzymes (ALT, AST), erythrocyte antioxidant status or plasma adipocytokines. Two 8-week randomized, double-blind, placebo-controlled trials were conducted in overweight subjects under calorie-restricted diet (< 2000 kcal per day) that received the Ca/K-60 % HCA-salt with a HCA dose of 2800 mg per day [93, 94]. The observed adverse effects are presented in a summarizing publication [95]. An increased incidence of headache was observed in the group receiving HCA in comparison to the placebo group, whereas other mild symptoms were comparable between the groups (see Table 2). Notably, a statistically significant increase in serum serotonin levels was observed under HCA treatment compared to the placebo group [95]. Case reports observed in conjunction with use of HCA-containing supplements

A series of recently published cases of (hypo-)mania (typical symptoms include irritability, aggressive behaviour, decreased sleep and delusions) were reported in association with the use of HCA- containing supplements [96-98]. In each case, the affected individuals developed manic symptoms while taking a supplement containing HCA in form of Garcinia cambogia extract over several weeks prior to onset of symptoms. According to these reports, two persons had no history of psychiatric illness [96, 98] whereas three other individuals [96, 97] had a diagnosis of bipolar disorder, but were stable (symptom-free) without or under treatment with mood stabilizers or neuroleptics until they started supplement ingestion. Though, due to the multifactorial mechanisms of mania and in some
cases concomitant use of prescribed drugs, it was not possible to establish HCA as causative principle
in most of these cases, it has been discussed that HCA may trigger mania in predisposed individuals due to its known serotonergic activity [96, 97]. In one case [97] the causal association between consumption of the Garcinia cambogia supplement and occurrence of hypomania was evaluated as probable/likely, as an improvement of manic symptoms was seen when the HCA-containing supplement was withdrawn.
In another recent report [99], a patient experienced two episodes of serotonin syndrome, each time after having taken a HCA-containing product (Garcinia cambogia extract with 60% HCA, 1000 mg daily) in addition to a prescribed antidepressant drug. The authors suggested that combining HCA- containing supplements with serotonergic drugs might increase the risk of serotonergic side effects.

3.6 Mode of action
Serotonin increase in brain due to ingestion of HCA was supposed as a possible mechanism for adverse psychiatric effects. Notably, HCA was suggested to influence appetite by promoting release and synaptic availability of serotonin [64]. Indeed, HCA was shown in vitro to induce increased serotonin release from the isolated rat brain cortex [100]. Elevated serotonin levels were reported in the brain tissues of male and female rats ingesting HCA [67]. Furthermore, a significant increase in serum serotonin levels was observed under HCA treatment in human clinical trials [95].

Possible mechanisms underlying testicular toxicity of HCA-preparation were addressed in one study, which investigated effects of zinc supplementation (0.01% and 0.05% in feed) on testicular toxicity of “Ca-type” Garcinia extract in F344 male rats (n=10) [101]. While a slight atrophy of spermatids and decreased inhibin-B plasma levels were observed in animals fed a diet with 5% extract, no signs of testicular toxicity were observed when 0.05% zinc was simultaneously supplemented to the extract- treated animals. The authors concluded, that zinc deficiency in diet or zinc depletion may be a possible mechanism of testicular toxicity of Garcinia cambogia extract [101].

3.7 Health risks of HCA: Assessment and discussion
Based on the available toxicological data, a main health concern associated with use of HCA preparations in food and sports supplements refers to male reproductive endpoints. However, rodent studies involving oral administration of various HCA preparations at high doses reported heterogeneous results (see Table 3).
At least three different HCA products (Garcinia cambogia extracts with 41% or 66% HCA and a preparation of a calcium salt of HCA) caused testicular atrophy and toxicity in male rats. The testicular toxicity included atrophy of seminiferous tubules, degeneration of germ cells, degeneration of Sertoli cells and impaired spermatogenesis.

These effects were reproducible and were observed in two different rat strains [74, 75, 77]. From animal data, obtained by testing the Garcinia cambogia preparation containing 41.2% HCA (no further specifications) a LOAEL of 778 mg HCA/kg/day and a NOAEL of 389 mg HCA/kg/day were identified. On the other hand, no testicular adverse effects were reported in animal studies testing another HCA preparation comprising a Ca/K- 60 % HCA-salt.

With this preparation, a NOAEL of 610.8 mg HCA/kg/day was identified for male rats in a reproduction toxicity study, which was the highest HCA dose tested [81]. In addition, in a 90-days toxicity study testing the Ca/K-60 % HCA- salt in rats, a NOAEL of 1500 mg HCA/kg/day was identified [79, 80]. It should be noted however, that in this study testicular atrophy and aspermatogenesis were reported in one animal (no information provided as to which group this animal belonged to; i.e. control or HCA group, or affected HCA-dose group, respectively) [79].

Taken together, results of the animal studies are not uniform and partly contradictory, and may depend on the Garcinia cambogia product or HCA preparation tested. Currently, it needs to be further elucidated to which extent observed adverse effects on the male reproductive system are to be ascribed to specific Garcinia cambogia extracts and constituents thereof or to HCA itself. However, adverse effects on the male reproductive system observed with high doses of certain Garcinia cambogia extracts/HCA preparations should be taken seriously and adequately addressed in risk assessment of such products.

Although the results obtained from several animal tests suggest serious adverse effects on the male gonadal system at high doses of certain Garcinia cambogia extracts/HCA preparations, the question of possible effects on human male reproduction associated with long-term oral ingestion of HCA- containing supplements has been addressed solely in one human study involving 24 male subjects. In this study [89], no physiologically relevant changes in plasma spermatogenesis markers (plasma levels of inhibin B and FSH) were detected following 12-weeks administration of 3000 mg HCA per day.

However, no determinations of classical markers of spermatogenesis, such as sperm concentration, total sperm count and morphology, were performed in male subjects following ingestion of HCA supplements. Thus, human data is currently insufficient to conclude on the safety of HCA with regard to the human male reproductive system. In addition, although no serious adverse effects were observed in clinical trials testing different HCA preparations at the daily doses up to 3000 mg HCA, no human data is available on the health effects of HCA ingestion over a period exceeding 12 weeks (see Table 3).

Garcinia cambogia is known to be used in India and Southeast Asia as a condiment. However, the “presumption of safety” approach as proposed by EFSA could not be applied for the derivation of the acceptable intake levels of HCA from food supplements, since adequate data on HCA intakes via traditional/conventional food that are not associated with adverse effects is lacking. Hence, the use of NOAEL values determined from animal toxicity studies as the “point of departure” is the only possible alternative for quantitative assessment/estimation of safe HCA levels in food supplements. In this case, the comparability of the given HCA preparation with that tested in animal studies would be of importance for choosing the appropriate NOAEL.

For HCA preparations that have not been further specified, rather the lowest determined NOAEL of 389 mg HCA/kg/day as a “worst-case scenario” should be applied. (For other HCA-preparations for which adequate animal data is available, other NOAELs may be applicable.) The use of daily HCA doses of 1000-3000 mg, which are commonly present in sports food supplements, corresponds to the ingestion of approx. 14-42 mg HCA/kg bw/day for a 70-kg person, resulting in intake levels that are only by a factor of 9 to 27 lower than the lowest NOAEL of 389 mg HCA/kg/day from animal studies.

It may be questioned whether such a margin to the NOAEL of only up to 27 is sufficient to ensure human safety, in view of the lack of adequate human data on the safety of HCA-preparations, particularly with respect to the human male reproductive system. It is noted that, in the context of risk assessment of chemicals, a safety factor of 100 is usually applied to derive human health-based guidance values from NOAELs observed in animal studies, to account for interspecies differences and variability in humans.

In conclusion, knowledge gaps and substantial uncertainties exist regarding the safety of HCA preparations found in commercially available food supplements, particularly with regard to the human male reproductive system. Considering the serious adverse effects on the testes observed in several animal studies with high doses of certain Garcinia cambogia extracts/HCA-preparations as well as in view of lack of the adequate human data on the safety of the long-term use of HCA-preparations, there are open questions regarding toxic effects on the male reproductive system of HCA-containing food supplements. In addition, in view of the recently published cases of mania/hypomania suspected of having been induced by the use of the supplements containing HCA in predisposed individuals or patients taking certain antidepressants, further research is needed to clarify the potential psychiatric effects of HCA- or Garcinia cambogia-preparations.

Concluding remarks
Many botanical supplements contain active ingredients that have the potential to elicit effects in the body, including unwanted or adverse effects. Thus, depending on the substances involved, their dose and possible combinations of substances within the product, such supplements may pose a risk to human health under certain conditions. Regarding products that contain phytochemical ingredients that possess sympathomimetic activity, severe health risks have been described for Ephedra herb- containing food supplements. There is concern that sports food supplements containing other sympathomimetics, such as synephrine, may also pose cardiovascular health risks, particularly when such phytochemicals are ingested in combination with caffeine. In the case of Garcinia cambogia extracts and HCA preparations, current safety concerns regarding possible testicular toxicity exist, which are based on toxicological data obtained for certain HCA-preparations in animals. Furthermore, as recent human psychiatric case reports suggest, use of HCA may pose a potential health risk to patients with bipolar disorder or those taking certain antidepressants.

A number of problems may be encountered in the context of risk assessment for supplements containing phytochemical preparations. Relevant information regarding the used botanical preparation(s), including the botanical origin, extraction procedure and the chemical composition is often not provided. Botanical preparations may contain a number of different biologically active substances which are often not sufficiently toxicologically characterized, neither individually nor with respect to possible combined effects and interactions. In addition, many products contain a number of botanical preparations of different species. For products containing mixtures of plant extracts, risks due to combined effects are difficult to assess.

Available toxicological data on the active ingredients or on the whole product are often scarce, and in many cases do not provide sufficient information on toxicokinetics or on relevant toxicological endpoints. In addition, studies, if available, often do not meet the requirements of existing guidelines (e.g. OECD test guidelines). Human studies involving such preparations often have shortcomings with respect to the number of participants and duration of the study and/or reporting of safety related data. Although case reports may provide indications for adverse effects due to consumption of supplements containing phytochemicals, the causality between the use of the product and the adverse effects is often difficult to establish, even though scoring systems are in place (e.g. [102]).

It should also be considered, that adverse effects resulting from use of botanical supplements may be under-reported [103]. Although implementation of post-marketing surveillance or nutrivigilance systems might be expected to contribute to improvement of risk assessments, a formal post-marketing surveillance system for food supplements is currently not in place in many countries.

Finally, in view of the present lack of data for risk assessment for many phytochemicals or botanical preparations used in food supplements, the importance of comprehensive systematic safety testing, preferably prior to product marketing, is emphasised, to improve the basis for risk assessment, and thus to ensure a high level of consumer protection.

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