Some Humifulvate® Science - Chapter 3: Biological Activity

Recently, a wide range of products containing Humifulvate as their active ingredient has become available. The classic product formula is the syrup (the HUMET-® syrup) supplemented with trace elements, also known as the Humifulvate Concentrate (HFC). Because of the sensitivity of the active ingredient, isolating it in a solid form posed substantial difficulties, and it took years of development to produce the solid product form. The solidified Humifulvate is designated as HF powder. More recently, producing a solid product that fully corresponds to the liquid HFC has also become possible, and it is going to be referred to as Enriched Humifulvate (EHF) hereafter.

References to the individual substances will be made hereafter in accordance with the nomenclature given below:

HUMET-® syrup = HFC: stabilised Humifulvate suspension supplemented with trace elements.

EHF = solid HFC without stabilising agent: enriched Humifulvate powder.

HF = Humifulvate powder.

As mentioned above the classic formula had been originally the HFC. In order to be able to generalise research data pertaining to this substance to apply also to the powder formulas, the two presentation forms of the products were subjected to a thorough comparison. It has been concluded that syrup made from EHF by adding water and stabilising agent to it would be identical in all physical and chemical parameters with the original suspension product (HFC).

Definition and origin

Humifulvate is a standardised peat-derived humic acid, fulvic acid and phenolic acid complex intended for oral consumption. Humifulvate is the base compound to be used in combination with minerals and trace elements as a dietary supplement. Since 1993, the standardised liquid concentrate (HFC) has been sold in numerous European countries as an OTC product called Humet®-R.

The Hungarian Humifulvate to be found in this product has been derived from geologically young Hungarian peat, estimated to be 3,000-10,000 years old [1]. The composition of this substance may be due to the botanical composition of the deposit growing at the time. This unique Humifulvate-rich material is derived from a specific type of peat contained within a square mile found only at a site near the northern embankment of Lake Balaton in Hungary. The peat deposit subjected to scientific research shows uniform geological and chemical characteristics. As it is located in a natural conservation area, no industrial or agricultural activities are performed in its environment.

It is known that peat is a highly organic deposit, or in other words, the accumulation of plants and vegetable matter that have humified over a period of thousands of years. Due to differences in the source and nature of the surrounding aqueous environment, peat varies in its botanical origin, extent of humification, and present flora [6]. However, its increasing degradation leads to the progressive evolution of humus first, then humic acids, and finally fulvic acids [2]. Peat is therefore a storehouse of nutrients, and as such, can be exploited to produce high quality vegetable crops [6].

Humic and fulvic acids are complex organic molecules, which comprise some 60-80% of peat and soil organic matter. The bacterial and chemical degradation of lignins (substances deposited in cell membranes that help give the plant support and rigidity) and other structural carbohydrates in plants are responsible for forming the intermediate products of humic and fulvic acids. [12, 13] These intermediate products are then polymerised in the presence of polyphenols, which are leached by rain, from the leaves and other plant components. Polyphenols are plant metabolites. They can be oxidised to quinones either spontaneously in the presence of molecular oxygen or enzymatically, mediated by a wide variety of microorganisms.

Since polyphenols from plant degradation are involved in the formation of humic substances, phenolic acids (pyrocatechic acid, vanillic acid, vanillin, resorcinol, ferulic acid, and benzoic acid) would then contribute an important part to the structure of these molecules. These phenolic acids have at least one carboxyl group (-COOH) and one phenolic hydroxyl (-OH) group [15]. These are the functional groups that are thought to possess the mineral chelating capacity of humic and fulvic acids. The phenolic compounds, quinones, and proteins condensate by the action of soil microorganisms on soil carbohydrates. This helps to form the structure and composition of humic and fulvic acids and thus humic substances [6, 12].

Figure 3 shows the mechanism of humic substance formation in the soil or peat.

Figure 3.

Structural and chemical properties

Humic substances represent an extremely heterogeneous mixture of molecules, which, in any given soil or sediment, may range in molecular weight from as low as several hundred to over 300,000 daltons [6]. Due to the complex nature of these biopolymers, determination of molecular mass, elemental composition and chemical moieties has been difficult [6, 16, 17]. Although research does indicate that each and every molecule in a given humic acid or fulvic acid fraction is most likely to have a different structure, the samples in one specific environment contain functional groups of similar types and in a similar number [1]. Elemental composition and functional group analyses have shown that peat humic acids tend to be similar to those from mineral soils [6].

Humifulvate is derived from well-defined, standardised peat using a thoroughly controlled technology. This manufacturing technology developed with a view to the stability of the raw material and the sensitivity of Humifulvate, and including interim and final product control, ensures the essential compositional consistency of the product.

Humifulvate contains oxygen-, nitrogen-, and sulphur-containing functional groups that made it very well suited as a metal-complexing ligand [6, 17]. At several sites and with varying strengths, metals are bound to the polypeptides and phenolic acids connected to the polynuclear heteroaromatic nucleus of Humifulvate. The properties of Humifulvate include its ability to bind heavy metals as an ion exchange agent [13] while acting as a carrier molecule of minerals and trace elements that are essential to human and animal health. This capacity has been attributed mainly to the presence of hydrogen ions in the aromatic and aliphatic carboxyl and phenolic hydroxyl groups of the humic substance [6, 12]. When metal ions and humic substances interact through ion exchange, protons on carboxylic acid and phenolic hydroxyl groups of the Humifulvate molecule are replaced by metal ions [13]. Evidence for this mechanism is supported by the fact that when these functional groups are masked by methylation and acetylation, the extent of activity and metal binding is drastically reduced [1, 2].

The percentage of the humus, which occurs in the various humic fractions, varies considerably from one soil type to another. The humus of forest soils is characterised by a high content of fulvic acids, while the humus of peat and grassland soils is high in humic acids. The humic acid/fulvic acid ratio usually, but not always, decreases with increasing depth of soil.

Practically all the cation exchange capacity of highly organic soils (peats), as well as the humus layers of forest soils, is due to organic matter. The greater the degree of humificaton, the higher is the cation exchange capacity. The contribution from humic and fulvic acids is due largely to the ionisation of carboxylic groups, although some contribution from phenolic hydroxyl and amine groups has been observed. The maximum amount of any given mineral ion that can be bound is approximately equal to the content of acidic functional groups, primarily carboxylic acid groups. Bonding mechanisms for the retention of organic compounds by humic substances in soil include ion exchange, hydrogen bonding, van der Waals forces (physical adsorption), and co-ordination through attached metal ion (ligand exchange) [6].

Composition of HFC

The organic material content of HFC is close to 50-70% (Table 1); it is made up specifically of carbon arranged in aliphatic chains and aromatic moieties, with hydrogen, nitrogen and oxygen contained in reactive functional groups. The humic substances found therein have peptide and saccharide chains with a protein content of about 10.5% [1]. Table 2 below shows the essential and nonessential amino acids contained in HFC.

The ash content or amount of inorganic minerals of HFC totals about 30%. The majority (10-18%) of the ash content includes calcium, aluminium, silicon, iron and magnesium. One to ten percent is made up of sodium and boron. Barium, lithium, tin, manganese, copper, nickel, potassium, lead, molybdenum, beryllium, and zinc make up the remaining 0.0001-1% [1]. Therefore, the peat used in the standardisation of the Humifulvate complex contains minute traces of naturally occurring minerals to which other minerals have been added for therapeutic purposes (Table 3).

Table 1: Composition of peat Humifulvate

Table 2: Distribution of amino acids

Table 3: Mineral composition added to Humifulvate

It is evident that humic substances can affect several biological processes [7]. HFC has been demonstrated to support the normal transport, absorption, and distribution of essential nutrients in the human body.

Humic and fulvic acids in water are thought to have a positive influence on biological growth in respect to phosphorus and nitrogen recycling, trace metal availability, and the limiting of potential metal toxicity [6]. Consequently, research has proposed that the standardised Humifulvate derived from peat could positively influence trace element absorption in animals and humans by its ion exchange capacity. This unique property of Humifulvate could potentially promote efficient uptake and incorporation of complexed essential minerals and trace elements into cells and tissues. Preliminary data suggest that this complex of humic substances derived from Hungarian peat in fact does affect the utilisation (absorption, transport, and distribution) of essential nutrients.

Elimination of heavy metals

Humifulvate has the capability of transferring metals to and from metalloproteins in vivo. [18]. These proteins play a role in metal storage and sequester excess metal ions, preventing toxicity. Metalloprotein concentrations are the highest in the liver where metals accumulate in the metallothionein portions of this organ. Metalloproteins can be found in many other human tissues, including small amounts in the blood plasma, which suggests that these proteins play a role in the transport of metals as well. [9].

When the free metal binding capacity of Humifulvate gets saturated, or contains a high concentration of a metal humate (attachment of metal to humifulvic acid), then Humifulvate will transfer this metal to the protein-type molecules that are able to bind and utilise it. On the other hand, if the free metal binding capacity is high, then Humifulvate will form complexes with metals that are free or attached to metalloproteins, helping in the excretion of these metals (i.e. in the case of toxic heavy metals like cadmium). Therefore, it may be concluded that Humifulvate may act somewhat like metalloproteins due to its chelating activity and ion exchange capacity. When metals are a part of a metalloprotein, they can modulate its biochemical reactions [19].

Primarily, investigations have focused on the ability of a microelement liquid concentrate, containing standardised Humifulvate (HFC), to deliver essential minerals while also eliminating toxic heavy metals like lead, cadmium, and mercury. Oral consumption of HFC administered daily for six weeks significantly decreased blood cadmium levels and increased urine cadmium in 31 adult workers continuously exposed to occupational cadmium [10]. In the majority of subjects, initial abnormally low serum iron levels increased, and markers of kidney and liver function improved.

Research indicates that absorption of cadmium from the gastrointestinal tract and its toxicity are influenced by the supply of element such as Zn, Cu, Fe, Se, Ca, and Vitamin C [8, 10, 19]. The ability of HFC, as an ion exchanger, may free its trace elements bound in chelate form for uptake into the tissues and bind other elements that are readily available, such as cadmium. At the same time, a number of essential elements are provided that may decrease the ability of cadmium uptake and absorption in the gastrointestinal tract. The improvement of liver and kidney enzymes could be attributed to the effect of the preparation on the microelement status and balance in the body, which would then play a role in the functioning of these enzymes. HFC was studied for its effect on the metabolism of trace elements in 51 healthy adult volunteers [53]. Following two-weeks of oral administration of HFC, blood lead and cadmium levels decreased significantly. Furthermore, HFC decreased absorption of cadmium and lead from food or environmental exposure based on urine measures of these metals. HFC had no significant effect on blood parameters studied (i.e. haematocrit, haemoglobin, leukocyte count; SGOT, GGT, ALP; and, Na, K, Ca, and P).

Further pieces of evidence of the beneficial effects of HFC have been documented in clinical trials evaluating occupational and environmental heavy metal exposure. In a three-week clinical observation with subjects screened for routine occupational health check-ups, 21 subjects were found to have higher than usual Pb levels (exceeding 1.0 micromol/l, the health risk limit being 1.5 micromol/l) and 26 subjects had Cd levels exceeding the accepted health limit (0.08 micromol/l). Subjects given HFC showed a significant decrease in their blood Pb and Cd levels following the daily oral intake of HFC. No significant or pathological changes were observed in the blood chemistry of these subjects [54]. Additionally, HFC was administered orally to six adult subjects with moderately elevated lead levels that did not require penicillamine. HFC was administered to each subject for three weeks. Four of these six subjects (66%) had significantly lower blood lead levels following three weeks of daily administration. The rate of decrease in lead levels in the subjects was similar to that reported for penicillamine [11]. Two patients in the HFC group reported mild side effects and therapy was discontinued. The results from these clinical observations indicate that reducing toxic levels of heavy metals in humans is apparently influenced by treatment with the HFC multimineral liquid concentrate following its administration.

Two open clinical trials examining the effects of HFC in volunteers exposed to lead have provided further documentation of the beneficial effects of HFC [55, 56]. Twenty individuals with high occupational lead exposure were given 20 ml per day of HFC for six weeks. Blood levels of lead decreased markedly and significantly from the beginning of the study when compared to the control group. None of the clinical or haematological parameters changed during the course of the treatment. Two subjects reported mild and transitory diarrhoea, which normalised without stopping treatment. Four subjects reported moderate nausea and one a transitory headache. Another open clinical trial in 60 subjects has demonstrated a similar but not as profound outcome [56]. At the end of a 12-week administration period, the change in serum lead parameters became significant compared to pre-administration values. The results of this trial are not as profound as the six-week administration of the HFC in volunteers exposed to lead [55]. Although the reduction in blood lead levels was significant, a longer treatment time was needed due to the smaller dosage of HFC that was administered to the individuals. The examined laboratory parameters (i.e. serum blood, routine laboratory tests, liver and kidney function, and urine examination) exhibited no significant changes, which supported the safety of HFC in the recommended dosage. Data from the two former studies indicate that the higher the serum or blood lead level, the more significant reduction in this parameter can be observed. Furthermore, doses of 20 ml per day of HFC appear more effective in the treatment of occupational lead exposure.

Studies in animals have confirmed the beneficial effects of HFC on heavy metal chelation. Some studies using isolated humic acid have demonstrated that it does affect cadmium speciation in the intestine and thus absorption and distribution of this heavy metal (see the Biological Role of Humic Acid section). Additional studies using HFC in animals provide support for the ability of HFC to chelate heavy metals. Adult pigs were fed varying doses of HFC or a control supplement and the excretion of a mercury radioisotope previously administered, was examined. Those animals that were fed HFC excreted more of the mercury isotope, than did the control animals. Although the data was not significant, due to the small number of animals, this study warrants further research to document the efficacy of HFC in alleviating mercury accumulation. [57].

The effect of HFC on the absorption and incorporation of isotope-labelled strontium chloride has also been documented. Not only did HFC slow the strontium absorption and its incorporation, it also affected the urinary excretion of this toxic element [58]. The urinary excretion of strontium was less intensive in the animals fed with HFC. The authors concluded that a lower amount of the toxic element complex was absorbed when HFC was present. This same effect has been documented in humans exposed to cadmium and lead [54]. Based on urine measures of these metals, HFC decreased the absorption of cadmium and lead from food or environmental exposure. Further data indicate that cadmium and lead urinary excretion increased in humans during the administration of HFC [10, 54], indicating the removal of this toxic element. Although it is premature to state the exact mechanism of action occurring in these animals and humans exposed to various heavy metals, it is safe to presume that the absorption and urinary excretion of heavy metals is affected by HFC.

Evidence for the protective effect of HFC bound with microelements against environmental exposure to irradiation has also been provided in the literature. The radioprotective effect of standardised HFC was tested in female Wistar rats. HFC was given in one dose of 240 mg/animal (960 mg/kg body weight) and the rats were subjected to whole body irradiation. Baseline and outcome data (white blood cell, erythrocyte, platelet counts, and total serum iron binding capacity) were taken to substantiate claims of efficacy of the HFC treatment. The results showed improvements in platelet counts (leukocytes and thrombocytes) which had markedly decreased after irradiation. Platelet counts began to normalise in the control group one week earlier than in the untreated control group of rats with just one dose of the HFC formula [59]. No side effects or toxicities were noted while administering HFC to this group of animals.

As indicated by the previous data, the standardised HFC appears to be an effective chelator of offending heavy metals. Furthermore, it shows a protective effect against radiation in vivo. Its benefits could be utilised in the prevention of heavy metal contamination in workers in hazardous occupations, by decreasing the absorption and increasing the elimination of toxic heavy metals like cadmium. Furthermore, this standardised HFC would be beneficial in eliminating heavy metals that can be accumulated throughout a lifetime of environmental exposure, and alleviating the physiological consequences that occur with irradiation. Animal studies show a similar mechanism of action when comparing them with the studies in humans. Both indicate that HFC may work to decrease the absorption of these heavy metals as indicated by its effects on the excretion of these toxic elements in the urine.

Iron restoration

Nutritional anaemias, of which iron deficiency is the greatest cause, constitute the second most prevalent nutritional deficiency in the world, second only to protein-energy malnutrition [60]. Iron deficiency anaemia affects primarily women and children and individuals with chronic disease. This nutritional deficiency respects neither social class nor geographic situation, as it is present in both developed and underdeveloped countries. Iron deficiency anaemia is a condition in which the haemoglobin levels of red blood cells are lowered, and the red cells become smaller and deformed, thus reducing their oxygen-carrying capacity. The most common cause is nutritional, including inadequate absorption of iron due to poor iron intake and reduced bioavailability. Iron loss from internal bleeding, low stomach acid and malabsorption are also important factors [19]. The standardised HFC may be an effective way to treat iron deficiency anaemia and maintain adequate amounts of necessary minerals in proper balance for optimal health.

The ability of the standardised HFC to restore iron levels and improve haematological parameters has been documented. Serum iron levels improved in fourteen adult volunteers given oral doses of HFC during a three-week period [61]. Serum ferritin levels approached the desired physiological range within three weeks. It was reported that for subjects with low iron values at the beginning of the study, their iron levels increased to within the desired range for iron status; conversely, those subjects who began the study with elevated iron status, their iron levels decreased to within the desired physiological range. This finding demonstrated that HFC could facilitate homeostasis of iron status in humans.

HFC was given orally as an adjuvant during cytostatic therapy to tumour patients [62]- [64]. Cytostatic therapy is used for the prevention of the growth and proliferation of cancer cells; however, damage may also occur to normal cells such as erythrocyte cells (red blood cells), which may lead to anaemia (the deficiency of red blood cells, haemoglobin, and blood volume). Therefore, iron therapy is needed, because iron functions as a part of haemoglobin and thus red blood cell function. One group of patients showed significant enough improvement in their erythrocyte counts so that no further need for iron therapy was required [65]. Further subjective evidence of benefits experienced by these cancer patients included: improved appetite, weight gain, reduced need for analgesics, increased general stress resistance, reduced nausea, reduced fatigue, and restoration of the capacity to work. No adverse side effects were reported that could be attributed to HFC.

The standardised HPC formula was used for the treatment of anaemias and for faster recovery from illnesses in children. Nineteen paediatric subjects with iron deficiency anaemia were studied to determine if HFC given orally would improve their general well-being, appetite, and serum iron levels [66]. Subjects reported improvements in appetite and well-being after treatment with HFC. A rise in serum iron levels was seen as early as two weeks after administration had begun. After three weeks, HFC caused a significant increase in the serum iron level. Haemoglobin levels were variable, with some rising and others decreasing, but within desired physiological levels [66].

This same effect (variable haemoglobin levels) was also manifest in elite athletes. Haemoglobin levels were studied to determine if the oral administration of HFC would affect stress resistance, and the ability to increase the intensity of exercise, following oral administration of HFC in 25 elite adult athletes [67]. Haemoglobin levels in the athletes remained within the desired physiological range. Athletes reported a subjective improvement in stress resistance and their ability to focus during exercise periods. From the two previous studies, it appears that the standardised HFC may have the ability to normalise iron, serum ferritin, and haemoglobin levels. Evidence for the effect of HFC's iron normalising capabilities has been described in the literature. Protocatechuic acid (a phenolic monomer of HFC) can form Fe+2-polyphenol complexes when excess amounts of iron are available. This occurs so that excess iron (Fe+2) cannot react with oxygen molecules and form reactive oxygen species [68]. This provide further support for the metal chelating activity of HFC and implies that it has the ability to normalise iron levels so that excessive oxidation does not occur in the presence of higher than usual amount of iron.

In vivo studies have also demonstrated the effectiveness of the standardised HFC for improving iron deficiency anaemia in rats and pigs. HFC was tested on an iron deficient rat model by rearing the mothers and their offspring on an iron free diet. Iron deficiency was signified by severe microcytic, hypochromic anaemia, and high zinc protoporphyrin (ZP) levels indicating the lack of iron at tissue level in the bone marrow. The iron deficient rat pups also exhibited a decreased weight at birth, decreased body mass gain, and increased lethality compared to controls [69]. HFC was compared to the effectiveness of an official medicinal preparation, Aktiferrin syrup, which is commonly used in the treatment of iron deficiency anaemia. Regarding the haemopoietic and hepatic effects, measured by red blood cells (RBC), mean cell volume (MVC), haemoglobin (Hb), haematocrit (Hct), total iron binding capacity (TIBC), transferring saturation, and liver enzymes (ALAT, ASAT, GOT, GPT) respectively, HFC exhibited equal effects compared to the Aktiferrin [69]. However, HFC proved to be superior in that body mass gain of the pups was better in this group as compared to the Aktiferrin group. Additionally, serum triglyceride levels were measured, and decreased concentrations normalised in the standardised HFC formula group but not in the Aktiferrin treated group.

Further support for the beneficial effects of the standardised HFC in the treatment of iron deficiency anaemia has been demonstrated in iron deficient pigs. Pigs of iron deficient sows that were fed the standardised HFC while pregnant exhibited significantly higher haemoglobin levels than did the pigs of iron deficient sows that were given the standard parenteral iron supplement treatment or no treatment [5]. These results and previous in vivo data indicate that the standardised HFC offers an effective treatment for iron deficiency and may help restore impaired metabolic processes due to iron deficiency anaemia.

Mineral supplementation

A number of factors have been associated with the occurrence of mineral deficiencies in humans: deficiency in the soil, water and plants; mineral imbalances; processing of water or soil; and inadequate dietary intake [19]. Mineral deficiencies can result in a multitude of conditions, such as hair loss, eczema, fatigue, and illness. A dermatological study of head hair growth was conducted in 29 adult subjects experiencing hair loss related to suspected trace element deficiencies. HFC decreased hair loss and actually increased the regeneration of hair in some subjects [70]. This was attributed to improved trace element status in subjects, particularly for iron status. Serum iron levels rose in those patients who experienced improvements in hair growth and regeneration, but not in subjects with little or no improvement. The same author reported on similar results in children but the data was inadequate to reach a conclusion.

HFC has also been shown to produce a positive response in another condition associated with mineral and trace element deficiency, notably chronic eczema. Eczema is an acute or chronic inflammatory condition that causes itching and burning of the skin. Eczema has various etiologic factors, such as allergic reactions, and nutrient deficiencies. For example, protein deficiency is thought to be a casual factor in chronic eczema, and manganese deficiency produces scaly dermatitis [71]. Severe zinc and magnesium deficiency may produce skin lesions [9]. It has also been reported that nutrients may be beneficial in the treatment of eczema. Selenium sulphate lotions inhibit different forms of dermatitis. Free form amino acids, manganese, magnesium, zinc, and selenium have all been implicated in the treatment of eczema. The response to oral administration of HFC over a three-week period was studied in nine paediatric subjects with chronic eczema. [66] After the study was concluded and subjects no longer received HFC, their eczema returned. As a result, the study was continued for an additional period of two to three months with the same subjects, and again during administration of HFC the amount of eczema decreased. HFC was then continued for six more months in the same subjects and again the amount of eczema decreased. Thus, the possibility of treating chronic eczema with HFC should be examined further for its potential role as a long-term treatment for this condition. The effects seen in this study could possibly be due to the combination of naturally occurring amino acids attached to HFC as well as the added minerals and trace elements to the liquid concentrate.

Research in a population of 51 healthy adults supports the role of the standardised HFC in improving microelement parameters [53]. The product significantly raised the level of copper in these individuals and improved iron metabolism. HFC had no significant effect on blood parameters studied (i.e. haematocrit, haemoglobin, leukocyte count; SGOT, GGT, ALP; and, Na, K, Ca, and P). All of these laboratory parameters were still within the normal range after the administration of the HFC. Isolated humic acid has been shown to facilitate the transport of several trace elements, including copper and iron, across the intestinal membrane of rats [7]. Therefore, this data in animals provides support for the mechanism of action of humic acid and the HFC containing humic acid in improving microelement parameters in humans.

Since humic substances have existed in nature well before human existence, research continues today to determine if humic substances pose a threat to human health. Some researchers in China have attempted to link humic substances in well water with two different endemic diseases: Blackfoot disease and Keshan Beck disease. Those endemic conditions are associated only with well water humic substances, which are ingested in extremely high amounts and are also contaminated with high levels of arsenic and other toxic compounds. The standardised HFC preparation derived from Hungarian peat has been documented not to contain toxic materials and so it should not be compared to the humic substances from China. Still, as the Hungarian preparation is to be marketed as a dietary supplement, its acceptable intakes should be determined.

The lack of toxicity of the ingredients used in this product is evident knowing that the product has been used in Europe for more than six years without any adverse event reports. Furthermore, the amounts of minerals and trace elements used in this product are considered safe, for which estimated safe and adequate daily dietary intakes and recommended dietary allowances are available. A cumulative body of evidence points to the safety of each ingredient in the standardised Humifulvate based multimineral liquid concentrate for its use as a dietary supplement by humans.

Acute toxicity testing in rats demonstrated that the lethal dose of the standardised HFC is extremely high, at more than 10 gm/kg body weight of the animals used in the study. Cumulative and subacute toxicity and mutagenicity studies have also documented the safety of HFC.

Furthermore, a review of human clinical studies indicates a lack of significant side effects from the ingestion of HFC. The amounts of humic substances in HFC are extremely low and have been documented as safe in animal and human studies.

A series of acute, cumulative and mutagenicity toxicological studies of HFC-containing Humet®-R have been carried out by investigators in Hungary. As recently as 1999, the manufacturer of Humet®-R commissioned an independent review of all data available to date on the toxicology and safety of Humet-R. This review confirmed the safety of this product for use as an oral multimineral supplement. The documentation of safety data in animals is considered adequate and applicable to humans, as is implied the same mechanism of action that is thought to occur in both animal and human studies.

Most importantly, clinical documentation of both the short-term and long-term use and safety of HFC in humans is available. All animal and human toxicology studies have used HFC as found in Humet®-R to study its safety. The substance tested complied with Good Laboratory Practices (GLP) methods and was performed by independent laboratories using reproducible analytical methods (IR spectroscopy and fingerprinting). Furthermore, elemental analysis by an independent laboratory (Flora Research Laboratory (San Juan Capistrano, CA, November, 1999) has documented that the levels of each mineral and trace element combined with HFC are well within safe ranges. The same independent laboratory has also reported that HFC contains non-toxic levels of aluminium, lead, cadmium and arsenic. Laboratory analysis performed by the National Institute of Food Hygiene and Nutrition (OÉTI) (the authority regulating foods) in Budapest, Hungary, in 1991, did not find detectable concentrations of polycyclic aromatic hydrocarbons (PAH) in Humet®-R, the product containing Humifulvate.

Acute toxicology studies

In a preliminary study, 84 Wistar rats were followed for two weeks following varying doses of the standardised HFC for evidence of acute oral toxicity. The rats were both male and female and were given up to 10 gm/kg body weight of the HFC formula. No death occurred even in the highest dose administered, nor were there any signs of toxicity reported based on macroscopic alterations seen in the organs of the test animals. The LD50 value was determined to be higher than 10gm/kg body weight. The standardised HFC was classified as belonging to the "practically non-toxic" category.

An additional oral acute toxicity study was designed as a 'limit test'. A limit test is often performed for relatively non-toxic chemicals. Twenty male and female Wistar rats were administered 20 ml/kg (300 mg/kg) of HFC two times a day in 24 hours [73]. All animals were continuously observed for six hours initially after the treatment and then twice a day during the post treatment. Clinical observations included, the state of the skin, fur, eyes, and mucous membranes; respiratory function, circulation, autonomic nervous system function; somatomotor activity, trembling, convulsions, salivation, diarrhoea, and somnolence.

There was no evidence of weight loss in either of the groups and no macroscopic alterations of the animals' organs were found. However, in the control and treatment groups, the researchers observed a few cases of haemorrhage and emphysema in the lung, haemorrhage in the thymus, and hyperaemia of the spleen, in which there was no significant difference in the number of occurrences between the two groups. The authors noted that these conditions were associated with agony. The few cases of hyperaemia and hydrometra of the uterus were connected with the neurohumoral regulation of sexual function or the cyclic physiological state of the uterus.

Results of the study indicate that the standardised HFC caused no toxic symptom or lethality during a fourteen-day post treatment observation period. Therefore, the maximal tolerable dose (MTD) to be administered within 24 hours was determined to be >40 ml/kg, (>600 mg/kg). This study gives a more precise demonstration of the safety profile of the standardised HPC formula, thus providing a base of evidence that this product is non-toxic in applicable physiological doses.

Cumulative toxicity test

The initial cumulative toxicity test with ten male Wistar rats involved their treatment with 10gm/kg (LD5O) of the standardised HFC for four successive days in increasing percentages of the test substance for a time interval of 24 days. Upon completion of the study, the animals' organs were measured and investigated for pathological signs. Additionally, the researchers documented body weights, haematological values, and thyroid hormones before and after treatment. Histological tests of tissues were administered after the treatment with the HFC. No significant differences between the control and treatment groups were found for any of the before mentioned parameters. However, the histological examination of the spleen did reveal an increase in the concentrations of tissue iron and additional stored metals in the treated group versus the controls. There was no mention of total body iron indicating if tissue injury would be possible at this particular dosage. In a few cases for both the control and treated group, examiners noticed a moderate change in lung tissues noted as peribronchial lymphocytic infiltration, which could not be explained [74].

A subsequent repeated dose toxicity study was conducted in order to clarify the possible side effects that could occur after prolonged administration of the standardised HFC. Food containing the HFC was fed to Wistar rats for 28 days in treatment doses of 1, 3, 10, 30, and 100 mg/200 g body weight per day. Control animals were fed normal rat food. Animals were observed daily, body weights taken weekly, and parameters of clinical chemistry, haematology and organ weights were measured at the time of necropsy. Two groups after week three of treatment with the HFC (doses of 30 and 100 mg/200 g body weight) showed a decrease in weight, which the authors attributed to a decrease in appetite influenced by the joint quantity of certain trace elements in the formula. [75]. However, there was no mention of a decreased amount of food intake for these animals. Examination of organs showed no significant change from the controls except at the dose levels of 30 and 100 mg/200 gram body weight per day, with organ weight loss in the liver and kidneys of these two treatment groups.

The results indicate that a four-week long dose of 1, 3, and 10 mg/200 g body weight per day of standardised HFC does not influence the development of the tested organs. No death occurred in any animals and no significant differences were seen in tested chemical parameters such as haematological indices and enzyme functions. Although a more complete picture could have been achieved by measuring food consumption and performing histological examinations, this study provides additional evidence that the standardised HFC is a non-toxic substance especially when used in relative doses for administration in animals and humans.

In a 60-day toxicology study involving rats fed with powdered Humifulvate in doses of 60mg/animal/day (~300 mg/kg body weight per day) and 240 mg/animal/day (1200 mg/kg body weight per day), respectively, no deaths were reported. The general condition and physical parameters of the animals did not change. The haematology parameters did not change, either.

No deaths were observed in another 180-day subchronic toxicology study on dogs, in which the animals were fed with EHF powder even at doses fourfold the usual human dose. The dose with no observable adverse effects (NOAEL) was established as 15 mg/kg body weight, as at higher doses the expected clinical adverse effects (nausea, diarrhoea) could be observed with dose-dependent frequency.

Mutagenicity studies

AMES-test

The standardised HFC containing the Hungarian humic substances has also been subjected to four mutagenic studies, and under the AMES test criteria exhibited no mutagenic activity. Five Salmonella typhimurium strains were used in the presence and absence of rat liver fraction with colony number in control plates and test plates being practically the same. The results indicate that the standardised HFC had no mutagenic activity and no bactericide effect using £ 7500 mcg of the test substance per plate [76].

Anti-clastogenic test

The effect of the standardised HFC on a known mutagen, ionising radiation, has been studied using human peripheral blood lymphocytes. A preliminary study was conducted to confirm that the HFC was not mutagenic and an additional study was administered to determine its anti-clastogenic characteristics (ability to reduce the number of chromosome aberrations) against the known mutagen [77]. No chromosome aberrations were induced by any of the standardised HFC concentrations as compared to controls, with all of the concentrations being much higher than any physiological dose. Therefore, it was concluded that the standardised HFC is not clastogenic even in very high concentrations of 200 mcl/ml. In the subsequent study concerning the anti-clastogenic effect of HFC, the resulting data was somewhat inconsistent. A significantly lower value of aberrant cells (abnormal cells) induced by irradiation was found when the cells were treated with the standardised HFC at a level of 5 mcl/ml. These results imply an in vitro anti-clastogenic effect of the standardised HFC. However, the number of di-centric ring aberrations (diagnostic value in detecting radiation effects and thus chromosome aberrations) decrease as the HFC concentrations decrease. The interpretation of mechanisms responsible for these effects in vitro was not attempted because of the illogical results of this data collection. The results do suggest that the standardised HFC may have potential anti-clastogenic effects; however, this cannot be stated as fact due to the variable results [77]. Considering that the standardised HFC was found to have no mutagenic activity in two studies using high doses of this substance, it is appropriate to suggest its relative safety for ingestion as a dietary supplement.

Micronucleus test

Potential mutagenic effects of the EHF powder was studied in a standard micronucleus test on mice receiving a dose of 2000 mg/kg compared to a control group. The test substance given in a dose of 2000 mg/kg demonstrated a negative, in other words non-mutagenic effect in this test.

Table 4. The Safety of HFC and/or Humic Acid in Animal Studies

A series of studies of HFC and humic acid given to mice and rats have evaluated the safety of Humifulvate. The following table provides a summary of this literature.

Clinical observation of HFC given to 514 patients under medical supervision for an average of 4.3 months of administration under controlled conditions have been reported. All case reports were collected, reviewed and summarised. These case reports represent patients who sought out medical treatment for specific conditions or diseases. Physicians and qualified public health workers supplied the outcome data.

Table 5 illustrates the rarity of adverse events that have been noted during the administration of the standardised HFC to humans. Although no significant adverse events were reported for any patients, 30 out of 514 individuals (5.8%) reported transient symptoms while taking HFC, including: headache, nausea, heartburn, diarrhoea, or skin reactions. Since these types of transient events may be due to random chance, it is not possible to attribute them to HFC consumption.

Furthermore, there have been no documented incidences of adverse event reports in Europe where it has been used for eight years as a non-prescription drug. Some consideration should be taken in choosing the population in which this supplement should be used. For example, individuals with iron storage diseases could have detrimental side effects from taking a supplement that contains iron or that may affect iron utilisation.

In 2001 a tolerance study using EHF powder (capsules), a preparation equivalent to HFC, was conducted involving 40 healthy volunteers who took 1-2-3 times the human dose for three weeks. No adverse effects were observed during the trial, and thus it may be safely assumed that the capsule containing the EHF powder is well tolerated.

Table 5: Adverse Events Reported in HFC Human Clinical Trials and Case Reports
(Observed in less than 6% of the population in which HFC was administered).

Human clinical trials

Case reports

Of the altogether 514 individuals treated with HFC, adverse effects were observed in the case of only 30. The Average treatment period was 4.3 months. A comprehensive analysis of trials with the HFC conducted under clinically controlled circumstances was carried out in 2001. The analysis of the data of 1141 subjects revealed a very low occurrence of adverse effects, representing 2.6% of the cases. Most of the adverse effects observed were gastrointestinal in nature (i.e. nausea and diarrhoea).

Long-term use of HFC

Long-term use of HFC has been documented in 194 individuals. The average time frame for long-term treatment (defined as greater than 4 months' consumption) with HFC was 12.0 months (range: 4 months to 5 years). Three patients under treatment for cancerous tumours consumed HFC for 5 years [63, 64]. None of these three subjects required cytostatic therapy during the period in which HFC was consumed and no significant adverse events were reported for any of these subjects consuming HFC over a prolonged period. Improvement in well-being was reported by many of the individuals taking the HFC, even during times of cytostatic therapy which may cause immuno-suppression and general malaise. Therefore, one can conclude that this supplement may be a roborant during times of illness and disease.

An open clinical trial was conducted to evaluate the long-term use of HFC in the treatment of nine paediatric eczema patients [66]. The patients were given HFC for two to three months. There was a relapse of the symptoms after the treatment was stopped; therefore, treatment with HFC resumed for an additional six months. Not only did the eczema improve in these patients, but no significant adverse reactions were reported due to the administration of HFC for greater than six months. One child reported an allergic skin reaction and one other child reported abdominal complaints and diarrhoea. Therefore, the long-term use of the standardised HFC is both beneficial and safe as documented in children and cancer patients.

When considering supplementation with a particular mineral or trace element, evidence regarding its biochemical fate in the organism including absorption rate, retention time, excretion route, competition with other minerals, and any potential risks for side effects must be evaluated. For example, the valence (or number of bonds an element usually forms) will affect the absorption and complexation of that particular element or mineral. In addition, the binding of elements to the metal proteins in the liver will ultimately affect its ability to become absorbed, retained, and excreted. This means that regardless of the absolute levels of an element or mineral in a product, only a fraction of this amount will enter into circulation. Furthermore, competitive site absorption occurs when several minerals are administered together in an organic complex such as with the standardised HFC.

In addition, dietary intake of many minerals is below the Recommended Dietary Allowance (RDA) or estimated safe and adequate daily dietary intake as developed by the Hungarian Academy of Sciences. Therefore, supplementation with particular minerals and trace elements is essential for health maintenance. The following is a table representing the elemental amounts of each essential mineral contained in HFC.

Table 6: Mineral composition of standardised HFC

Independent laboratory analysis of HFC

Independent laboratory analysis by Flora Research Laboratory (San Juan Capistrano, CA, November, 1999) reported that HFC contains non-toxic levels of aluminium, lead, cadmium and arsenic: 20.7 ppm, 0.07 ppm, 0.02 ppm, and 0.07 ppm, respectively. This has been translated into the amount that could occur in a single oral dose of the standardised HPC formula (Table 7).

Table 6: Trace Elements in the HFC formula

The levels of trace toxic metals in HFC were compared to the amounts of each metal found in the daily diet. Food can contain about 10 ppm of aluminium. Conservative estimates indicate that at least 2-3 mg of aluminium are consumed a day [9]. 20.7 ppm found in HFC is the same as 0.188 mg of aluminium in one 10 ml serving of the standardised HFC. This amount is less than one-tenth the amount of aluminium a person would consume in the diet on a daily basis. The amounts of lead and arsenic found in HFC are insignificant.

Cadmium toxicity is generally based on oral inhalation of ambient cadmium. Therefore, other data must be utilised to determine its safety in the amounts found in HFC. Average daily intakes from food in most areas not polluted with cadmium are between 10-40 mcg [104]. Therefore, the estimated amount of cadmium in a typical diet is ~5.5 to 25 times that found in the standardised HPC formula.

It has been proposed that humic substances may bind with or absorb mutagens rendering them less toxic or less mutagenic [20]. Among these mutagens are polycyclic aromatic hydrocarbons (PAH). PAHs are generated through inefficient or incomplete combustion of organic matter and, while initially released largely into the atmosphere, they are subsequently deposited in soil and water [105]. Stream humic substances have been documented to interact with PAHs [6]; these aromatic compounds have also been extracted from the deeper layers of peat (2.5 m) [2]. Therefore, PAHs are widely distributed in the environment and human exposure to them is unavoidable [106]. The food chain appears to be the dominant pathway of human exposure to toxic and mutagenic PAHs. While many hydrocarbons are non-carcinogenic and efficiently removed from the body, small fractions of some hydrocarbons are converted to eloctrophilic metabolites which are not effectively further metabolised and which are probably responsible for the carcinogenic properties of these hydrocarbons [105]. There is speculation that humic substances, due to their binding with these compounds, may ultimately affect the fate of these carcinogenic products.

Uptake and bio-concentration factors of benzo(a) pyrene (a toxic PAH) in Atlantic Salmon were determined in water containing natural aquatic humic substances and control water. Uptake and bio-concentration of these toxic compounds were observed to significantly decrease in the presence of aquatic humic substances compared to the control water [107]. Therefore, it is apparent that humic substances in water do have a beneficial effect in vivo. However, more research is needed to determine whether this is also true with terrestrial humic substances.

Some attempts have been made to understand the mechanisms responsible for the effects of humic substances on PAH biodegradation. Various PAHs could be degraded by activated sludge [20]. Furthermore, it is thought that the bacteria present in the humic acids and activated sludge may decompose the absorbed mutagens. Humic compounds were observed to be able to contribute to the enzymatic activity in activated sludge [108]. Other investigators have stated that the sorption of PAHs to organic matter renders the PAHs non-biodegradable, which may in turn affect bio-toxicity [109]. However, these observations and speculations will require additional data to document the specific mechanisms responsible for humic substance-PAH complex biodegradation, bioavailability, and toxicity.

Laboratory analysis performed by the National Institute of Food Hygiene and Nutrition (OÉTI) in Budapest, Hungary found non-detectable concentrations of polycyclic aromatic hydrocarbons (PAH) in Humet-R that contained Humifulvate. None of the following PAHs were detected by OÉTI: benzo-(a)-pyrene, benzo-(b)-fluoro-anthene, indeno-pyrene, benzo-(k)-fluro-anthene, fluoro-anthene, or benzo-(ghi)-perylene.

To sum it up, when considering the toxicological data compiled from in vitro and in vivo laboratory tests and the lack of a significant amount of side effects reported in human subject studies, one can conclude that the standardised HFC taken in the recommended dosage of 10ml per day is safe. Furthermore, laboratory analyses indicate that HFC contains insignificant and non-toxic amounts of aluminium, lead, arsenic, and cadmium and is free of any carcinogenic compounds.

Chapter 1, Chapter 2, Chapter 4