1 Introduction

Proanthocyanidins (PAs, also termed condensed tannins) are colorless, oligomeric, and polymeric plant secondary metabolites formed from flavan-3-ol molecules (Khanbabaee and Van Ree 2001; Santos-Buelga and Scalbert 2000), which can be also substituted by methyl, acyl, galloyl, or glycosyl moieties (He et al. 2008). PAs, being considered the second most abundant group of natural phenolics after lignin (He et al. 2008), were discovered in 1947 by French doctor Jacques Masquelier (Masquelier 1987; Passwater 1991). Masquelier’s research on PAs was inspired by finding that crew of famous explorer Cartiers was cured from scurvy with tea made from needles and bark of pine, served by native Canadians (Carper 1998). During his work for PhD thesis, Masquelier extracted a colorless fraction from the red-brown skin of peanuts and confirmed its biological activity as vitamin P. He also proposed that major components of this fraction are oligomers of flavan-3-ol units (oligomeric procyanidins, OPCs) (Weseler and Bast 2017). OPCs derived from peanut skins were then successfully marketed as a blood vessel protectant, and in 1969, Masquelier obtained a US patent for the extraction of OPCs from pine bark and grape seeds and for its medical application (Masquelier 1987; Passwater 1991). Since that time, PAs were extensively studied in terms of structure elucidation and biological activities and over 200 oligomers with degree of polymerization (DP) up to 5 have been described (He et al. 2008). In plant material OPCs are accompanied by PAs with higher DPs reaching even 30 building blocks in grape seeds or black soybean seed coat (Takahata et al. 2001).

Proanthocyanidins are widely distributed in roots, wood, bark, leaves, fruits, and seeds of many herbaceous and woody plants and, most importantly, in variety of edible plants consumed in a daily diet (Engström et al. 2014). Fruits (apples, apricots, avocado, banana, berries, cherries, dates, grapes, kiwi, mango, pears, plums), cereals and beans (barley, beans, cocoa, sorghum, pea), and nuts and spices (cinnamon and curry) contribute to the everyday intake of PAs. Also, snacks and beverages (chocolate, fruit juices, red wine, and beer) are good sources, especially, of OPCs (Prior and Gu 2005). The most important plant species containing PAs are presented below.

1.1  Pinus pinaster Aiton (syn. Pinus maritima Lam)

  • Pinus radicata D.Don (syn. Pinus insignis Douglas ex Loudon)

  • Pinus massoniana Lamb

The bark and leaves of pine species and also of other conifers were used as a natural remedy since ancient times. Pine bark served as a food source in emergencies, while its decoction cured scurvy indicating that some of active ingredients correspond to the action of vitamin C. Application of the bark of spruce, pine, and juniper for wound healing was also known at that time (Li et al. 2015). The bark, needles, pollen, and turpentine of P. massoniana have been used in traditional Chinese medicine as a remedy for a treatment of hemorrhages, rheumatism, arthralgia, inflammation, and cancer (Cui et al. 2005). Due to the presence of flavonoids, catechins, phenolic acids, but mainly proanthocyanidins (non-conjugated procyanidins B1 and M1 (formed in vivo from catechin polymer by gut microbiota)) (Jerez et al. 2007), the extract of pine bark has found wide application in the fields of nutrition and health. Over the past few decades, it has been used in poor blood vessel conditions (Li et al. 2015). The procedure of preparation of standardized water extract from pine bark was patented and product obtained from P. pinaster was marketed worldwide under the trademark Pycnogenol®, while extract from P. radiate was named Enzogenol®. The variety of properties (antioxidant, anti-inflammatory, anticarcinogenic, cardioprotective and neuroprotective) was studied for these marketed extracts and strong synergistic activity of a mixture was observed comparing to individual components (Yoshida et al. 2011) underlining beneficial properties of OPCs.

1.2 Theobroma cacao L.

Theobroma cacao L. is the most widely cultivated among over 20 species in genus Theobroma. It is a source of cacao beans, which were of historical importance in Mesoamerican culture. Cacao drinks were known and popular in Aztec royalty, while beans were used as currency well into the nineteenth century in remote parts of Mesoamerica (Steinberg 2002). Cacao drinks served as a primary remedy or as a vehicle to deliver other medicines. Among over hundred documented traditional uses of cacao, the application for improvement of digestion and elimination, for stimulation of nervous system and induction of weight gain in emaciated patients was the most common (Dillinger et al. 2000). Lower rates of hypertension, cardiovascular disease, obesity, diabetes mellitus, myocardial infarction, stroke, and cancer were observed among native people drinking daily higher amounts of cacao (Katz et al. 2011). The beneficial effects of cacao consumption are related to the action of OPCs (e.g., dimeric procyanidins B1 to B7, trimeric procyanidin C1, tetrameric procyanidin (cinnamtannin A2), and pentameric procyanidin (cinnamtannin A3)) (Esatbeyoglu et al. 2015), which were proved to be more potent than monomeric and polymeric cocoa procyanidins (Dorenkott et al. 2014). The astringent and bitter taste of cacao is also related to the high content of PAs.

1.3 Vitis vinifera L.

The primary and traditional use of Vitis vinifera is wine-making. During this process the pomace containing grape seeds and skin is produced. As being a side product pomace usually was discarded, however, it is a rich source of PAs. Pomace major fraction constitute polymeric procyanidins (PPCs) (65% and 83% in grape seeds and skins, respectively) which are accompanied by lower content of monomeric and OPCs (35% and 17% in grape seeds and skins, respectively) (Luo et al. 2018). The medicinal uses of V. vinifera date back to ancient times. European folk treated sore throats with unripe grapes, while dried fruits were used to heal constipation. For such complaints like cholera, smallpox, nausea, eye infections, skin, kidney, and liver diseases, fresh, ripe grapes were used (Badet 2011). Nowadays grape seed extract composes the majority of dietary supplements in the market containing phenolics. It is usually added in the quantity from 50 to 100 mg per capsule, tablet, or granule. Grape seed extract also found application in cosmetics dedicated to skin care and is widely added into different kinds of foods. The pharmacological effects exerted by grape seed extract such as antidiabetic, anti-inflammatory, lowering blood pressure, or reducing plasma cholesterol are related to the presence PPCs and OPCs with B-type linkages (Ashraf et al. 2015; Choy et al. 2013).

1.4 Vaccinium macrocarpon Aiton

Wild cranberries were discovered by Native Americans, who used its fruits as a food, fabric dye, and a traditional medicine. The primary medical application of cranberries was treatment of bladder and kidney ailments by American Indians (Boon and Smith 2004). Sailors used berries to prevent scurvy. Wounds and blood poisoning were also cured with cranberry fruits poultice (McKay and Blumberg 2007). Till now, cranberries are commercially grown in Canada, the USA, South America, and Europe. The fruits are usually processed before consumption due to low content of sugar and high amount of organic acids and PAs giving berries characteristic tart and astringent taste. Dried cranberries are popular snack and can be found as an ingredient in baked goods, bars, smoothies, trail mixes, cereals, and juice blends. Fresh berries are used for production of juices, jams, and sauces. The folk knowledge about V. macrocarpon usefulness in the treatment and prevention of urinary tract infections has been supported by numerous scientific evidence. The bioactive compounds found in cranberry juice are A-type proanthocyanidins, which were proved to inhibit the adhesion of Escherichia coli to the epithelium in urinary tract. The direct consequence of the interaction between bacteria and PAs is the inhibition of bacterial biofilm formation (Howell et al. 2005).

1.5 Cinnamomum verum J.Presl (syn. Cinnamomum zeylanicum Blume)

Cinnamomi cortex (the inner bark of Ceylon cinnamon) is known as a spice since the Middle Ages. Earlier, in ancient Egypt, it was used to embalm mummies and to burn in temples and during funerals. In the Middle Ages, cinnamon and other spices were transported by Arabs to Alexandria, Egypt, and then shipped to Europe (Gunawardena et al. 2014). In that time, cinnamon powder was an ingredient of medicines for sore throats and coughs. It was traditionally applied to heal toothaches, dental problems, and bad breath. The known folk application of cinnamon was intestinal spasms, nausea, stomach cramps, indigestion, loss of appetite, or diarrhea (Gunawardena et al. 2014). The effectiveness of cinnamon in prevention and treatment of type 2 diabetes, cancer, and inflammation has recently been shown. Its hypotensive and cholesterol-lowering effects were also promising (Mateos-Martín et al. 2012). Besides its medicinal usefulness, essence and aroma industries use cinnamon in cosmetics and perfumes and in different varieties of foodstuffs due to its characteristic fragrance and flavor. Some activities of cinnamon are attributed to volatile compounds present in the bark; however, majority of studied health benefits was associated with proanthocyanidins, which are main polyphenolic fraction found in commercial cinnamon. Cinnamon PAs are polymers with a degree of polymerization up to 11 and with a high proportion of A-type linkages (Mateos-Martín et al. 2012).

1.6 Sorghum bicolor (L.) Moench

Sorghum is a drought-tolerant crop, nowadays commonly produced in semi-arid regions of Africa, Asia, Australia, and North and South America. The earliest findings of wild examples of sorghum grains comes from circa 800–600 BCE from Qasr Ibrim in Egyptian Nubia. It was domesticated no earlier than CE 100 (Zohary and Hopf 2000). Traditionally sorghum was grown for grain; however, besides this primary need of food, now sorghum is also used for forage and sugar (sweet stalk) production. The bioactive compounds (polyphenols and lipids) are mainly located in the bran fraction of sorghum grains. These compounds are accompanied with the abundant cell wall polysaccharides and contribute significantly to the health benefits attributed to whole grain intake (Girard and Awika 2018). Consumption of sorghum grains is related to reduction of oxidative stress and chronic inflammation, prevention of cancer, improvement of glucose metabolism, prevention of insulin resistance, and improvement of lipid metabolism (Girard and Awika 2018). The PAs fraction in sorghum constitutes up to 5%, what is tenfold higher than in other grains. It is composed of catechin/epicatechin oligomers and polymers with the degree of polymerization up to 20 (Girard and Awika 2018).

2 Bioactive Constituents

PAs present a wide structure variability which is determined by stereochemistry at the chiral centers and the hydroxylation pattern of flavan-3-ol units. 2R-type flavan-3-ols are predominantly produced by plants whereas SR-type flavan-3-ols can rarely be found in monocotyledons and in selected dicotyledonous families like Rhus, Uncaria, Polygonum, Raphiolepis, and Schinopsis (He et al. 2008). The location and stereochemistry of the interflavan linkage between extension and end units as well as degree of polymerization (DP) also influences the complexity of final oligomeric and polymeric structures (He et al. 2008). Two main types of linkages between monomer units can be observed: A-type, when C2 position of the upper unit is linked to the oxygen at C7 or C5 position of the lower unit and C4 position of the upper unit is linked to the C8 or C6 of the lower unit (Fig. 1), and B-type, when C4 position of the upper unit is linked to the C8 or C6 of the lower unit (Fig. 2). Mixed-type PAs combine both kinds of linkages, which can be either α or β (Fig. 3) (He et al. 2008). The clear nomenclature of PAs includes corresponding flavan-3-ol monomers and indicates the configuration (described as α or β), location, and direction of interflavan linkage using parentheses with an arrow (→). The examples of names of oligomers are presented on Figs. 1, 2, and 3.

Fig. 1
figure 1

The structures of A-type proanthocyanidins

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Fig. 2
figure 2

The structures of B-type proanthocyanidins

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Fig. 3
figure 3

The structures of mixed-type proanthocyanidins

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The chemical term proanthocyanidins suggests they can produce anthocyanins. Indeed, catechin- and epicatechin-based oligomers or polymers are known as procyanidins because upon acid hydrolysis they yield anthocyanin aglycon cyanidin. Similarly, gallocatechin- and epigallocatechin-based polymers (prodelphinidins) are the source of anthocyanin aglycon delphinidin. The other subgroups of PAs classified according to the hydroxylation pattern are presented in the Table 1. Among these subgroups, procyanidins are the most common one. Mixed polymers composed of several types of units can also be found in nature (He et al. 2008; Sieniawska and Baj 2016; Wallace 2010). The main bioactive constituents of plants rich in PAs are presented in the Table 2.

Table 1 The hydroxylation pattern of proanthocyanidins
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Table 2 Bioactive constituents of plants rich in proanthocyanidins
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3 Bioavailability and Metabolism

Although the beneficial effects have been observed as a result of dietary PAs intake, a more in-depth knowledge of their metabolic pathways in human and animals is still necessary. The low absorption rate of PAs is well-known and can lead to favorable direct activity in the gastrointestinal tract. PAs are found in both forms of oligomers and polymers, but polymers are the most abundant in foods. While oligomers are absorbable in vivo, the high molecular weight of the polymers determines their poor absorbability in the gastrointestinal tract.

After ingestion procyanidins undergo metabolic modifications in the gastrointestinal tract. These modifications take place from mouth to colon and result in smaller oligomeric molecules or metabolites with higher bioavailability and thus higher in vivo bioactivity.

3.1 Influence of Saliva and Gastric Juice

It is known that the first organ involved in the modification and digestion of PAs is the small intestine. However, the role of the two fluids encountered before reaching the small intestine (saliva and gastric juice) must be investigated.

Even though PAs are not metabolized in the oral cavity, however, studies on the role of saliva in altering their structure have been conducted (Zanotti et al. 2015). PAs interactions with salivary proteins were investigated in a study using synthesized procyanidin dimers, C1 trimer, (−)-epicatechin O-gallate, and B2-3″-O-gallate from grape seeds. The results showed a higher tannin-specific affinity to saliva proteins of (+)-catechin, as well as of procyanidin dimers linked through a C4–C8 interflavan bond (de Freitas and Mateus 2001).

In a study conducted on green tea extracts of 5.0 mg/ml administrated as mouth rinse solution for caries prevention, it was found that, eight catechins were retained in saliva at μg/ml levels after 1 h (Tsuchiya et al. 1997).

Because the pH of the gastric juice generally varies between 1 and 3, it is necessary to know the behavior and stability of PAs in such an environment. Two hypotheses may be taken into account: acidic conditions lead to the degradation of PAs to monomers, and as a result, they would not get intact in the small intestine, or PAs are stable under acidic conditions, and in this case, they can be absorbed into the small intestine and detected in the plasma (Zhang et al. 2016). In vitro studies have been initiated to simulate the conditions encountered in the stomach, thus constituting a good indicator of the potential bioavailability of procyanidins in vivo (Spencer et al. 2001). The results showed that at pH ranging from 1.8 to 2, (−)-epicatechin was stable, while B2 dimer and procyanidins oligomers degraded into (−)-epicatechin, and dimers and monomers, respectively; on the contrary, while procyanidins oligomers were stable at alkaline pH, (−)-epicatechin and B2 dimer were unstable, and at nearly neutral pH, the latter was stable (Zhu et al. 2002; Kahle et al. 2011; Spencer et al. 2000; Zhang et al. 2016).

The results obtained in in vitro studies have not been completely confirmed in vivo. On the one hand, the number of such studies is still very small, and on the other hand, besides pH influence, the food matrix may play an important role in PAs digestion as well. In a study conducted by Rios et al. (2002), six human subjects consumed 500 ml of cocoa beverage containing 733 mg of OPCs. At pH of 6.5 monomers, dimers B2 and B5, C1 trimer, as well as oligomers had similar HPLC profiles in the gastric content extracts and in the cocoa beverage indicating that most of the ingested procyanidins reached the small intestine as intact molecules. This could be explained by the higher pH value (5.4) determined by the food bolus.

3.2 Metabolism in Small Intestine and Colon

The pathways of PAs absorption can be through the stomach or intestine. However if not absorbed at this level, PAs reach colon and undergo the modification by the microbiota. During this process, PAs come under catabolic or conjugation reactions, then pass into blood, and are subsequently eliminated either through the urinary bladder or through the bile; some unabsorbed ones are eliminated through feces.

Although the small intestine is considered the first organ involved in PAs digestion, there is no evidence of mammalian enzymes that can degrade this type of high molecular weight molecules. It has been estimated that only 5–10% of the total ingested polyphenols are absorbed in the small intestine. The rest is accumulated almost intact in the large intestine (Zanotti et al. 2015). In fact, only procyanidin B2 was found to be absorbed intact. In a study investigating the possibility of quantifying the plasma levels of procyanidins after consumption of a flavanol-rich cocoa, Holt et al. (2002) detected procyanidin dimer in the plasma of human subjects after 0.5 h. The concentration reached a maximum at 2 h after consumption (16 ± 5 nmol/L, 41 ± 4 nmol/L, respectively). The other oligomers, with higher polymerization degree, reached colon and were metabolized by the colonic microbiota (Williamson and Clifford 2017).

Because PAs are absorbed in the small intestine in small proportions, a massive accumulation of several hundred micromoles/l is obtained in the colon (Choy and Waterhouse 2014). The behavior of procyanidins oligomers and polymers in the colon is not fully elucidated, and the results of the studies are sometimes contradictory. However, it has been demonstrated that metabolites with lower molecular weight, such as phenyl valerolactone, phenylacetic acids, and phenylpropionic acids, result after procyanidins are catabolized by colonic microflora (Zhang et al. 2016). The potential biological effects of procyanidins are attributed to these gut microbiota metabolites, rather than parent compounds. The metabolites described are presented in Table 3.

Table 3 Metabolites produced by colonic bacteria
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In a study conducted on rats by Baba et al. (2001), urine absorption and excretion of (−)-epicatechin was monitored. After administration of both, cocoa powder (in different doses) and equivalent doses of the (−)-epicatechin, the sum of metabolites of (−)-epicatechin in plasma and urine, excreted after 18 h postadministration, was measured. The results showed a linear dose increase and, moreover, an equal level of the amount of (−) epicatechin metabolites in the urine for both types of administration.

Opposite results have been obtained by Donovan et al. (2002), in a study in which the rats were fed with catechins, procyanidin B3 dimer, and grape seed extract containing catechin, epicatechin, and a procyanidin mixture at a single meal. The results showed the presence of catechin and epicatechin conjugated forms, but the absence of procyanidins or their conjugates in plasma and urine. Also, the mixture present in the grape seed extract showed no bioavailable monomers or any effects on plasma levels or urinary excretion.

The microbial metabolism of two A-type procyanidins, procyanidin A2 and cinnamtannin B1, was studied by incubation in a pig cecum model. It was found that both A-type procyanidins were degraded by the microbiota; the procyanidin A2 was degraded by about 80%, while cinnamtannin B1 about 40% after 8 h of incubation. The main identified metabolites were 3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid, 3- hydroxyphenylpropionic acid, and 3,4-dihydroxyphenylacetic acid, respectively (Zhang et al. 2016; Engemann et al. 2012).

In a comparative study conducted in vitro and in vivo, Serra et al. (2011) investigated colonic metabolism for catechin, epicatechin, B2 dimer, epicatechin gallate (EGC), and epigallocatechin gallate (EGCG) by incubating each individual standard with rat fecal suspension for 48 h, by fermenting a cocoa cream with high-procyanidins content, previously digested by in vitro digestion, and also by in vivo analysis of rat large intestine and intestinal content after a single intake of the same nuts – cocoa cream. The results allowed the assumption of the existence of two metabolic pathways – dehydroxylation and rupture of the 1–2 bond of the C ring – considering the presence of diarylpropan-2-ol and 5-(3,4-dihydroxyphenyl)-c-valerolactone in the catechin and epicatechin fermentation mediums. The metabolism of ECG and EGCG did not yield the equivalent valerolactone with three hydroxylations, and the metabolites of dimer B2 differed from the ones of epicatechin, phenylacetic, and 4-hydroxyphenylacetic acids being the only common metabolites. In vivo verification of the metabolic pathways led to the quantification of phenylacetic acid, 3-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid as main metabolites and of two valerolactones 5-(hydroxyphenyl)-c-valerolactone and 5-(3,4-dihydroxyphenyl)-c-valerolactone in lower levels.

Several in vivo studies were conducted in order to detect the microbial-derived metabolites in urine, after intake of procyanidins-rich diets. As a result, an increase in 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenyl-γ-valerolactone was found in rats’ urine after being fed with procyanidin B3, but neither parent compound nor catechin derivatives could be detected (Choy and Waterhouse 2014; Gonthier et al. 2003). In case of procyanidin trimer C2 intake, the main metabolites found in urine were 3- hydroxyphenylvaleric, 3-hydroxyphenylpropionic acid, and m- coumaric acids (Gonthier et al. 2003). In another study, the absorption of procyanidins was investigated, after apple peel extract was administrated to rats catechin, epicatechin, procyanidin B1 and B2, and procyanidin C1 were detected in rat’s plasma, with a maximum level registered at 2 h after administration, and decreasing after until 24 h (Shoji et al. 2006).

In a study investigating the structurally related –(−) epicatechin metabolites present in human systemic circulation, after the consumption of a cocoa dairy-based drink containing –(−) epicatechin, Ottaviani et al. (2012) identified –(−)epicatechin –glucuronide, −(−)epicatechin sulfates as the main metabolites in humans, reaching a maximal plasma levels at 2 h after consumption of the test drink (589 ± 85 nM, and 331 ± 26 nM, respectively). Also unmetabolized –(−)epicatechin was detected in circulation but at low concentration of about 4 nM at 1 h after drink consumption.

Having complicated structures and high molecular weight, the absorption of procyanidins in the small intestine is very ineffective, and therefore the colon remains the main organ responsible for the bioavailability of the dietary procyanidins.

4 Bioactivities (Animal Aspects)

4.1 Extracts Rich in Procyanidins

The activity of procyanidins present in such plants as cocoa, grape, maritime bark, etc. is intensively studied in animal models (usually rats). Researches in this field are mainly focused on study of diabetes, coronary, or gastrointestinal diseases. Some other effects of procyanidins on other affections are less studied.

The activity of extracts and the main isolated compounds discussed in this chapter is presented in Table 4.

Table 4 Extracts and plants sources with the main compounds
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4.1.1 Antidiabetic Activity

Pinent et al. (2012), in their review article Procyanidins Improve some Disrupted Glucose Homoeostatic Situations: An Analysis of Doses and Treatments According to Different Animal Model, analyzed the potential beneficial effects of PAs, in situations in which glucose homeostasis is disrupted. They observed that several authors have assayed the ability of procyanidin-enriched extracts to ameliorate the physiological state caused by inability to synthesize and/or secrete functional insulin (hyperglycemia in type 1 diabetes). Maritim et al. (2003) used the commercially available Pycnogenol product (at a dose of 10 mg/kg) for the treatment of streptozotocin-induced diabetes in female Sprague Dawley rats (14 days treatment). A decrease of serum glucose level at day 15 was observed. At the same time the safety of tested product was confirmed on normal or diabetic rats. The results obtained by the authors (elevated levels of reduced glutathione and glutathione redox enzyme activities) demonstrated the ability of Pycnogenol (standardized extract of Pinus maritima consisting of a mixture of procyanidins) to scavenge free radicals altered oxidative stress and to increase the activity of hepatic-glutamyltranspeptidase, which normally hydrolyzes glutathione and thiol derivatives and conserves cysteine levels.

Lee et al. (2007) evaluated the antidiabetic and antioxidant potential of PAs obtained from persimmon peel on male Wistar STZ-diabetic rats. At 24 h from the last dose, the decrease in serum glucose and glycosylated protein was observed.

The probable mechanisms involved in beneficial effects of PAs in diabetes mellitus are limitation of post-prandial glycemia increases, inhibition of α-glucosidases and α-amylases, and inhibition of transporters involved in glucose uptake (e.g., glucose transporter type 2 – Glut2) (Kwon et al. 2007).

In long-term treatments, procyanidins which are administered in a daily treatment are more effective than when given with food, which indicates the presence of mechanisms of action other than those inhibiting carbohydrate digestions and/or glucose absorption. Thus these compounds are acting as an insulin-mimetics (Pinent et al. 2012).

Other authors suggest that procyanidins have insulin-mimetic effects in adipose tissue and muscle. Montagut et al. (2009) proved that oligomeric PAs of a grape seed extract interact and induce the autophosphorylation of the insulin receptor in order to stimulate the uptake of glucose, however, in different manner than insulin. It was found that Akt (protein kinase B) and MAPK proteins are essential for PAs signaling mechanisms. The ability of PAs to mimic insulin effects in insulin sensitive targets was demonstrated also in healthy animal models when insulin is scarce or absent. Wistar rats and Zucker lean rats used as healthy model simultaneously with diabetic animals demonstrated no changes in glucose level after 8 and 24 weeks of treatment, respectively (Agouni et al. 2009; Li et al. 2009).

4.1.2 Anticancer Activity

Yamagishi et al. (2002) observed that cacao liquor procyanidins inhibit in vitro mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and rat pancreatic carcinogenesis in the initiation stage. However such promising results were not confirmed in rat mammary carcinogenesis induced by PhIP model. The same authors evaluated also chemopreventive properties of cacao liquor proanthocyanidins against lung carcinogenesis in F344 male rat multi-organ carcinogenesis model. PAs were effective as chemopreventive agents in the lung cancer; however, none protecting influence in other major organs was observed (Yamagishi et al. 2003).

Mittal et al. (2003) studied how grape seeds procyanidins can prevent UVB radiation-induced photocarcinogenesis and malignant conversion of benign papilloma to carcinomas in SKH-1 hairless mouse model. They introduced PAs into the diet of hairless mice in order to demonstrate the effect of the compounds on different stages of photocarcinogenesis, such as UVB-induced tumor initiation stage, UVB-induced tumor promotion stage and UVB-induced complete carcinogenesis. The used composition was based on dimers (containing procyanidin B1, procyanidin B2, procyanidin B3, procyanidin B4, and procyanidin B5) - 6.6% of total grape seed procyanidins, trimers (containing procyanidin B5-3′-gallate and procyanidin C1) – 5.0%, tetramers – 2.9%, and oligomers – 74.8%. The results presented by the research group showed that this diet prevented photocarcinogenesis, in terms of tumor incidence, tumor multiplicity, and tumor size. PAs showed also protective action against UVB-induced skin tumorigenesis in all the three stages of multi-stage carcinogenesis. These compounds have inhibitory effect on malignant conversion of benign skin papilloma to carcinomas in SKH-1 hairless mice. The observed positive effects were explained by probable antioxidant activity of PAs (Mittal et al. 2003).

4.1.3 Neurological Diseases

The administration of procyanidins in neurological diseases was also studied. PAs were showed to effectively protect ischemic neurons, but the mechanism remains poorly understood. The intraperitoneal administration of ginkgo proanthocyanidins in Sprague Dawley rats mitigated neurological disorders, shortened infarct volume, increased superoxide dismutase activity, and decreased malondialdehyde and nitric oxide contents. PAs inhibited inflammatory reaction and activated survival pathways after ischemia/reperfusion injury (Cao et al. 2016).

4.1.4 Metabolic Syndrome

Ibars et al. (2017) studied the effects of procyanidins from grape seeds on rats with diet-induced obesity. Dietary obesity is usually linked with hypothalamic leptin resistance, and the authors presented the role of proanthocyanidins on hypothalamic leptin/STAT3 (signal transducer and activator of transcription 3) signaling and pro-opiomelanocortin gene expression on male Wistar rats fed either a standard chow diet or a cafeteria diet for 13 weeks, followed by treatment with grape seed proanthocyanidin extract. The treatment activated the hypothalamic leptin receptor-STAT3 pathway, ameliorated food intake but did not reverse the obesity and hyperleptinemia. Rats treated with procyanidins did not display a significant body weight reduction indicating that the doses were not sufficient to totally reverse leptin dysfunction induced by a high-fat diet.

Osakabe and Yamagishi (2009) investigated cacao procyanidins (CP) effects on plasma lipid levels in high cholesterol-fed rats. The experiments were developed on 9-week-old male Sprague Dawley rats. They demonstrated that ingestion of cocoa procyanidins reduced plasma total cholesterol levels in rats fed with high cholesterol diet, and the accumulation of cholesterol and triglyceride in liver was significantly decreased. The PAs precipitated micellar cholesterol, and this ability was dependent on the molecular weight of the compounds (monomers to tetramers).

The effect of an extract enriched in the flavan-3-ols procyanidin dimmers on obesity-related disorders via estrogen receptor alpha (ERα) was investigated by Leonetti et al. (2018). The investigated obesity-related cardiovascular and metabolic disorders with a particular interest in the role/contribution of ERα revealed that procyanidins reduced adiposity, plasma triglycerides, and oxidative stress in the heart, liver, adipose, and skeletal tissues, but did not improve the vascular dysfunction in a 2-week treatment. The heart structure and function were not affected by the diet, nor the grape seed extract supplementation and reactive oxygen species production in aorta was not significantly modified. The treatment improved muscle function by activating β-oxidation and by increasing mitochondrial functionality. ERα was identified as an important target involved in the reduction of fat accumulation.

4.1.5 Antithrombotic Activity

Zhang et al. (2011) studied antithrombotic effect of grape seed proanthocyanidins. Deep vein thrombosis was induced in rats, and a recipe based on 400 mg/kg procyanidins was applied. The authors observed that active compounds reduced thrombus length and weight and protected the integrity of the endothelium, inhibited thrombogenesis-promoting factors, and promoted thrombogenesis-demoting factors CD34, vascular endothelial growth factor receptor-2, and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type one motif, member 13). The antithrombotic properties of proanthocyanidins were associated with endothelial protection and regeneration, platelet aggregation, and inhibition of inflammatory cell and thrombus adhesion.

4.1.6 Other Activities

Schizophrenia is mental disorder with disturbances in emotion, perception, cognition, and social function. Although this illness affects 1% of the population worldwide, fundamental neuropathology of schizophrenia remains unexplained. Tian et al. (2018) demonstrated how procyanidin B2 protects myelin integrity in cuprizone-induced schizophrenia in mice. They evaluated the effects of procyanidin B2 widely available in food on behavioral impairment in mice exposed to cuprizone for 5 weeks. Behavioral impairment tests, myelin integrity assay, and myelin basic protein (MBP) expression revealed that procyanidin B2 could mitigate behavioral impairment and protect myelin integrity via regulating oxidative stress by activating nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling.

Yamakoshi et al. (2002b) studied the effect of PAs from grape seeds on cataract formation on a 39 male ICR/f group of rats. The diet was based on mixture of procyanidin oligomers (DP between 2 and 10) but also polymers in total concentration of 38.5% and 2.4% monomeric flavanols such as (+)-catechin. PAs were found to significantly prevent and postpone the development of cataract formation in rats. The comparison of effectiveness of procyanidin structures with different degrees of polymerization resulted in conclusion that procyanidin pentamer to heptamer and procyanidin oligomers more than decamer groups were significantly more efficient in lowering plasma cholesteryl ester hydroperoxide levels comparing to the procyanidin dimer to tetramer group. The antioxidant properties of PAs and their metabolites were suggested to contribute to the inhibition of progression of cataract formation.

5 Benefits (Human Studies)

The 67 clinical trials involving PAs can be found in Cochrane Database of Systematic Reviews. Some of them are exemplified in the next subchapters, being classified according to the treated diseases.

Additionally, a summary of clinical trials investigating procyanidins and meta-analysis of these is presented in Table 5. An important source of PAs used in such cases are fruits and berries (blueberries, cranberries, and black currant), some legumes (peas and beans), hazelnuts, pistachios, almonds, walnuts and cocoa, and spices as cinnamon. From the fruits we can mention apples, chokeberries, strawberries, and green and red grapes (especially grape seeds) and the product obtained from grapes: the wine.

Table 5 Summary of clinical trials investigating procyanidins
Full size table

5.1 Cancer

As a disease of the century with a particular severity, cancer is considered the second leading cause of death worldwide. The major concerns of the scientific and medical world are to find cures for this disease, due to the fact that conventional cancer therapies result in serious side effects and, at best, merely extend the patient’s life span by a few years. Formulations based on natural products can be considered good alternatives to traditional treatments.

Procyanidins obtained from grapes and grape seeds are considered good candidates for the anticancer formulations. Santos-Buelga and González-Manzano (2011) considered that excessive alcohol consumption increases the risk of liver cirrhosis and cancers; however, low to moderate red wine consumption has been associated to health-promoting properties. Among the wine phenolics, the authors assign the claimed health effects to procyanidins (such as oligo- and polymeric flavan-3-ols) and to resveratrol.

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world, with a high percent of incidence among all types of cancers. Jin et al. (2012) have studied the effect of dietary flavonoids on the incidence of colorectal adenoma and CRC. At the start of Jin’s study, only few researchers investigated the association between procyanidins and colorectal neoplasms, while others reported the effects of six flavonoid subclasses (flavonols, flavones, flavan-3-ols, procyanidins, flavanones, and phytoestrogens) (Theodoratou et al. 2007). Theodoratou’s results showed a statistically significant decreased risk of CRC with a high intake of procyanidins (22% reduction in CRC risk associated with the highest quartiles of intake versus the lowest quartile, odds ratio 0.78, Ptrend = 0.031). Their research method was based on the analysis of metadata up to July 2011 in the Cochrane Library, PubMed, EMBASE, and other CINAHL databases and reference lists of previous reviews. Eight studies with 390,769 participants were included. Five studies used a prospective cohort design, two case-control studies, and one a randomized controlled trial (RCT). The methodological quality was measured using the Newcastle Ottawa scale (NOS).

In the clinical trial developed by Hamilton et al. (2015), PAs were evaluated for their potential to attenuate the adverse effects of cancer radiotherapy. The authors used commercially available standardized cranberry capsules, containing 72 mg of proanthocyanidins, for the evaluation of their effect in prevention and treatment of radiation cystitis in prostate cancer patients. The authors reported decrease in cystitis incidence in treated patients compared with control group, recommending the use of cranberry capsules as a support treatment in prostate cancer patients experiencing common side effects, such as radiation cystitis and inflammation of the bladder.

Rossi et al. (2013) examined the relation between dietary biologic active compounds and endometrial cancer, from an Italian case-control study including 454 incidents, histologically confirmed endometrial cancers, and 908 hospital-based controls. They estimated the intake in proanthocyanidins from fruits and vegetables through statistical tests and concluded that high consumption of proanthocyanidins may reduce endometrial cancer risk. In their study, the authors identified, for the Italian population, as major source of proanthocyanidins monomers and dimers the wine, apples, pears, peaches, apricots, and prunes while for trimers and superior proanthocyanidins the apples, pears, wine, vegetables/bean soups, chocolate, pulses, and grapes.

5.2 Cardiovascular Disorders

The use of biologic compounds from plants and fruits offers potential benefices in human health. In the literature there are many papers which describe the role of dietary food, also clinical trials which prove an improvement of health. In the comprehensive review paper, Schroeter et al. (2010) listed several studies on the potential health benefits of dietary flavanols and procyanidins, especially in the context of cardiovascular health. The work was revisited 8 years later (Ottaviani et al. 2018) by authors describing significant developments in understanding of dietary flavanols and procyanidins in the human health and nutrition. In spite of these findings, the recent progress was considered by the authors insufficient for the development of dietary guidance on the flavanols and procyanidins intake or for the establishment of a minimum intake necessary to achieve health benefits.

The effects of cocoa procyanidins on vascular function were investigated in the trial NCT02728466 (Heiss and Kelm 2016). The study started in 2014 involved group of 45 participants. The tests were performed with cocoa-based supplement containing flavanols (monomers) and procyanidins (dimers to decamers) and a placebo comparator (flavanol and procyanidin deprived supplement). The study evaluated the influence of the sustained intake (2x daily over 1 month) of the macro- and micronutrient matched supplement over the baseline endothelial function. Post-consumption, endothelial function, plasma flavanol metabolites, urinary flavanol metabolites, urinary valerolactone metabolites, pulse wave velocity, blood pressure, and high- and low-density lipoproteins were measured at different times. The obtained results showed that the consumption of cocoa extract containing 130 mg (−)-epicatechin and 560 mg procyanidins led to the increase of flow-mediated vasodilation and of the concentration of (−)-epicatechin metabolites in the circulatory system and to a decrease of the pulse wave velocity and blood pressure. The total cholesterol decreased for both the above described recipe and for another extract (containing 20 mg (−)-epicatechin and 540 mg procyanidins).

De Palma et al. (2016) assessed the potential therapeutic value of a high dose of cocoa flavanols in patients with chronic heart failure. The reductions in N-terminal pro-B-type natriuretic peptide (NT-proBNP) were used as an index of improved cardiac function. The study was a single-center randomized double-blind placebo-controlled investigation with a crossover design with exclusion criteria: age <45 years, diabetes mellitus; LV (left ventricular) dysfunction not related to systolic HF (heart failure) or ischemic heart disease; exertional angina; atrial fibrillation; cardiac surgery, percutaneous coronary intervention, acute coronary syndrome, or stroke within 6 months of the study; active psychiatric or psychological illness; and life expectancy. The patients’ diet was rich in flavanols, especially procyanidin dimers and trimers to decamers. This study indicates that combining cocoa flavanols with guideline-directed medical therapy has potential for improving cardiac function in chronic heart failure, changing blood pressure, decreasing flow-mediated dilatation, using reductions in N-terminal pro-B-type natriuretic peptide (NT-proBNP) as an index of improved cardiac function.

Another clinical trial indicates apples as a source of compounds which can be associated with source of reducing risk of cardiovascular disease (CVD) (Kroon 2016). The effect of the ingestion of epicatechin-rich flavanol extract and isolated apple procyanidins on systolic blood pressure (BP) and other cardiometabolic biomarkers was studied in the cited paper. Low epicatechin and procyanidin doses, high epicatechin and procyanidin doses, high procyanidin only and as control, no epicatechin, and procyanidin were administrated for a group of 42 participants. The results of the study showed that none of the isolated flavanol treatments significantly changed systolic or diastolic BP (peripheral and aortic), plasma nitric oxide (NO) reaction products, or measures of arterial stiffness, suggesting that, in isolation, neither monomeric flavanols nor PCs affect BP, blood lipid profiles, endothelial function, or glucose control.

Some studies demonstrated that moderate daily consumption of wine is associated with a lower risk of coronary heart disease. Khan et al. (2015) described the effects of oligomeric procyanidins (OPCs) on vascular endothelial function. They provided an explanation for the reduced incidence of coronary heart disease in red wine drinkers. OPCs were proved to induce atheroprotective changes in vascular function through oxidant signaling mechanisms originating from the mitochondrial electron transport chain.

The efficacy of procyanidins obtained from grape seed extract to decrease blood pressure was investigated by Belcaro et al. (2013). In this study, a controlled group formed by 119 healthy pre- and mildly hypertensive subjects were involved. After 4 months of treatment with two dosages of procyanidins (150 and 300 mg/day), microcirculation state and plasma oxidative status were evaluated. The treatment had beneficial cardiovascular effects that complement current intervention strategies in the hypertension area. The effect on blood pressure (decrease of blood pressure, decrease of the diastolic pressure) and oxidation of membrane lipids (endothelial dysfunction, formation of oxidized LDL, and activation of phagocytic cells) were observed. The compounds improved endothelial function and promoted microcirculation decreasing the plasma oxidative status.

Valls et al. (2016) reported the effects of Oligopin® (extract from French maritime pine bark with low molecular weight procyanidins) in a clinical trial formed by 24 participants on cardioprotective effects. The randomized patients received a placebo 2 times a day or Oligopin® quantified extract also 2 times a day for 5 weeks each. The measured parameters were blood pressure, lipid profile, anthropometric variables, and other CVD risk biomarkers. The consumption of Oligopin® improved lipid cardiovascular profile and represents one of the scarce ways to increase HDL-c in stage-1-hypertensive subjects. In addition, it also tends to improve systolic BP and LDL oxidation.

5.3 Diabetes

Diabetes is a disease characterized by the increase of blood glucose levels. Gonzales-Abuin et al. (2015) suggested that procyanidins help in the hemostasis of glucose in various tissues, reducing the lipogenesis and modulating the secretion of insulin in pancreatic cells. Sun et al. (2016) reported proanthocyanidins as retina protector against early diabetic injury by activating the Nrf2 pathway. Even though the exact mechanisms involved remained unclear, the authors present several previous studies suggesting mechanisms responsible for the protective effect of grape seed procyanidin extracts.

Vanschoonbeek et al. (2006) showed no significant reduction in glucose values using cinnamon diet in postmenopausal type 2 diabetes patients, finding no time × treatment interactions for whole-body insulin sensitivity or oral glucose tolerance, nor changes in the blood lipid profile of fasting subjects following cinnamon supplementation. At the same time, the study of Mang et al. (2006) on the effects of cinnamon aqueous extract in adult diabetes patients on oral hypoglycemic treatment revealed a moderate decrease of fasting plasma glucose, but no effect on glycated hemoglobin A1c, serum lipids, or blood coagulation parameters.

Akilen et al. (2010) studied the effect of cinnamon administered daily (2 g) over a period of 12 weeks in a placebo-controlled double-blind clinical trial. The study demonstrated a significant reduction in blood pressure, fasting plasma glucose, and body mass index, suggesting the potential of cinnamon supplementation in a daily diet to regulate blood glucose and blood pressure levels.

Anderson et al. (2015) demonstrated a significant decrease of fasting insulin, glucose, total cholesterol, and LDL cholesterol and the increase of insulin sensitivity at the end of a 2 months trial after treatment with 500 mg cinnamon water extract daily.

5.4 Urinary Infections

Several clinical trials describe the effect of procyanidins on urinary infections. In a randomized controlled 12-month follow-up trial, Kontiokari et al. (2001) described the effect of administration of 50 ml of cranberry-lingonberry juice concentrate daily for the reduction of urinary tract infection recurrence. The results showed a 20% reduction in absolute risk in the cranberry group compared with the control group (16% recurrence in the cranberry group, compared with 36% recurrence in the control group).

The study of Foxman et al. (2015) evaluated the therapeutic effect of cranberry juice capsules in preventing urinary tract infections (UTI) after surgery. The results showed a decrease by half of the UTI for women undergoing elective benign gynecological surgery involving urinary catheterization (19% vs. 38% for the control group).

The trial developed by Wan et al. (2016) studied the protective effect of highly concentrated cranberry juice against urinary tract infections, using 55 uncircumcised boys and 12 circumcised boys, from 6 to 18 years old, with histories of urinary tract infections. The results showed that prophylactic treatment with cranberry juice led to the reduction of bacteriuria incidence (especially E. coli) to 25%, compared with the negative control group (37%) and positive control group (33.3%). The protective effect of the cranberry juice was assigned to its proanthocyanidins content.

5.5 Other Diseases

Rumex acetosa L. (garden sorrel), known as a plant with high content of oligomeric and polymeric proanthocyanidins and flavonoids, was used in the clinical trial NCT02039648 in order to establish the prophylactic potential as mouth rinse on periodontitis (especially on Porphyromonas gingivalis, one of the major pathogens associated with the onset and progression of periodontitis) (Beikler 2014). Changes of the intraoral prevalence of Porphyromonas gingivalis, Approximal Plaque Index and Sulcular Bleeding Index were observed from baseline to 7 and 14 days; changes of cytopathological appearance of the mucosal tissue were observed from baseline to 7 days.

Heinrich et al. (2006) evaluated the contribution of procyanidins-rich cocoa to endogenous photoprotection. Their results suggested that a 12 weeks diet containing high doses of procyanidins (having as main compounds the monomers epicatechin – 61 mg/day and catechin – 20 mg/day, total procyanidin oligomers – 247 mg) provided photoprotection against UV-induced erythema, significant decrease of skin roughness and scaling.

The procyanidin mixture Pycnogenol® (extracted from pine tree bark) was evaluated for application in treating attention deficit hyperactivity disorder (ADHD). The product was reported to decrease the hyperactivity and impulsivity in a one-patient study (Heimann 1999). The study of Trebaticka et al. (2006) on 61 patients with ADHD found beneficial effects of Pycnogenol® at a dose of 1 mg/kg and an administration period of 1 month. A very interesting conclusion of the study of Trebaticka et al. was that the effect was sex-related (being more effective in boys). Administration of Pycnogenol® caused a significant reduction of hyperactivity, improved attention and visual-motoric coordination, and concentration of children with ADHD. At the same time, addition of Pycnogenol® to the classic ADHD treatment with dextroamphetamine resulted in superior effects.

6 Application in Food (Including Correctly Cooking Foods Rich in Phytochemicals)

Proanthocyanidins in general (among which procyanidins are the most common) can be found in a series of foods, such as fruits (avocado, bananas, blackberries, grapes, plums, etc.), vegetables (broad beans, etc.), nuts (almonds, peanuts, pistachios, etc.), grains (barley, buckwheat, red beans, etc.), spices (cinnamon, curry, etc.), and beverages (beer, grape juice, tea, wine, etc.). A very useful database regarding the procyanidin content of different foods is maintained by the United States Department of Agriculture (Haytowitz et al. 2018). Table 6 presents the content of proanthocyanidins in common fruits and vegetables.

Table 6 Maximum total proanthocyanidins (PAs) content in common foods (monomers not presented) (Gu et al. 2001; Haytowitz et al. 2018)
Full size table

Several procyanidin-rich products are also commercially available. Cranberry extract powder is considered as food supplement which can be used in fruit-flavored and isotonic drinks, tea drinks, vitamin-enhanced waters, and yogurts. Grape seed powder also exists on the market. It which can be added to food directly, used in cooking and baking, diluted in water or other drinks, or used as an ingredient in more complex products such as protein powders, dietary supplements, functional foods and drinks, and sports nutrition. Another source rich in procyanidins is cocoa; it can be used as powder in cookies, chocolate, etc., and unheated and untreated at temperatures allow it to keep its nutrient-dense qualities.

Due to their antimicrobial and antioxidant properties, procyanidins could be successfully applied for extending the shelf lives of different meat products (Jeong et al. 2015). The cited study presents the effect of procyanidins (extracted from grape seeds, no further details on composition provided) on samples of pork patties: the samples had higher redness and yellowness values, as well as lower registered values for volatile basic nitrogen, 2-thiobarbituric acid reactive substance, and total bacterial counts assays.

The correct cooking of foods rich in procyanidins represents the subject of only a few research papers. Generally speaking, the processing step reduces the higher procyanidin content. Rodriguez-Mateos et al. (2014) showed that the thermal treatment has more pronounced effect on the higher procyanidins (nonamers and decamers disappeared, in the same time, the dimers and trimers having increasing values), with no significant changes in the total procyanidin content. Similar behavior was observed after extrusion of freeze-dried blueberry pomace at 180 °C or after processing berries into juiced, canned, and pureed products.

The study of Stahl et al. (2009) on the influence of preparation of cocoa-containing food on recovery of PAs revealed that the temperature was not the key factor in procyanidin loss but the leavening agents used. Comparing the effect of baking soda (sodium bicarbonate) and baking powder (mixture of baking soda and acidic ingredients) used as leavening agents, the authors showed that the key factor in procyanidin loss was represented by the pH level (higher recoveries of procyanidins being recorded at lower pH values reached using baking powder).

The literature data suggests that, due to the reduction of higher procyanidins on processing, the consumption of raw products it is recommended, if possible.

7 Safety: Toxicity and Side Effects

The intake of PAs can be beneficial for different diseases, but the most important is to find the optimal doses at which the biologic compounds are safe and have no adverse reactions. Ottaviani et al. (2015) investigated the effects of cocoa flavanols (CF) (including procyanidins) intake amount and intake duration on blood pressure, platelet function, metabolic variables, and potential adverse events. The limitations of their study lie in its duration and the number of volunteers studied; however, due to the lack of the studies on human subjects (up to 2015), the authors suggested that their study provides relevant and needed information for current safety assessments. The intake of cocoa flavanols (including procyanidins) up to 2000 mg/day for 12 weeks was well tolerated in healthy adults; the consumption was not associated with significant changes in blood pressure or platelet function.

Safety assessments of grape seeds extract (rich in procyanidins) have been conducted using animal models by Ray et al. (2001) and Yamakoshi et al. (2002a). Ray et al. (2001) conducted acute oral toxicity, dermal toxicity, dermal irritation, and eye irritation studies administrating doses of PAs from grape seeds extract. LD50 was found to be greater than 5000 mg/kg in albino rats via gastric intubation and greater than 2000 mg/kg when administrated dermal. The examination of other organs like the brain, duodenum, heart, kidney, liver, lung, pancreas, and spleen did not reveal any abnormalities. The serum chemistry was also not changed in female mice at chronic administration. The acute and subchronic oral toxicity of PAs from grape seeds extracts was also performed on Fischer 344 rats (Yamakoshi et al. 2002a). The authors evaluated mutagenic potential by the reverse mutation test using Salmonella typhimurium, the chromosomal aberration test using CHL cells, and the micronucleus test using ddY mice. No signs of toxicity were found indicating the safety of preparation. Lluís et al. (2001) confirmed the previous results describing LD50 of PAs from red grape marcs (variety Syrah) as being higher than 5000 mg/kg.

Due to the lack of consistent data regarding evaluation of the safety and tolerability of continuous intake of oral grape seeds extract, Sano (2017) conducted a 4-week toxicity test with daily doses of 1000–2500 mg proanthocyanidin-rich grape seeds extracts in healthy Japanese volunteers. Measured physical parameters as body weight, blood pressure, and pulse rate showed that tested doses were well tolerated, and subject compliance was 100%.

The potential systemic toxicity of Oligopin® was evaluated by Segal et al. (2018). The preparation was showed to be not acutely toxic via oral administration at up to 2000 mg/kg and was well tolerated following repeated oral administration to Sprague Dawley rats, with a NOAEL (no-observed-adverse-effect level) of 1000 mg/kg/day.

Cranberry extract powder is considered a food supplement and that’s why it was emitted an opinion pursuant to Regulation (EC) No 258/97 of the European Parliament and of the Council. None of the clinical trials conducted on this product indicated adverse reactions. The Panel (Turck et al. 2017) which analyzed the safety of this product took into consideration the studies of Cos et al. (2003), and Prior and Gu (2005), regarding absorption, distribution, metabolism, and excretion of this biologically active compounds, and some clinical studies on human subjects (Valentova et al. 2007).

The no-observed-adverse-effect level (NOAEL) of a chronic toxicity study of procyanidins from edible plant extracts is different according to the study or clinical trial which is reported.

The analytical standards available have safety data sheets, covering some aspects regarding their toxicity. For example, the monomers catechin and epicatechin have oral LD50 values (mouse and rats) >10,000 mg/kg and 1000 mg/kg, respectively, while for the dimers, trimers, and higher procyanidins, there are no acute toxicity data available. The GHS (Globally Harmonized System of Classification and Labelling of Chemicals) hazard statements usually presents the hazard statements H315 Causes skin irritation, H319 Causes serious eye irritation, and H335 May cause respiratory irritation and several precautionary statement codes for the monomers and other available procyanidins (e.g., Procyanidin B2, Procyanidin B3, etc.). The hazards and precautionary statements should be read carefully and understood, while the general safety regulations in the laboratory should be respected when working with procyanidins fractions or purified procyanidins.

8 Marketed Products

Several products based on PAs are present on the market. These are mainly herbal drugs (containing extracts), herbal products, dietary supplements, and functional foods: Pine Bark Extract 95%®, Enovita®, Oligopin®, Pycnogenol®, Leucoselect®, Endotélon®, Masquelier’s®, Anthogenol®, Enzogenol®, etc.

Enovita® is a food-grade grape seed proanthocyanidin extract (seed extract from Vitis vinifera L.) specifically designed for the food market, developed by Indena (Milan, Italy), with a content more than 95.0% of proanthocyanidins determined by spectrophotometry, 5.0–15.0% of catechin, and epicatechin determined by high-performance liquid chromatography (declared by the company). Belcaro et al. (2013) developed a registry study on 19 healthy, pre- and mildly hypertensive subjects regarding the efficacy of a standardized grape seed procyanidins extract (GSPE, Enovita®) to decrease blood pressure when associated with nondrug intervention. According to Belcaro report, Enovita contains ca. 8.6% monomeric procyanidins (catechin, epicatechin, and epicatechin gallate) and ca. 91% proanthocyanidins (OPC), of which 9% are of the dimeric type. A decrease of systolic blood pressure was observed at month 1, but the decrease was significantly higher in the treatment group.

French Maritime Pine Bark extract (FMPBE) rich in procyanidolic oligomers was tested as Oligopin® (PureExtract, DRT, France) in different clinical trials or studies on animals, where the systemic toxicity and mutagenicity were evaluated. Segal et al. (2018) have evaluated systemic toxicity of Oligopin® (obtained from the pine tree Pinus pinaster) in an acute oral limit test and a 90-day repeated dose oral toxicity study with Sprague Dawley rats. The researchers assessed potential mutagenicity in a bacterial reverse mutation assay and in vitro mammalian chromosome aberration assay with human lymphocyte. The results of their tests indicate that Oligopin® was nongenotoxic in both bacterial and human cell assays, was not acutely toxic via oral administration at up to 2000 mg/kg, and was well tolerated following 90 days of oral administration to rats, with a no observed adverse effect level of 1000 mg/kg/day. The composition of the recipe used is procyanidolic oligomers, out of which dimers constituted 15–20%, trimers 15–20%, and tetramers and higher oligomers 30–40%.

The effect of Oligopin® consumption on blood pressure of randomized group of 24 people with mild/moderate degree of hypertension was evaluated in the clinical trial NCT02063477 conducted by Technological Center of Nutrition and Health, Spain (Valls and Sola 2014). The differences in the time of evolution of blood pressure in both two arms of intervention were observed. Other measured parameters were endothelial function, biomarkers related with endothelial function, and biomarker related with inflammatory processes.

Poussard et al. (2013) reported that heat-shock protein beta-1 (HSPB1) is modulated by Oligopin®. Oligopin® prevented the stress-induced phosphorylation of HSPB1 and its subsequent structural modification. This supports the therapeutic usefulness of preparation for preventing the age-related muscle mass loss and for protecting muscle cells from oxidative stress. Valls et al. (2016) reported Oligopin® as being characterized by a practical absence of other tannins (<1%) and a high content in low molecular weight oligomeric procyanidins (OPCs >70%; dimers about 20%).

Pycnogenol® (Horphag Research Ltd., UK, Geneva, Switzerland) is a standardized extract with many benefices for human health due to its chemical composition. It is a standardized plant extract obtained from the bark of the French maritime pine Pinus pinaster Aiton (formerly known as Pinus maritima), subspecies Atlantica des Villar. With a reach composition in procyanidins comprising of catechin and epicatechin subunits with varying chain lengths, it is now utilized throughout the world as a nutritional supplement and as a phytochemical remedy for various diseases ranging from chronic inflammation to circulatory dysfunction, including several impaired psycho-physiological functions (D’Andrea 2010). This supplement is good in the treatment of attention deficit hyperactivity, osteoarthritis, type 2 diabetes, and cardiovascular diseases. Pycnogenol® can be used to slow the aging process, maintain healthy skin, improve athletic endurance, and improve male fertility. Jessberger et al. (2017) reported cellular pharmacodynamic effects of Pycnogenol® in randomized controlled pilot study with 33 patients with severe osteoarthritis. Grether-Beck et al. (2015) reported Pycnogenol®’s effects on human skin (photoprotection, reducing hyperpigmentation, improving skin barrier function, as well as extracellular matrix homeostasis).

Leucoselect® is a standardized extract from grape seeds developed by Indena (Milan, Italy) with a content of 95.0–100% of proanthocyanidins, determined by spectrophotometry, and catechin and epicatechin between 13.0 and 19.0% evaluated by HPLC. Nuttall et al. (1998) reported Leucoselect® as a placebo treatment in a clinical trial aiming to evaluate the effects of a capsule formulation of an antioxidant polyphenolic extract of grapes on serum total antioxidant activity and vitamin C and E levels. As results they reported that the extract had no effect on serum vitamins C and E levels but increased total serum antioxidant activity. To further improve their bioavailability, Leucoselect® has been formulated with soy (non-GMO) phospholipids (1:3 w/w), thus generating Leucoselect® Phytosome®.

Several other extracted from lychee fruits and grape seeds rich in PAs are currently commercialized under different names (Oligonol®, Vitaflavan®, MegaNaturalTM).

Besides those marketed products, several foods and beverages with high PAs content are available (as previously presented in Sect. 6) and could provide the necessary daily intake of PAs for a healthy and equilibrated diet (Bhagwat and Haytowitz 2015).

9 Patents

One of the first patented recipes based on PAs was Jack Masquelier patent (patent number 4.698.360 from 1987) “Plant extract with a proanthocyanidins content as therapeutic agent having radical scavenger effect and use thereof,” where the author’s invention relates to the use of a plant extract with a PAs content as therapeutic agent with radical scavenging effect as well as the use of pharmaceutical compositions containing this extract as active ingredient. The recipe is based on bark conifers (Pinus maritime Lam.) rich in biological active compounds.

The invention of Soulier et al. (2012) relates to the process of extraction involving the use of cranberries (Vaccinium macrocarpon) as a raw material, crushed fruits, or already pre-purified extracts rich in PAs. This invention allows to obtain extracts containing more than 10% PAs, using hydroalcoholic extraction and absorption on a macroreticular polymeric resin. Venkatramesh et al. (2013) describe in their patent the production and extraction of procyanidins from plant cell cultures of Theobroma or Herrania. Schmitz et al. (2017) patented “Compositions and methods of use of A-type procyanidins.” The authors extracted procyanidins from peanut skin and investigated characterized compounds for their effect on nitric oxide (NO) production and vasorelaxation using serum-free human umbilical vein endothelial cell (HUVEC) culture system in vitro and rabbit aortic ring ex vivo models. Additionally, the effects of A1 dimer on platelet count in whole blood were measured. Hammerstone and Chimel (2003) patented the method of extraction of procyanidins from cocoa. They used defatted, unroasted, or unfermented cocoa beans and optimized extraction parameters (solvent used, extraction temperature, extraction pH) in order to obtain higher yields of PAs. A preferred extraction technique was countercurrent extraction chromatography. The patent of Howard et al. (2012) described a method of extraction of PAs from any plant pomace (apples, pine bark, cinnamon, cocoa, grapes, bilberry, black currant, green tea, black tea, chokeberry, blueberry, and sorghum) by alkaline hydrolysis. Subsequently the active compounds can be used in dietary supplements or added to products to enhance health benefits. The invention of Rull et al. (2004) related to vegetable extracts containing at least 15 and preferably 20–25% of OPC of A2 type. The authors patented the isolation of the compounds from litchi fruits shell involving extraction with methanol and liquid chromatographic purification step.

Patent describing preparations containing A-type procyanidins and their derivatives for treatment or prevention of certain tumors/cancers (especially tumors/cancers overexpressing cycloxygenase-2 (COX-2)) can also be found (Schmitz and Kwik-Uribe 2007). Rohdewald and Ferrari (2003) patented the use of proanthocyanidins and arginine in the treatment of erectile dysfunction. The patent obtained by Danoux et al. (2002) described the use of procyanidin oligomers in the field of cosmetics for skin treatment products, especially products with the ability to counteract skin aging effects.

Several other patents referring to commercial products are described in Sect. 8.

10 Perspectives

In order to cope with an increasing global population and a quantity of wastes obtained from agricultural industry, to conform to the principles of sustainable development and bioeconomy, it is a must to find new methods to obtain added value products from plant waste.

This is the case of plants and fruits with a high amount of biological active compounds, as procyanidins. Based on the United Nation Food and Agriculture Organization (FAO) statistics, at the end of the year 2016 (most recent available), huge productions of plants rich in procyanidins, which processed will produce waste, were reported worldwide: cocoa beans, 4,466,575 t; grapes, 92,281,609 t; tea, 8,368,892 t; strawberries, 12,920,201 t; raspberries, 795,249 t; nuts, 1,087,750 t; cranberries, 683,672 t; cocoa beans, 4,466,575 t; cinnamon, 300,630 t (from 13 countries); apples, 133,777,757 t; apricots, 3,955,025 t; etc. The growing demand of green materials and renewable resources is due to the fact that wastes from agroindustry can be recycled into food/feed/regenerable fuels, fertilizers, pharmaceutical, and cosmetic products. All over the world, the pomace remaining after fruits extraction is produced. It is not valued as highly profitable waste, but instead leads to several environmental and waste disposal problems.

Also, in the present eco and bio rush, materials such as essential oils, pharmaceuticals, supplementary foods, cosmetics, etc., are obtained from plants. From this perspective, the use of active compounds obtained from plant waste is encouraged, especially that agro-horticultural waste is cost-effective natural sources. Initiatives regarding the valorization of plant wastes are developed channeling on different types of compounds or plants pomace. Kolodziejczyk et al. (2007) considered by-product deriving from apple juice pressing as a source of dietary fiber (about 50% of dry weight) and phenolics (from 1200 to 4000 mg/kg dry weight), including flavanols (catechin, epicatechin, procyanidins), hydroxycinnamates, and dihydrochalcones. Altiok et al. (2007) investigated the possibility of recovering of proanthocyanidin from the by-product of Turkish traditional product (molasses). Teixeira et al. (2014) presented in their review paper the types of residues produced by the wine industry and the types of compounds that can be obtained from grape waste. Being one of the most important and widespread agro-economic activity, grape industry produces organic residues (nine million tons/year) and inorganic one (diatomaceous earth, bentonite clay, and perlite).

Nevertheless, implementation of waste management from agro-industry is a challenging task, making the development of innovative and effective capitalization procedures necessary in order to recycle, reuse, and recover these residues. Food wastes should no longer be regarded as a waste to be disposed, but as a renewable source of valuable molecules that should be fully exploited.

Adequate procedures to obtain the target compounds will provide successful achievements for phytochemical recovery and obtaining added value products. At the same time, scientific advances are necessary toward the separation and purification of procyanidin compounds, as well as for the in vitro and in vivo evaluation of the potential effects of those compounds. Moreover, supplementary scientific data are necessary to support the various claims of beneficial effects of regarding procyanidins arising either from folk medicine or from industrial companies in the area of food supplements and related products.

11 Cross-References