*Review* **Botanicals in Functional Foods and Food Supplements: Tradition, E**ffi**cacy and Regulatory Aspects**

#### **Francesca Colombo 1, Patrizia Restani 1,2,\*, Simone Biella <sup>1</sup> and Chiara Di Lorenzo <sup>1</sup>**


Received: 3 March 2020; Accepted: 27 March 2020; Published: 1 April 2020

#### **Featured Application: The review aims to provide guidance on the definitions and regulation of products containing botanical ingredients, which are at present quite complex.**

**Abstract:** In recent decades, the interest in products containing botanicals and claiming "functional" properties has increased exponentially. Functional foods, novel foods and food supplements have a special impact on the consumers, who show significant expectation for their well-being. Food supplements with botanical ingredients are the food area that has witnessed the greatest development, in terms of the number of available products, budget, and consumer acceptability. This review refers to and discusses some open points, such as: (1) the definitions and regulation of products containing botanicals; (2) the difficulty in obtaining nutritional and functional claims (botanical ingredients obtaining claims in the EU are listed and summarized); (3) the safety aspects of these products; and (4) the poor harmonization between international legislations. The availability of these "new" products can positively influence the well-being of the population, but it is essential to provide the consumers with the necessary recommendations to guide them in their purchase and use.

**Keywords:** botanicals; food supplements; nutritional claims; functional food

#### **1. Introduction**

The sentence "Let thy food be thy medicine and medicine be thy food", commonly attributed to Hippocrates (400 BC), indicates the importance that nutrition has always had in the concept of health and prevention of disease. This relationship has obviously undergone changes and historical recurrences with an evolution parallel to that of human civilization with its variable habits in social behavior and diet. More than 2500 years after Hippocrates' claim, the concept of diet has evolved to include aspects based on research in the field of nutrition and on knowledge relating to active molecules contained in foods with potential benefit to human health.

The vegetables entered massively in the diet of humans with the Agricultural Revolution that occurred about 12,000 years ago. Cereals, only rarely consumed in the Paleolithic era, have become the basis of human nutrition with milk derivatives [1]. However, considering the use of plants for their healing properties, archaeological excavations place their use up to 60,000 years ago. These were plants not commonly consumed as food: poppy (opium), ephedra and cannabis.

The possible placement of the plants among food and products with therapeutic properties is therefore ancient history and reappears, with its contradictions, in the current legislation, as described below. The aim of this review was the clarification and relative discussion of the most critical aspects regarding products containing botanicals, in order to promote the correct use of plant food supplements among consumers.

#### **2. Definitions and Regulation**

There is no internationally shared definition of the term "botanical". However, it is possible to cite what was defined by European Food Safety Authority (EFSA) in its guidelines published in 2009 [2]. The term *botanical* includes whole, fragmented or cut plants, plant parts, fungi and lichens. The term *botanical preparations* include all preparations obtained from botanicals by various processes, such as pressing, squeezing, extraction, fractionation, distillation, concentration, drying up and fermentation. A similar definition is reported by a group of food and beverage producers: "*botanicals* are fresh or dried plants, plant parts or plants' isolated or collective chemical components, extracted in water, ethanol, or other organic solvents, plus essential oils, oleoresins, and other extractives used for flavoring, fragrance, functional health benefits, medicine, or other uses" [3].

Based on the definitions listed above, it is clear that botanicals include numerous possible ingredients with extremely variable composition and characteristics. First of all, the presence and concentration of active molecules can vary greatly, with possible effects on the beneficial effect expected on the consumers' health.

Hundreds of plants are ingredients commonly used in the food industry as such or derivatives, or food additives (flavorings, colorings, preservatives, etc.), and botanicals can be included in different types of products regulated by the food law as illustrated in Figure 1.

**Figure 1.** Categories of food containing botanicals.

#### *2.1. Fruits and Vegetables*

Fruits and vegetables have been included in the diet of humans since ancient times; they can be consumed as such or as household or industrial derivatives (jams, juice, purée, etc.). Fruits and vegetables are included in the EC Regulation N. 178/2002, which defines any unprocessed or processed substances or product designed for human consumption [4]. Generally speaking, consumers prefer products from the geographical area of belonging, but with the increasing globalization it is possible to expand the choice to imported products.

#### *2.2. Enriched or Functional Food*

Although there is no international agreement, according to the consensus document on "Scientific Concepts of Functional Foods in Europe" of the European Commission Concerted Action on Functional Food Science in Europe (FUFOSE), functional food is usually considered a product to which one or more ingredients have been added (or more rarely subtracted) with a positive consequence on the functionality of human organs or systems [5]. It is, therefore, a food that has not only the function of providing calories and nutrients but intends to carry out a favorable action on the consumer's health. This effect must be reached with the quantity of food normally consumed, it must be in "traditional" form (and not in pharmaceutical form) and must guarantee the safety of the subjects taking the product, even if they present common pathologies. As an example, the prebiotic ingredients are frequently added to food; they are soluble fibers capable to promote the growth of the positive intestinal microbiota; among others, the most important fibers in this field are inulin and fructo-oligosaccharides (from chicory or Jerusalem artichoke). Further details will be reported in Section 4.1.2.

#### *2.3. Novel Food and Traditional Food from Third Countries*

At the European level the regulation of new food, or novel food, was amended in 2015 (EU Regulation 2015/2283). Compared to the previous regulations, the definition of this products was maintained: "novel food means any food that was not used for human consumption to a significant degree within the Union before 15 May 1997". Some of the categories listed in novel food include botanicals: (1) food consisting of, isolated from or produced from microorganisms, fungi or algae; (2) food consisting of, isolated from or produced from plants or their parts. Novel food category also includes products obtained with new technologies. Before marketing them, appropriate documentation must be provided in order to demonstrate their safety. Examples of novel food include: new sources of vitamin K (menaquinone) or some extracts from existing food (Antarctic krill oil rich in phospholipids from *Euphausia superba*) [6].

The aforementioned regulation has introduced a new authorization procedure for "traditional foods deriving from Third Countries", where a food with proven tradition of safe use in a third country for at least 25 years is included. Compared to the novel food procedure, this new category must present less documentation for their approval. Examples of foods belonging to this category are the dried aerial parts from *Hoodia parviflora* N.E.Br. (hoodia) and seeds from *Salvia hispanica* L. (chia) [6].

#### *2.4. Food*/*Dietary Supplements*

Botanicals are widely used in the formulation of food (or dietary in US) supplements, that can be defined as "products intended to supplement the common diet and which constitute a concentrated source of nutrients, such as vitamins and minerals, or other substances having a nutritional or physiological effect, in particular, but not exclusively, amino acids, essential fatty acids, fibers and extracts of vegetable origin, both single- and multi-compound, in pre-dosed forms" [7]. The role of food supplements is limited to correcting nutritional deficiencies, maintaining the recommended intake of some essential nutrients and supporting specific nutritional needs such as pregnancy, breastfeeding and menopause. They must not show therapeutic activity or significantly modify the physiological functions [8]. Among the most frequently botanical ingredients used in food supplements, there are *Ginkgo biloba* L. (ginkgo), *Oenothera biennis* L. (evening primrose), *Cynara scolymus* L. (artichoke) and *Panax ginseng* C.A. Meyer (ginseng) [9].

#### *2.5. Other Categories*

Botanicals can be regulated by drug law. In fact, their use can enter into conventional medicine, or, as in some European countries (e.g., Germany), in "Traditional Herbal Medicinal Products (THMPs)". THMPs must guarantee their safety in the expected conditions of use and their biological activity/efficacy must be plausible on the basis of a chronic use and of available experience [10].

Botanicals can also be ingredients of products belonging to the category of medical devices; among others: (1) ophthalmic solutions containing chamomile (*Matricaria recutita* L.), calendula (*Calendula o*ffi*cinalis* L.), cornflower (*Centaurea cyanus* L.), etc.; (2) some liquids used in contact lens care, which include arabinogalactans and plant extracts; (3) some solutions used for oral hygiene containing mainly chamomile, aloe (*Aloe vera* (L.) Burm.f.) and marshmallow (*Althaea o*ffi*cinalis* L.). Since this chapter is about food, only examples of the use of botanicals in functional foods and food supplements will be described.

#### **3. Claims**

One of the most interesting aspects associated with functional food and food supplements is the possibility to boast on the label their health properties, the so-called "claims".

The European Commission allows health claims when they can be supported by the scientific literature and if they can be understood by consumers. In the EU, the European Food Safety Authority (EFSA) is in charge of evaluating the scientific evidence [11] and provides the guidelines, which describes in detail the studies required for the approval of nutritional/physiological claims [2,12].

There are two types of health sentences, those related to art. 13, and those relating to art. 14 of the European Regulation 1924/2006. Article 13 claims refer to: (1) growth, development and functions of the body; (2) psychological and behavioral functions; (3) slimming or weight control. Article 14 claims are associated with: (1) a reduction of risk factors in the development of diseases; (2) support of normal children's development and health. Table 1 lists the most important health claims allowed for vegetable/botanical ingredients in the EU [13].


ListofmainclaimsallowedintheEUforplant(botanical)ingredientscontainedin,oraddedto,functionalfoodorfood


of coronary heart disease


#### *Appl. Sci.* **2020**, *10*, 2387



#### **4. Botanicals in Functional Foods**

Some botanical derivatives have been successfully included in functional food for their healthy properties. Among others, soluble and insoluble fibers, beta glucans and phytosterols/phytostanols deserve a more in-depth description.

#### *4.1. Soluble and Insoluble Fibers*

The beneficial properties of dietary fiber intake have been known for a long time and have been publicized since the 1920s. Over the years, the importance of fiber for human health has been fluctuating to be re-evaluated in the second half of the 20th century [14]. With the progress of knowledge, dietary fiber has been classified in various ways; the first subdivision concerns the difference between fiber, which are soluble or insoluble in aqueous medium. In addition to the obvious difference in "chemical" behavior, the solubility (or less) of the fiber in water greatly conditions the activity on humans' health. Insoluble fiber acts mainly on the intestinal transit, while the soluble fibers mediate beneficial properties based on different biological mechanisms.

#### 4.1.1. Insoluble Fiber

Insoluble fiber includes mainly cellulose, hemicellulose and lignin, and it is characterized by its "inertia" at the level of human organism. In other words, it does not undergo any metabolic modification during the transit in the gastro-intestinal tract. Products containing insoluble fiber from different plant sources can use the health claim "the fiber contributes: (1) to normal bowel function; or (2) to increase faecal bulk; or (3) to an acceleration of intestinal transit" (see barley, oat, rye, sugar beet, and wheat fiber in Table 1). Insoluble fiber increases fecal bulk, that leads to a speeding of intestinal transit with the consequent beneficial effects (elimination of waste containing toxic molecules, less contact of fecal waste with the intestinal mucosa, etc.). These effects have been recognized as protective factors versus some cancers including the colon cancer.

#### 4.1.2. Prebiotic Fibers

The prebiotic fibers fall into the category of soluble fibers. The definition of the term "prebiotic" has undergone a certain number of changes since the 1990s, a period in which they were identified as beneficial ingredients of the human diet. In agreement with the International Scientific Association for Prebiotics and Probiotics, a prebiotic fiber is "a substrate, that is selectively used by host microorganisms conferring a health benefit" [15]. Similarly, the Food and Agriculture Organization of the United Nations (FAO) published the following definition in 2008: "a nonviable food component that confers a health benefit on the host associated with modulation of the microbiota" [16].

In recent decades, the awareness of the critical role played by the intestinal microbiota on human health has progressively grown. Studies to support this knowledge have stimulated the formulation of food products capable to contribute to the modulation of microbiota towards positive bacterial strains (mainly lactic bacteria) at the expense of pathogenic ones. These foods, mostly milk-based, contain particular strains of live bacteria (probiotics) or soluble fibers capable of promoting their growth. In functional foods, the most commonly used prebiotic fibers are: inulin, fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS); the latter are mostly included in products for infants. Inulin is a linear polymer of 2-60 fructose molecules linked by β-(2-1)-bonds.

The nature of chemical bonds prevents inulin from the usual digestion of caloric sugars. FOS are similar to inulin for chemical bonds, but the number of units ranges between 2 and 10 [17]. Inulin and FOS are storage carbohydrates contained in several plants; the most important sources are chicory, sugar beet, leeks, asparagus, and Jerusalem artichokes. Inulin and FOS reach the colon undigested where they are fermented by the "positive" microbiota. A health claim for "native chicory inulin" is allowed in EU; it states that "chicory inulin contributes to normal bowel function by increasing stool frequency". This claim must be associated with those products, providing at least a daily intake of 12 g

of inulin, as a non-fractionated mixture of monosaccharides (<10%), disaccharide, inulin-type fructans and inulin, with an average of polymerization > 9 (see Table 1).

Although from animal origin, galacto-oligosaccharides (GOS) are considered here to describe the possible healthy effect due to prebiotic fiber. GOS are oligosaccharides containing 2-10 molecules of galactose and one molecule of glucose. The purity and degree of polymerization largely influence the prebiotic properties of these oligosaccharides, as shown in clinical studies [18,19]. GOS are widely used in lactating formulas to improve the microbiota colonization and modulate the intestinal disorders such as colic. The effects of GOS added to infant formulas were evaluated in a randomized, double-blind, controlled, parallel-group clinical trial including healthy term infants [20]. Three groups were considered: breastfed, formula-fed, and GOS-supplemented formula-fed infants. The effects of supplemented formula were assessed considering four bacterial targets: *Bifidobacterium*, *Lactobacillus*, *Clostridium*, and *Escherichia coli*. The effect of the prebiotic-supplemented formula was very close to that of breast-feeding in stimulating *Bifidobacterium* and *Lactobacillus* growth and in inhibiting *Clostridium* growth. The whole result was a significantly lower incidence of colic [20].

#### 4.1.3. Beta-Glucans

Beta-glucans are soluble fibers contained in cereals, such as barley, oat, rye, wheat and some mushrooms [21]. They are linear polymers of glucose linked by β-(1-4) and β-(1-3) bounds. Two claims for beta-glucans received positive opinion by EFSA and were included in the list of wordings allowed in the EU (Table 1) [22].

The first claim is "consumption of beta-glucans from oats or barley as part of a meal contributes to the reduction of the blood glucose rise after that meal" (Table 1). In fact, beta-glucans modulate the post-prandial glycemic response by increasing the meal bolus viscosity, with a consequent delayed absorption of nutrients (including glucose) in the small intestine [23].

On the basis of intervention studies performed in healthy subjects, EFSA concluded that quantities of about 4 g of beta-glucans/30 g of available carbohydrates are capable to decrease the post-prandial glycemic response without modifying the insulin release [22]. The claim must be associated with the recommendation to use beta glucans during meals.

The EFSA received from the European Commission a second request for an opinion regarding the claim associating beta-glucans with the positive modulation of normal blood cholesterol levels [24]. On the basis of a certain number of randomized-controlled trials in adults with normal or mildly increased cholesterolemia, the EFSA Panel formulated the following wording "regular consumption of beta-glucans contributes to the maintenance of normal blood cholesterol levels". The beneficial effect is linked to a daily consumption of 3 g of beta-glucans in one or more servings. The proposed mechanism of action is based on the fact that dietary beta-glucan forms, in the small intestine, a viscous mixture that reduces the intestinal absorption of dietary cholesterol and the re-uptake of bile acids. As a consequence, new bile acids are synthetized from circulating cholesterol [25].

#### *4.2. Phytosterols*/*Phytostanols*

Phytosterols and phytostanols are compounds of plant origin, which are commonly part of the human diet. Phytosterols can be transformed in phytostanols by hydrogenation and both class of molecules can be esterified with fatty acids from vegetable origin to the corresponding esters [26,27]. Phytosterols and phytostanols are structurally similar to cholesterol, apart from their side chain. The most common phytosterols and phytostanols are sitosterol, sitostanol, campesterol, campestanol, stigmasterol and brassicasterol [26].

In the EU, the use of phytosterols, phytostanols and their esters in foods refers to the Regulation (EC) No. 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods/novel food ingredients.

These molecules reduce the intestinal absorption of cholesterol and, as a consequence, cause a decrease in blood cholesterol levels. For this property, phytosterols and phytostanols, in free or esterified form, can be added to foods for their properties to maintain normal (claim under Art. 13) or reduce blood cholesterol levels (claim under Art. 14). The first wording is allowed for food providing 0.8 g of plant sterols/stanols with the daily dose and the second for daily doses ranging from 1.5 to 3 g. Doses of 1.5–2.4 g/day are associated with a cholesterol level reduction of 7–10%, while 2.4–3 g/day can produce a decrease of 10–12.5% [28].

#### **5. Botanicals in Food Supplements**

The category of food/dietary supplements is that in which the presence of ingredients from plant origin is more frequent (botanicals). Food supplements containing plants or derivatives have found an increasing diffusion and acceptance by the consumers, which consider the word "natural" synonymous with "non-chemical" and for association with safe. This belief explains the success of these products and therefore their consistent market presence.

The plants contained in food supplements are many and most of them derive from the tradition of use, that is the preparation of infusions or decoctions. The same plants have been then used by the industries to prepare extracts, in order to enrich the products in active molecules and enhance the expected positive properties. Supplements may contain a single plant ingredient or a mixture of them. It is clear that the greater the number of botanicals present in the finished product, the greater the problems in the correct use by the consumer and in the quality control especially when the product is already on sale.

Another critical aspect derives from the fact that there is no harmonization at the international level (even in Europe) as regards the lists of plants allowed in food supplements. Many countries of the European Union, but also of other continents, have published positive and/or negative lists of botanical ingredients, which are allowed or prohibited in food/dietary supplements. Unfortunately, there is few correlations between them.

The inconsistent situation of the European market of products with botanical ingredients (presence both in products regulated by food or drug laws, see Section 2) leads to several consequences, including the difficulty in discriminating "healthy" or "therapeutic" information for the same plant used in different product classes. As the researchers well know, the dose makes the difference, but discriminating the dose with physiological effects from the one that is suitable for clinical applications is a complex objective even for plants of more ancient tradition. These difficulties were reflected in the very small number of claims approved by EFSA in the field of botanicals.

To boast a nutritional or health claim as a guide for the consumer, it is necessary to refer to the European Regulation n. 1924/2006, according to which: "Scientific substantiation should be the main aspect to be taken into account for the use of nutrition and health claims and the food business operators using claims should justify them". To obtain authorization to associate a nutrition/health claim with a botanical in EU, the manufacturer must submit a dossier to EFSA, which evaluates the scientific evidence of the studies provided and publishes the relative opinion. The guidelines provided by EFSA [2,8] describe in detail the studies required for the approval of nutritional/physiological claims.

The guidelines published by EFSA, although extremely well-articulated, have nevertheless led to immediate difficulties in the creation of a dossier that could meet all the listed requirements. Among the most critical and important aspects:

(a) speaking of dietary supplements, food industries cannot claim any therapeutic effect, but only physiological ones; as a consequence, apart from few exceptions, it is very difficult to prove a healthy activity that aims to maintain homeostasis or reducing disease risk factors in the long term;

(b) to obtain statistical significance, it is necessary to recruit a very large "healthy" population that is willing to take a certain product for very long periods. All this is economically unsustainable, and the results could still be affected by the dietary habits (and not only) of the subjects considered;

(c) it seems unreasonable that the tradition of use has been accepted for traditional medicine drugs and not for food supplements.

Considering the list of claims listed in Table 1, only one case can be clearly associated with food supplements: *Monascus purpureus* (red yeast rice); in the remaining cases, claims are related to ingredients generally used in all foods.

Red Yeast Rice (RYR) is a rice fermented by the red yeast *Monascus purpureus*, which contain a certain number of active polyketides named monacolins, that are secondary metabolites deriving from the fermentation process [29]. The most abundant active molecule is monacolin K.

RYR obtained the approval of the claim: "Monacolin K from red yeast rice contributes to the maintenance of normal blood cholesterol levels" [30]. This wording was supported by two randomized controlled studies, where RYR preparations providing 10 mg of monacolin K (with the daily dose) were responsible of a reduction of blood Low-Density Lipoprotein- cholesterol (LDL-cholesterol) level in subjects suffering from hypercholesterolemia. EFSA considered also the fact that the effect of pure monacolin K (chemically identical to the drug lovastatin) on blood LDL-cholesterol concentration was well known and based on the inhibition of HMG-CoA (3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A) reductase [31].

#### **6. Safety Aspects**

This chapter deals with food, the safety of which should be able to be taken for granted. Actually, when botanicals are food supplement ingredients, some distinction has to be made. Compared to common or functional food, food supplements containing botanicals have no or little role from the nutritional point of view, as these do not contribute significantly to the intake of calories and nutrients. In fact, normally these products do not contain caloric nutrients (sugars, fats and proteins) and the presence of vitamins and minerals (not mandatory) covers, with a few exceptions, very limited percentages of the recommended daily dose. On the other hands, food supplements with botanicals are frequently promoted for their possible healthy properties, leading the consumer to prolonged use over time. Furthermore, as mentioned above, the growing popularity among consumers of "natural products" has led to an increasing number of producers and thousands of food supplements on the international market, without certainties relating their composition, activity and safety. While biological activity may be at least partly based on the "tradition of use", the risk/benefit assessment still remains orphaned of sufficiently reliable scientific data.

Botanicals, especially if in their extract form, can have biological effects which, in relation to the dose and quality of the ingredient, can be functional, therapeutic and also toxic [32]. Different factors could be involved in the incidence of adverse effects such as the quality of the raw botanical material, the presence of environmental contaminants, the specific technological process applied (extraction and concentration), misidentifications of the plant ingredients, adulterations, counterfeits. The phytochemical fingerprint of a botanical can vary significantly, when the biological variability or the different processes applied are considered. Different factors (extractant solvent, the temperature and time, the time from harvesting, etc.) can modulate the quality of botanical ingredients, with consequent changes in the relative abundance of active molecules present in the final product. Consumer's age and gender, unsuitable use of botanical food supplements, genetic factors and specific pathological or physiological conditions are further possible sources of unexpected adverse reactions.

Confirming the particularity of the supplements with respect to usual food, there are cases in which limits on the content of molecules naturally present in botanicals are regulated by national and international directives. The established values must also be indicated on the label, such as in the case of bitter orange (*Citrus aurantium* L.) for which it is necessary to declare the content of synephrine and other active amines with the daily dose (Figure 2). Limits are established for molecules that can lead to unwanted side effects at high levels and therefore for which safe daily doses for consumers need to be "standardized".

**Figure 2.** Active amines of *Citrus aurantium* L. regulated for their content in food supplements.

#### *6.1. Adverse Events*

Even in the case of adverse events, supplements containing botanicals show specific characteristics. Excluding the accidental presence of toxic xenobiotics, the adverse events associated with common foods are mostly due to allergies and intolerances, which concern restricted groups of at-risk populations. On the contrary, in the case of supplements with botanicals, the consumer must be informed that adverse events can occur. Moreover, pregnant, lactating women and children should avoid food supplements or strictly follow the doctor's/pediatrician's indications.

Since botanicals are "natural", the average consumer considers them safe and rarely communicates their use to doctors in the case of short- or long-term therapy. This point is probably the most important factor leading to unexpected adverse effects. Botanicals could modify the efficacy of other molecules (pharmaceutical drugs or other dietetic compounds) reducing or increasing their plasmatic concentration. To give an example of the possible adverse events to supplements containing botanicals, the case of RYR is described here.

#### *6.2. The Case of RYR*

As described previously (see Section 5), in 2011 RYR received a positive opinion for the claim supporting the reduction of LDL-cholesterol plasma level, if the daily intake of monacolin K was 10 mg. In 2018, the EFSA was asked a second time in relation to RYR, with the request being to evaluate the safety of the monacolins contained in it and to provide advice on the daily dose of monacolins that does not give rise to concerns for harmful effects [33]. Since the lactonic form of monacolin K has the same chemical structure of lovastatin (a pharmaceutical drug), the EFSA, after consulting the scientific material provided, concluded that the intake of 10 mg of monacolin K with a food supplement would lead to the intake of a therapeutic dose. Moreover, the evaluation of the data presented showed that several adverse events were reported following the consumption of RYR. These adverse events involved musculoskeletal system (the most severe clinical event associated was rhabdomyolysis) and

liver. The clinical symptoms appeared after consumption of RYR, with an intake of 3–10 mg/day of monacolin K.

Two problems have therefore been highlighted for RYR:


#### **7. Conclusions**

Since the last century, a progressive lengthening of the average life of the population has been observed; this social phenomenon is in itself a positive fact, but inevitably entails an increase in the occurrence of chronic-generative diseases more frequent in elderly: cardiovascular diseases, tumors, dementias, etc. At the same time, new data on the role of nutrition in human health has stimulated important international researches and industrial activities:


These premises explain the great interest of the food industry for products that present, in addition to nutritional ingredients (proteins, carbohydrates and fats), new "functional" properties, which have special impact on the consumer. In this area, particular interest has been paid to ingredients of vegetable origin, the botanicals. Most products containing botanicals are regulated by food law: fruits and vegetables, functional foods, novel foods, traditional foods from third countries, and food supplements. Some botanical ingredients have received positive opinion by EFSA to support their health claims; fibers, beta glucans and phytosterols/phytostanols are among the most known functional ingredients. Food supplements with botanical ingredients are the sector that has witnessed the greatest development, in terms of both the number of products on the market and consumer acceptability. The availability of these "new" functional products can positively influence the well-being of the population, but it is essential to provide the consumer with the necessary information to guide him in the purchase and use of food supplements.

The difficulties in demonstrating scientifically the supposed physiological/healthy properties of botanicals are the reasons of the limited number of claims approved by EFSA. In addition, safety aspects should be considered in relation to the high number of products available on the market and their growing popularity among consumers: among other things, the quality of raw materials, possible adulterations, presence of active molecules regulated by national/international directives and factors associated with consumers (age, gender, concomitant diseases/drugs). These factors can contribute to adverse effects occurring, underlying the need for tools and intervention to promote consumers safety.

In conclusion, this review provides an overview on the actual knowledge about products containing botanicals aimed to promote consumers' wellbeing. The main purpose is to provide useful information to consumers in order to guide their choices. In fact, although much progress has been made in the field of botanicals, there is still a lack of data, information, and guidelines to ensure the suitable use of these products.

**Author Contributions:** Investigation, F.C. and S.B.; Writing-Original Draft Preparation, P.R.; Data curation, S.B. and F.C.; Writing-Review & Editing, C.D.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This paper has been prepared in the framework of the MIUR Progetto di Eccellenza.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review Cucurbita* **Plants: From Farm to Industry**

**Bahare Salehi 1, Javad Sharifi-Rad 2,\*, Esra Capanoglu 3, Nabil Adrar 3,4, Gizem Catalkaya 3, Shabnum Shaheen 5, Mehwish Ja**ff**er 5, Lalit Giri 6, Renu Suyal 6, Arun K Jugran 7, Daniela Calina 8, Anca Oana Docea 9, Senem Kamiloglu 10, Dorota Kregiel 11, Hubert Antolak 12, Ewelina Pawlikowska 11, Surjit Sen 12,13, Krishnendu Acharya 12, Moein Bashiry 14, Zeliha Selamoglu 15, Miquel Martorell 16,17,\*, Farukh Sharopov 18, Natália Martins 19,20,\*, Jacek Namiesnik 21,\* and William C. Cho 22,\***


Received: 25 July 2019; Accepted: 14 August 2019; Published: 16 August 2019

**Abstract:** The *Cucurbita* genus, a member of Cucurbitaceae family, also known as cucurbits, is native to the Americas. Genus members, like *Cucurbita pepo* and *Cucurbita maxima*, have been used for centuries in folk medicine for treating gastrointestinal diseases and intestinal parasites. These pharmacological effects are mainly attributed to their phytochemical composition. Indeed, *Cucurbita* species are a natural source of carotenoids, tocopherols, phenols, terpenoids, saponins, sterols, fatty acids, functional carbohydrates, and polysaccharides, that beyond exerting remarkable biological effects, have also been increasingly exploited for biotechnological applications. In this article, we specifically cover the habitat, cultivation, phytochemical composition, and food preservative abilities of *Cucurbita* plants.

**Keywords:** *Cucurbita* plants; cucurbits; pumpkin; phytochemical composition; food industry

#### **1. Introduction**

Natural products are a rich source of chemical diversity that has boosted pharmaceutical industry over the centuries [1,2]. Plants and herbs have been applied in both prevention and treatment of human disorders since ancient times [3–5]. Indeed, many investigations on herbs and plants have been conducted, and their efficacy proved as a compelling source of antioxidant, antimicrobial, anti-inflammatory [6], anticancer and neuroprotective agents [7,8].

The Cucurbitaceae (pumpkin) family contains many species as human foods. This family frames a huge gathering with roughly 130 genera and 800 species. Cucurbitaceae plants are commonly known as cucurbits, extensively cultivated in tropical and subtropical countries. Cucurbit species include pumpkins, squashes, gourds and melons [9]. In addition, it is an economically important family with a rootage of various valuable products, such as edible vegetables, fruits, seed, seed oils, and drugs. Indeed, they include a large variety of vegetables, often used through various forms for cooking, pickling, salad, dessert fruits, candied, confectionary, etc. In addition, *Cucurbita* products have a high nutritional value, being also an important source of vitamins, and widely used in culinary for biscuits, bread, desserts, soup and beverages production [10–12].

Overall, this article aims to provide an update on the *Cucurbita* plants sources, as well as historical culinary usage, habitat and cultivation, phytochemical composition and industrial purposes.

#### **2.** *Cucurbita* **Plants: An Historical Culinary Usage**

*Cucurbita* plants were first explained by Linnaeus in the middle of the eighteen century and are among the earliest known plants that have been grown by mankind. Primarily, they are classified by their shape or harvest season (summer or winter), although there are both female and male types. The male flowers produce pollen and the female ones produce the fruit. *Cucurbita* species have bristly and prickly stems and leaves. Broadly, leaves are large, occasionally lobed or spotted, and spiraled clinging tendrils are frequently given at the leaf axils. Both leaves and stems can be rough or prickly. Yellow or orange trumpet shaped flowers develop into variously shaped, sized, marked, and colored fruits [13]. However, these plants are also cultivated for their edible and ornamental fruit, also having therapeutic and wholesome advantages. The juvenile natural products are often expended as a vegetable, but some assortments of natural products are used with beautiful purposes in the Halloween party. They can be consumed as raw or cooked, and also for animal feed, but due to their high nutraceutical value, *Cucurbita* plants have been widely used in many countries as constituents of many commercial products. For instance, in Indian cuisine, squashes (ghia) are traditionally cooked with seafood, such as prawns. French people use marrows (courges) as a gratin. In Italy, many of regional dishes are prepared from squashes. Japanese people use *Cucurbita moschata* Duchesne pumpkins (kabocha) for preparation of different Japanese foods. *C. moschata* fruits can be round, oblate, oval, oblong, or pear-shaped, variously ribbed, 15–60 cm in diameter, and weigh up to 45 kg. In African countries soup is prepared from squash, and soft alcoholic drinks are made from *Cucurbita ficifolia* Bouché. *C. ficifolia* fruits are oblong with a diameter of 20 cm, weighs 5 kg to 6 kg, and its skin can vary from light or dark green to cream-colored.

Specifically, the commercially produced pumpkin is mainly used for production of pumpkin pie, bread, biscuits, cheesecake, desserts, donuts, granola, ice cream, lasagna dishes, pancakes, pudding, pumpkin butter, salads, soups, and stuffing. The seeds, wealthy in oil, likewise are used in Mexico, with nectar to make pastries, known as palanquetas. Blossom buds and blooms are used in Mexico to cook quesadillas. Indeed, *Cucurbita* products contain carbohydrates, protein and are also rich in vitamin C, pyridoxin (B6) and riboflavin (B2). The content of fat is negligible. Fruit and root of *Cucurbita foetidissima* Kunth is also rich in saponin, which can be used as soap, shampoo and bleach [10–12,14].

#### **3. Habitat and Cultivation of** *Cucurbita* **Plants**

#### *3.1. Habitat*

The *Cucurbita* genus is native to the Americas. Archaeological observations have explained that, for more than 8000 years, *Cucurbita* domestication has moved from the southern parts of Canada to Argentina and Chile [10–12,14]. Five species of the genus, namely *Cucurbita argyrosperma* C.Huber, *C. ficifolia*, *C. moschata, Cucurbita maxima* Duchesne, and *Cucurbita pepo* L. were habituated and cultivated in several areas of North and South America [15–17]. *C. pepo* is considered as one of the most seasoned and developed varieties, by Mexican archeological evidence of 7000 BC. Along these lines, it was extensively developed through aboriginal people through Central, North America and Mexico, earlier the landing of Europeans. *C. pepo* is local to Northern Mexico and Southwestern and eastern USA [18]. *C. pepo* fruits can be oval, cylindrical, flattened, globular, scalloped, fusiform, and/or tapering to a curved or straight neck on one or both ends. They can be up to 5 times longer than wide and their weight varies from 30 g to 50 kg. The skin can be smooth, warty, wrinkled, furrowed, or/and have shallow to deep longitudinal ridges. Often there is more than one color to the soft to hard skin: white, yellow, light to dark green, nearly black, cream, and/or orange. Subsequently, *Cucurbita* species were cultivated in all tropical, subtropical and temperate regions of the planet and are now considered as one of the valuable foods for most of the world's inhabitants [12,19].

*Cucurbita* species also show diverse habitat use, growing in terrestrial and wetland environments, as monoecious climbers or annuals; they may be found in meadows, fields, and on the shores of rivers or lakes [20].

#### *3.2. Cultivation*

#### 3.2.1. Climate

In general, *Cucurbita* species prefer warm weather. Temperatures of 18–27 ◦C are ideal for maximum crop production. So, a long warm season is important to obtain a quality production. For seed germination, soil temperatures above 16 ◦C are essential and it takes about 14 days for the crop to leaves at this temperature, and it was also reported that when soil temperature rises to 20 ◦C seed emerge within a week [21]. *Cucurbita* species requires a continuous water supply, but overwatering also lead to crop spoiling. Uniform moisture supply is, therefore, important during the growing season of the crop. *C. argyrosperma* is generally cultivated in regions with little arid climate with proper watering or in regions with a distinct rainy season [22]. *C. ficifolia* grows in specific environmental parameters, such as not in a high frost condition, but it prefers heavy rain agricultural systems [23]. *C. maxima* are mainly grown in regions with temperate climate, and exceptionally grown in warm and moist area [24]. The shape of *C. maxima* fruit can be an elongated cylinder, oval, flattened, globular, heart-shaped, and/or tapering to a curved neck on one or both ends. The length (from 5.8 cm to 71.6 cm), width (from 11.2 cm to 48.6 cm) and weight (usually ranging from 0.3 kg to 50 kg, some pumpkins can grow over 90 kg) is very variable. The skin can be smooth, warty, wrinkled, or/and have shallow to deep longitudinal ridges. Often there is more than one color on the soft to hard skin: red, white, gray, black, green, cream, and/or orange. *C. moschata* is reported mainly from regions with low altitude, hot climate

and high humidity [25,26]. *C. pepo* can adapt to different ecological conditions, but prefers low altitude and warm/humid places [27].

#### 3.2.2. Soil

*Cucurbita* spp. can be grown in a wide range of soil varieties, but favors well-drained fertile ground [22]. A well-drained soil is preferred, and the crop roots can penetrate up to a meter deep into the soil. The ideal soil pH is in the range of 6.0 to 6.5, but the crop can withstand both slightly acidic and alkaline soils. In areas with low soil pH, the application of lime or dolomite is essential to allow better uptake of nutrients. Well-drained loamy fertile soils or sandy loam soils are good for commercial cultivation. However, heavier soils (clay) can also be used as long as drainage is adequate.

#### 3.2.3. Propagation and Planting Method

Cultivated *Cucurbita* spp. are usually propagated by planting seeds in the ground. Sometimes, seeds are germinated in small pots and seedlings are transplanted to the field when climatic and edaphic factors are favorable [22]. Transplant seedling are often used to establish an early season crop or when using permanent beds [21]. It has been reported that *Cucurbita* species that are developed from transplanted seedling have luxuriant growth, larger fruit size and significantly higher seed yield compared to direct sowing method [28,29]. Commercially cultivated varieties are sometimes grown with traditional crops, like maize, beans or even in vegetable gardens along with other species. Plant density affects fruit size, yield and its number per plant. Napier [21] reported that in *C. maxima* and *C. moschata* higher plant densities resulted in smaller fruit size, higher total yield and fewer fruit per plant.

#### 3.2.4. Irrigation

The first irrigation is important just after planting, and subsequent irrigation is given at a weekly basis or depending upon growth of the plant and soil condition. Waterlogging should be avoided throughout the cultivation process. However, in the absence of rain, the crop should be regularly watered. Irrigation is vital during flowering, fruit set and fruit fill, but should be minimized at the time of fruit maturity. Various types of irrigation methods are practiced in *Cucurbita* species cultivation, such as furrow, drip and overhead irrigation. Furrow irrigation needs the type of soil which passes water to reach laterally, without penetrating very deep into the soil, and drip irrigation is practiced in permanent bed systems which help to minimize weeds in the field [21].

#### 3.2.5. Fertilizer

Proper fertilizer application is essential for *Cucurbita* species cultivation, but the excessive use of nitrogen fertilizer, early in the growing period, results in huge foliage growth, delayed fruit set and lower crop yield [21,30–32]. For *C. maxima* production, mineral fertilizers are used, which consists of NPK-nitrogen (N), phosphorous (P) and potassium (K)-applied either manually or with sub-soiled in a row. A nitrogen side-dressing in the form of urea, is also applied about two weeks after crop emergence [33]. In case of *C. maxima,* the recommended amounts of fertilizers are about 150 kg nitrogen/ha, 95 kg phosphorus/ha, 80 kg potassium/ha and 10 kg gypsum/ha [33]. Bannayan et al. [34] reported that, in *C. pepo*, the optimum nitrogen rate not only can increase crop growth, but can also boost up its resistance to higher temperatures.

#### 3.2.6. Pest and Disease Management

*Cucurbita* species are very prone to pests and diseases, and some pathogens attack this economically important crop. Thus, it is important to protect them to obtain a good quality and high commercial yield *Cucurbita*. Several notorious fungal pathogens are associated with *Cucurbita* species, e.g., *Cladosporium cucumerinum* which causes a scab or gummosis [35], while *Choanephora cucurbitarum* causes fruit rot of *C. pepo* [36]. This disease, also known as "wet rot" and "blossom end rot", can spoil many blossoms

and fruit during prolonged damp weather. *Pseudoperonospora cubensis* is an unusual organism of downy mildew *Cucurbita* species, mainly cucumbers, melons, squashes, gourds and watermelons [37]. *Phytophthora capsici* causes blight and mostly infect the seedlings, vines, leaves, and fruits of *Cucurbita* plants [38]. Cucurbits powdery mildew, caused by *Erysiphe cichoracearum*, is the most critical disease of cultivated *Cucurbita* spp. [39,40]. *Fusarium oxysporum* is a soil borne fungal pathogen that causes a damping off and wilt disorder [41]. Powdery mildew is caused by *Sphaerotheca fuliginea*, which forms pads of whitish mycelium on upper and under leaf sides, petioles, and stems [42]. Important bacterial diseases of *Cucurbita* species are across leaf spot evoked by *Pseudomonas syringae* pv. *Lachrymans* [43,44]. Bacterial wilt occurs in *Cucurbita* species infected with *Erwinia tracheiphila*. This pathogenic bacterium is imparted with striped (*Acalymma vittatum*) and spotted (*Diabrotica undecimpunctata*) cucumber beetles [45]. Several viral variants have also been identified, causing many problems to the crops due to the rate of disease expansion, asperity of infection and difficulty in controlling the diseases. The most important viral variants are *clover yellow vein virus* (CYVV), *papaya ringspot virus Type W* (PRSV), *squash mosaic virus* (SqMV), *tobacco tingspot virus* (TRSV), *tomato ringspot virus* (ToRSV), *watermelon mosaic virus* (WMV), and *zucchini yellow mosaic virus* (ZYMV) [22,46,47]. Many insects invade *Cucurbita* species, e.g., aphid species (*Myzus persicae* and *Aphis gossypii*) and beetle species (*A. vittatum* and *D. undecimpunctata*), which causes significant crop losses. Proper crop rotation is the best way to minimize pests and diseases. Thus, the application of appropriate fungicide, like copper oxychloride, bravo, cupravit, dithane, and dichlorophen, at specific doses can control fungal pathogens of *Cucurbita* species. However, the control of viral diseases is difficult. The breeding of disease resistant varieties by the hybridization technique or the application of innovative methods to promote resistant varieties is an additional way to control viral diseases. *C. pepo* cultivar contains unique transgenic forms that show resistance to viruses like WMV and ZYMV [22].

#### **4.** *Cucurbita* **Plants Phytochemical Composition**

Twenty-eight *Cucurbita* species are named in the literature [48,49], but some of them could just be hybrids or synonyms [49]. The relative lack of studies on some *Cucurbita* species may be linked to their rarity and endemic aspect, as is the case of *Cucurbita okeechobeensis* (Small) L.H.Bailey [50], also considered as an endangered species that must be protected [50,51]. The potential toxicity of the wild species may also be a limiting factor [52].

Edible kinds of pumpkins and squashes, like *C. ficifolia, C. maxima, C. moschata* and *C. pepo* can be a natural source of some bioactive components. Indeed, these species are rich in tocopherols and carotenoids, especially the seeds and the fruit peel, respectively [53]. Generally, it is complicated to make a straight qualitative or quantitative distinction between the species, because of the high variability within a single species depending either on their subsequent varieties or environmental effects and ripening stage. This aspect is also true for other phytochemicals. Depending on the edible part, *Cucurbita* species are found to be very rich in compounds with nutritious value, especially carbohydrates, proteins, minerals (Table 1), fatty acids (Table 2) and amino acids [53].



#### *Appl. Sci.* **2019** , *9*, 3387


**Table 2.** Seed fatty acid composition of some common *Cucurbita* spp. \*.

\* Results expressed in dry base of seeds; ND: Not detected; NQ: Not quantified.

The content of carbohydrates is highly variable within each species and edible parts, starting from 2.62% to 48.40% on dry basis. In general, the seeds and peels are richer than flesh, and *C. maxima* and *C. pepo* were described as containing higher levels of carbohydrates when compared to *C. moschata*. The mineral contents of *Cucurbita* fit the recommended daily intake values of FAO in various essential elements. In addition, it seems to be far from the upper limits [65], when the values are converted on a fresh matter basis. Seeds contain high amount of proteins and fats, ranging from 14.3% to 38.0% and 21.9% to 54.9%, respectively. In contrast, fruit seems to have relatively low and variable levels of proteins (0.20% to 23.95%), and a very low content of lipids (0.04% to 6.57%). *Cucurbita*, especially the seed parts, can also be a good source of amino acids [53]. Indeed, among the 20 amino acids which constitute the human proteins, 17 are present in *Cucurbita*, including the 8 + 1 essential amino acids [53,55]. In addition, Fang, Li, Niu, and Tseng [66] discovered for the first time a new amino acid in *C. moschata*, named as cucurbitine.

#### *4.1. Carotenoids and Tocopherols in Cucurbita*

Carotenoids are present in high amount in the fruit of these plants and their hybrids, α- and β- carotene; ζ-carotene; neoxanthin; violaxanthin; lutein; zeaxanthin; taraxanthin; luteoxanthin; auroxanthine; neurosporene; flavoxanthin; 5,6,5 ,6 -diepoxy-β-carotene; phytofluene; α-cryptoxanthin; and β-cryptoxanthin [53,67–70]. The total carotenoid content varies in range from 234.21 μg/g to 404.98 μg/g in *C. moschata* fruit [71], and 171.9 μg/g to 461.9 μg/g in *C. pepo* fruit [61]. The concentration of carotenoids is much higher (10 fold higher) in the peel of *C. moschata* than flesh [57]. Azizah et al. [72] investigated the effect of different baking procedure on β-carotene and lycopene contents of *C. moschata*. It was observed that boiling led to a 4 and 40-fold increase in β-carotene and lycopene contents, respectively. There are also several reports on carotenoid content of several *Cucurbita* plants, such as landrace pumpkins (*C. moschata*) [71], *C. moschata* and *C. pepo* [73,74], and *C. maxima* [75]. A summary of the major carotenoids found in *Cucurbita* species is given in Table 3.



\* Results expressed in dry base of edible flesh part; NQ: Not quantified; ND: Not detected.

Edible *Cucurbita* seeds are also rich in vitamin E (49.49 μg/g to 92.59 μg/g); γ-tocopherol is more abundant than α-tocopherol [53,62]. The amounts of α-, β-, γ- and δ-tocopherol from the cold pressed oil extracted from six samples of pumpkin seeds (*C. pepo*) from Serbia were reported by Rabrenovi´c et al. [78], which were in the range of 38.03 to 64.11 mg/100 g oil. *C. maxima* var. *béjaoui* seed oil was found as a rich source of tocopherols, where δ-tocopherol was the dominant tocopherol with 42% of the total [64]. However, for *C. pepo* seed oils, γ-tocopherol was stated as the remarkably abundant tocopherol (13–21%) compared to α- and δ-tocopherols [79].

#### *4.2. Phenolic Compounds in Cucurbita*

Table 4 presents the main phenolic compounds found in *Cucurbita* species and their structures. *C. moschata* fruit seems to have a low total phenolic content in comparison with other fruits [57]. No flavonoids were found in either the fruit nor seeds of *C. moschata* in the study of Eleiwa et al. [80]. However, Li et al. [81] have previously discovered five novel phenolic glycosides from *C. moschata*

seeds, named cucurbitosides A–E. Other new phenolic glycosides were discovered later from the same source [81,82]. In another work, Li et al. [83] isolated eight different cucurbitoside compounds (F–M).


**Table 4.** Chemical structures of phenolic compounds found in the *Cucurbita* spp. \*.


#### **Table 4.** *Cont*.

\* The data were collected from the Phenol-Explorer database which is an online comprehensive database on polyphenol contents in foods, http://phenol-explorer.eu/ (Accessed on 09.12.2018).

A study by Yang et al. [84] showed no flavonoid content (detection limit: 0.05 mg/100 g) in either the immature or mature fruits of *C. maxima*. Only the shoots and buds showed positive results. However, in accordance with [85], the total flavonoid and phenolic contents of this species were determined as approximately 2.7 mg quercetin equivalent/g extract and 8.8 mg gallic acid equivalent (GAE)/g extract, respectively. Sreeramulu and Raghunath [86] reported that average total phenolic content of *C. maxima* was 46.43 mg GAE/100 g. In another study, *C. maxima* was analyzed for its flavonoid content, and kaempferol was found to be the only flavonoid present in this species at a concentration of 371.0 mg/kg of dry weight [87].

*C. pepo* was also found to have low polyphenol content. However, Iswaldi et al. [88] have reported for the first time a list of 34 polyphenols, including a variety of flavonoids in the *C. pepo* fruit, in addition to other unknown polar compounds. Also, the *C. pepo* flowers may contain considerable amounts of phenolic compounds. A total polyphenol content ranging from 0.054 to 0.297 μg GAE/μg dry biomass has been reported to be dependent on the extraction conditions and flower sex [89]. Andjelkovic et al. [90] studied the phenolic content of six pumpkin (*C. pepo*) seed oils and identified the following compounds: tyrosol, luteolin, ferulic acid, vanillic acid, and vanillin. Among them, tyrosol was the most abundant compound, ranging from 1.58 mg/kg to 17.69 mg/kg.

Five major compounds in *C. ficifolia* fruit aqueous extract were identified by Jessica et al. [91] as: *p*-coumaric acid, *p*-hydroxybenzoic acid, salicin, stigmast-7,2,2-dien-3-ol and stigmast-7-en-3-ol. On the other hand, Periˇcin et al. [92] assessed the phenolic acids content of *C. pepo* seeds. *p*-Hydroxybenzoic acid was found to be the prevailing phenolic acid, with 34.72%, 51.80%, and 67.38% of the total phenolic acids content in whole dehulled seed, hulls, and kernels, respectively. Aside from *p*-hydroxybenzoic acid, the most dominant phenolic substances can be listed in a decreasing order of quantity as

follows: caffeic, ferulic, and vanillic acids in whole dehulled seeds. *Trans*-sinapic and protocatechuic acids, and *p*-hydroxybenzaldehyde were the abundant phenolic acids presented in kernels of hulled pumpkin variety; the hulls comprised *p*-hydroxybenzaldehyde, vanillic, and protocatechuic acids with considerable amounts.

#### *4.3. Terpenoids, Saponins and Sterols in Cucurbita*

Badr et al. [55] have demonstrated the existence of calotropoleanly ester and cholesterol in *C. pepo* fruit, and Younis et al. [62] also represented the cholesterol content in seeds (<0.2 to 3 mg/100 g). These plant parts were also reported as containing more β-sitosterol (383.89 mg/kg fresh weight) than those of *C. moschata* and *C. maxima* (277.58 mg/kg and 235.16 mg/kg fresh weight, respectively) [53]. Dubois et al. [93] have reported that, besides cucurbitacins, a new triterpenoid saponin, foetidissimoside A (3,28-bidesmosidic triterpenoid saponin) is also present in C. *foetidissima* roots. Ten years later, Gaidi et al. [94] discovered the foetidissimoside B in the same source. Also, Matus et al. [95] extracted sterols from seeds, and the most abundant were Δ7.22.25-stigmastatrienol, β-sitosterol, spinasterol and Δ7.25-stigmastadienol [61].

Cucurbitacin is a triterpenoid [96] with a bitter taste, isolated from members of the Cucurbitaceae family, and there are more than 18 types of cucurbitacin in the nature. Cucurbitacins are a group of distinctive highly oxygenated triterpenoid substances, having tetracyclic triterpenes with a cucurbitane skeleton. Figure 1 details the structure of 19-(10→9β)-abeo-10α-lanost-5-ene. They are cucurbitane triterpenes with double bonds between C4 and C5, a hydroxyl at C16, C20 and C25 and a ketone at C11 and C22 [20]. These compounds are well-recognized for their bitterness and toxicity [97].

**Figure 1.** Principal structure of cucurbitacins (19-(10→9β)-abeo-10α-lanost-5-ene).

Cucurbitacins are randomly divided into 12 groups, specifically cucurbitacins A–T, varying in their oxygen functionalities at different positions. The chemical structures of a few cucurbitacins (A, B, C, and D) are presented in Figure 2, and the cucurbitacin composition in different *Cucurbita* species is summarized in Table 5.

**Figure 2.** Structure of different cucurbitacin: (**a**) Cucurbitacin A, (**b**) Cucurbitacin B, (**c**) Cucurbitacin C and (**d**) Cucurbitacin D.

**Table 5.** Cucurbitacin content of *Cucurbita* spp. (mg/g fresh weight) \*.


\* Tr.: Trace, ND: Not detected.

Seedlings of the pumpkin *C. maxima* have been reported to involve high amounts of cucurbitacin B and small amounts of cucurbitacin D and E in radicles and cotyledons [98]. Eighteen *Cucurbita* plants species were analyzed for their cucurbitacin contents. Bitter species having comparatively high contents of cucurbitacins B and D and no detectable cucurbitacins E and I were identified as *Cucurbita andreana* Naudin, *Cucurbita ecuadorensis* Cutler & Whitaker, *Cucurbita radicans* Naudin, *Cucurbita lundelliana* L.H.Bailey, *C. argyrosperma* and *Cucurbita pedatifolia* L.H.Bailey. The other group of wild, bitter species having relatively large amounts of cucurbitacins E and I were *Cucurbita cylindrata* L.H.Bailey, *C. foetidissima*, *C. martinezii*, *C. okeechobeensis*, and *Cucurbita palmata* S.Watson, *Cucurbita pepo* var. *texana (Scheele) D.S.Decker* was found to contain nearly all cucurbitacins as cucurbitacin E-glycoside. However, no cucurbitacin could be identified in domesticated and sweet species, including *C. ficifolia*, *C. maxima*, *C. moschata*, and *C. pepo* [48]. The same group of investigators also determined the cucurbitacin and cucurbitacin glycoside content in fruits of two *Cucurbita* plant hybrids (*C. andreana* × *C. maxima* and *C. texana* × *C. pepo*), as well as in *C. foetidissima* roots. As a result, *C. andreana* × *C. maxima* fruits contained cucurbitacins B and D, whereas *C. texana* × *C. pepo* fruits and *C. foetidissima* roots presented cucurbitacins E and I [99]. With regards to *C. andreana* fruits extract, it was chromatographically fractionated, and cucurbitacins B, D, E and I, as well as cucurbitacin E and I aglycones and cucurbitacin B, E, and I glucosides were detected in this species [102]. *C. pepo* comprises the most investigated *Cucurbita* plant for its cucurbitacin content. In an earlier study, cucurbitacin E was detected as a primary bitter component in seedling *C. pepo* radicles, whereas cucurbitacins B and I were found in trace amounts in the same tissue. On the other hand, cotyledons of this species were found to include cucurbitacin D and E in moderate and low concentrations, respectively [103]. Freeze-dried samples of Blackjack cultivar of *C. pepo* were analyzed by two research teams in different years. Ferguson et al. [101] found 1.12 mg cucurbitacin E-glycoside/g fresh weight, whereas Hutt and Herrington [100] detected 0.6 mg cucurbitacin E-glycoside/g fresh weight. Wang et al. [104] investigated *C. pepo* cv dayangua for its phytochemical content, and cucurbitacin L and cucurbitacin K were isolated at concentrations of 2.13 mg/kg dry matter and 2.67 mg/kg dry matter, respectively. In a very recent study, cucurbitacin C and E glycosides in *C. pepo* fruit were reported for the first time. The corresponding concentrations of compounds were 105 μg/g fresh fruit for cucurbitacin C and 438 μg/g fresh fruit cucurbitacin E. The authors also demonstrated that gamma irradiation did not affect the cucurbitacins concentration, when compared to non-irradiated control group [105].

From a biological point of view, it has been demonstrated that cucurbitacins exert several bioactivities, such as antitumor, anti-inflammatory, anti-atherosclerotic, antidiabetic effects [106]. There is a large body of evidence suggesting that cucurbitacins hold very high orders of cytotoxicity towards a vast quantity of malignancies. Seed and fruit portions of few cucurbits are found to exhibit purgative, emetic and anthelmintic activities due to presence of cucurbitacin triterpenoids [107,108]. Jayaprakasam et al.[109] isolated cucurbitacins B, D, E, and I from *C. andreana* fruits and evaluated their effects at a level of growth suppression of human breast (MCF-7), colon (HCT-116), lung (NCI-H460), and central nervous system (CNS) (SF-268) tumor cell lines; cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes; and even on lipid peroxidation. All isolated cucurbitacins were able to abate the proliferation of investigated cancer cell lines in varying percentages. Among them, cucurbitacin B exhibited more than 80% proliferation inhibitory activity. Additionally, cucurbitacins B, D, E, and I inhibited COX-2 enzyme, an enzyme responsible for inflammation, by 32%, 29%, 35%, and 27%, respectively, at a concentration of 100 mg/mL. A cucurbitacin derivative, with a cucurbitacin D-like structure, was separated from the methanolic extract of *C. pepo* seeds and revealed to have antiulcer activity in a dose-dependent manner [110].

In contrast to their biological activities, cucurbitacins have toxic effects in mammalians. According to Le Roux et al. [111], 353 poisoning cases linked to *C. pepo* cucurbitacins were reported to French Poison Control Centers between 1 January 2012 and 12 December 2016. In 1980s, various human poisoning cases were stated due to the consumption of commercially produced zucchini (*C. pepo*) in Australia. The most common developed characteristic symptoms evidenced by exposed individuals include a bitter taste in the mouth, abdominal pain, diarrhea and rarely, collapse [101]. Pfab et al. [112] reported that, 81 symptomatic cases were registered to Poison Information Centre (PIC) Munich between 2002 to 2015. The primary symptom was colitis with bloody diarrhea after zucchini (*C. pepo*) consumption. As the authors stated, cucurbitacins, the responsible toxins of poisonings, are not often identified in cultivated fruits, but some fruits spontaneously produced them. In an in vivo experiment, mice were fed with *C. pepo* fruit of two cultivars, 'Blackjack' and 'Straightneck', and an accession of the bitter species, *C. texana.* As a result, mice having the diet with 1% *C. texana,* containing cucurbitacins E glycoside and I, exhibited poor growth, severe diarrhea, anemia and 40% mortality. When the *C. texana* percentage increased to 10 or 20%, mortality rate reached 100% within a few days. However, no detectable cucurbitacins in *C. pepo* cultivars were identified and, animals fed up to 20% freeze-dried squash in their diets displayed no toxicity-related symptoms [113].

#### *4.4. Functional Carbohydrates and Polysaccharides in Cucurbita*

*C. moschata* fruit is a rich source of polysaccharides, with a useful biological benefit, i.e., cytoprotective and antioxidative activities [114,115], that can reach 16.2% in dry matter, under optimised conditions [116]. Xia and Wang [117] reported high amounts of D-chiro-inositol, a hypoglycaemic molecule, in *C. ficifolia* fruit (without seeds). In addition to D-chiro-inositol, the plant was found to be rich in other carbohydrates, like myo-inositol, fagopyritols and sucrose [117].

#### *4.5. Fatty Acids of the Oil of Cucurbita Seeds*

Seeds of *Cucurbita* species are also rich in fat (around 45%) and a variety of fatty acids, such as saturated, unsaturated (around 55% of the oil), and conjugated fatty acids (CFA) [118,119], therefore, may be considered as a source of molecules with high pharmacological potential and health benefits. Specifically, CFA are polyunsaturated fatty acids containing conjugated double bonds with positional and geometric isomers, and have several beneficial effects [120].

#### *4.6. Other*

*C. ficifolia* can also be considered a safe source of proteinases, with a high potential to be used for bioactive peptides production [121,122]. Other enzymes, like peroxidases can be obtained from *C. moschata* [123]. No alkaloids were found in neither the fruit nor seeds of *C. moschata* [80]. In contrast, Chonoko and Rufai [124] have represented alkaloid contents both in the back peels and seeds of *C. pepo*. Elinge et al. [56] also reported the presence of phytate (35.06 mg/100 g), oxalate (0.02 mg/100 g), hydrocyanic acid (0.22 mg/100 g) and nitrate (2.27 mg/100 g) as antinutrient compounds in *C. pepo* seeds.

#### **5.** *Cucurbita* **Plants for Industrial Purposes: Key Role as a Food Preservative**

Three main pumpkin varieties, including *C. pepo, C. maxima,* and *C. moschata,* are considered both as nutritional and medicinal foods in many countries [125]. Some biological activities are reported in pumpkins, among them antimicrobial activity [126,127]. The reason why this plant show antimicrobial applications is related to its high vitamins content (mainly A and C), phenolic compounds, minerals, dietary fiber, amino acids and other advantageous compounds to humans. Indeed, antioxidant, antibacterial and intestinal antiparasitic activities are also part of the active function of this crop [125,128]. Pumpkin extracts are rich in steroids, flavonoids, tannins, alkaloids and saponins, showing momentous antimicrobial and antifungal activity against some microorganisms [124,129]. Based on Muruganantham et al. [130] study, the ethyl acetate extract of *C. maxima* flowers has meaningful antifungal and antibacterial activity against some microorganisms, like *Escherichia coli*, *Salmonella typhi*, *Bacillus cereus*, *Enterobacter faecalis*, *Candida albicans*, and *Curvularia lunata*. Nonetheless, the antioxidant and antimicrobial effects of pumpkin seeds have also been reported. The extracted oil from pumpkin seeds chiefly contains fatty acids, among them linoleic, oleic, palmitic and stearic acids. In addition, the oil is also full of tocopherols (δ-tocopherol, γ-tocopherol, β-tocopherol and carotenoids, like lutein and β-carotene), characterized by displaying strong antioxidant effects. It is

worth noting that pumpkin seeds oil can preserve lipids by having selenium, tocopherol, enzymes, hormones and vitamins. Adeel et al. [126] stated that pumpkin seed oil exhibits high antibacterial activity against *Staphylococcus aureus*, and pumpkin seeds are also able to markedly inhibit *Rhodotorula rubra* and *C. albicans* growth at 0.5 mg/mL and 1.0 mg/mL, respectively [129]. Other research showed that *Bacillus subtilis*, *S. aureus, E. coli* and *Klebsiella pneumonia* are easily affected by the antibacterial properties of the methanolic extract from seed oil at 1.0 mg/mL, 2.0 mg/mL, 2.0 mg/mL and 3.0 mg/mL, respectively [127]. Moreover, the authors stated that the antibacterial activity is not the only property of the oil, since it also exerts a remarkable antifungal activity against *R. rubra* and *C. albicans,* at concentrations of 0.5 mg/mL and 1.0 mg/mL, respectively. It has even been stated that there is a high susceptibility of *R. rubra* to the seeds' oil. Still, researchers have stressed that 1.0 mg/mL of pumpkin oil seeds was effective against *Penicillium chrysogenum* and *Aspergillus parasiticus,* and 2.0 mg/mL against *A. flavus* [127].

Pumpkin rinds and leaves contain some special bioactive proteins, which have shown preservative effects. For example, pumpkin leaves contain the PR-5 antifungal protein [131], highly homologous to thaumatin. In addition, the antimicrobial activity of this protein, besides the synergistic effect of nikkomycin against *C. albicans* development, has been proved. Moreover, the growth and survival of the phytopathogenic bacteria *Erwinia amylovora*, *Phytophthora infestans* and *Pseudomonas solanacearum* is prohibited by a ribosome-inactivating protein purified from *C. moschata* [132]. Black pumpkin seeds have another component, named cucurmoschin and identified as an antifungal peptide abundant in glycine, arginine, and glutamate residues. Additionally, Park and et al. [133] found two novel antifungal proteins (Pr-1 and Pr-2) from pumpkin rinds that showed strong in vitro antifungal activity against *Botrytis cinerea*, *Colletotrichum coccodes*, *Fusarium solani*, *F. oxysporum*, and *Trichoderma harzianum* at 10–20 μM.

#### **6. Conclusions and Future Perspectives**

Overall, *Cucurbita* species have chemical components with an intriguing impact in health promotion. Several squash and pumpkin species are a natural and rich source of potential bioactive compounds, such as carotenoids, tocopherols, phenols, terpenoids, saponins, sterols, fatty acids, functional carbohydrates and polysaccharides. Of these triterpenoids, cucurbitacins are particularly noteworthy for their multiple marked abilities. The presence of active phytochemicals in Cucurbitaceae species makes them a great matrix to be further exploited for both preventive and therapeutic purposes, beyond biotechnological applications. For an emphasis on *Cucurbita* plants' pharmacological potential, please refer to other review [134].

**Author Contributions:** All authors contributed significantly to this work. In addition, J.S.-R., M.M., N.M., W.C.C., and J.N., critically reviewed the manuscript. All the authors read and approved the final manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This work was supported by CONICYT PIA/APOYO CCTE AFB170007. N. Martins would like to thank the Portuguese Foundation for Science and Technology (FCT-Portugal) for the Strategic project ref. UID/BIM/04293/2013 and "NORTE2020-Northern Regional Operational Program" (NORTE-01-0145-FEDER-000012).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


Technología Agrícolas (ICTA) and Consejo Internacional de Recursos Fitogenétic, International Board for Plant Genetic Resources (IBPGR): Guatemala, 1986.


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