Quinoa Secondary Metabolites and Their Biological Activities or Functions

Quinoa (Chenopodium quinoa Willd.) was known as the “golden grain” by the native Andean people in South America, and has been a source of valuable food over thousands of years. It can produce a variety of secondary metabolites with broad spectra of bioactivities. At least 193 secondary metabolites from quinoa have been identified in the past 40 years. They mainly include phenolic acids, flavonoids, terpenoids, steroids, and nitrogen-containing compounds. These metabolites exhibit many physiological functions, such as insecticidal, molluscicidal and antimicrobial activities, as well as various kinds of biological activities such as antioxidant, cytotoxic, anti-diabetic and anti-inflammatory properties. This review focuses on our knowledge of the structures, biological activities and functions of quinoa secondary metabolites. Biosynthesis, development and utilization of the secondary metabolites especially from quinoa bran were prospected.


Introduction
Quinoa (Chenopodium quinoa Willd.), a dicotyledonous plant belonging to Chenopodiaceae family, is one of the oldest native crops in the Andean region of South America, with approximately 7000 years of cultivation [1]. It has been considered as a pseudo-cereal because of the grain characteristics [2]. Consumption of seeds is the most common use of quinoa. Once the bran (also called hull or seed coats) containing saponins has been eliminated, the seeds can be consumed as entire grains or milled to flour for preparation of bread and pastry. The other parts such as leaves and stems were used as feed [2,3].
Quinoa has been recognized as a complete food due to a variety of vitamins, significant amounts of minerals, unsaturated fatty acids, dietary fiber, abounding proteins, and excellent balance of essential amino acids. The year 2013 was named "The Internaitonal Year of Quinoa" by the UN. Quinoa has been introduced and cultivated all over the world in the past ten years [3][4][5][6][7][8].
To our knowledge, there are many reviews on quinoa, most of them are focused on the nutritional, functional and antinutritional aspects [16][17][18], abiotic stress responses [19], biodiversity and sustainability [20], or only a specific topic of quinoa secondary metabolites and their biological activities such as steroids [21,22] and triterpenoid saponins [23], but no review covers almost all secondary metabolites and their biological activities. In this review, we summarize and discuss quinoa secondary metabolites on their structural diversity, biological activities or functions during the past 40 years.
Phenolic acids can be released by acid, alkaline, and enzymatic treatments from the conjugated forms. It was reported that at least 19 phenolic acids were released in the residue of quinoa which can enhance bioaccessibility [25]. Bound phenolic acid derivatives in conjugated forms were not affected by environmental stresses [26]. Higher content of phenolic acids showed stronger antioxidant and inhibitory activities of α-glucosidase and pancreatic lipase [25].

Benzoic Acid Analogues and Their Biological Activities or Functions
At least 16 benzoic acid analogues have been identified from quinoa. Their biological activities are listed in Table 1, and the structures are shown in Figure 1. Benzoic acid derivatives include benzoic acid (1), gallic acid (8), protocatechuic acid (10), syringic acid (12), vanillic acid (13), and their analogues. They are rich in the leaves and seeds of quinoa [25,27]. Though the benzoic acid analogues from quinoa have not been evaluated for their biological activities, these metabolites from other plant species have been reported to have antimicrobial [28,29], allelopathic [30], antioxidant [31], and antifeedant [32] activities (Table 1). Table 1. Benzoic acid analogues and their biological activities or functions.

Name
Quinoa Part Used for Isolation Biological Activity or Function Ref.

Flavonols and Their Biological Activities or Functions
About 21 flavonols have been identified in quinoa. Most of them are present in the seeds. Their biological activities are listed in Table 4, and their structures are shown in Figure 4.
Both kaempferol (35) and quercetin (46) are two main flavonols. They are in the form of glycosides present in quinoa. Structure-activity relationship of their antioxidant activity showed that the ability to quench free hydroxyl radicals increased with the amount of hydroxyl groups in the ring B. For example, myricetin (45) was a stronger antioxidant than kaempferol (35) [118]. In addition, the compounds with 3',4'-dihydroxy substituents in the ring B had much stronger antioxidative activities than those without ortho-dihydroxy substitution in the ring B [119]. Quercetin (46) was the strongest antioxidant among the flavonoids. Both isorhamnetin (34) and kaempferol (35) were the most abundant flavonoids in quinoa leaves, and it also contained large amounts of rutin (54) [27]. Four kaempferol 3-glycosides (38-41) exhibited moderate antioxidant activity while two quercetin 3-glycosides (50,51) showed strong antioxidant activity, suggesting that quinoa could represent an important source of free radical inhibitors [120].
Many flavonoids are characterized by antibacterial, antifungal and antiviral activities, not only against plant pathogens, but also against the pathogens for humans and animals (Table 4). Kaempferol (35) and its derivatives showed antibacterial activity against Gram-positive and Gram-negative bacteria, as well as against the fungus Candida glabrata [121,122].

Name Quinoa Part Used for Isolation
Biological Activity or Function Ref.

Flavanols and Their Biological Activities or Functions
Three flavanols namely catechin (58), epicatechin (59), and epigallocatechin (60) were found in quinoa seeds. Their biological activities are listed in Table 6, and their structures are shown in Figure 6. They generally showed antioxidant [149,168] and antimutagenic [169] activities.

Terpenoids and Their Biological Activities or Functions
The terpenoids in quinoa mainly include monoterpenoids and triterpenoids which are biosynthesized through the isoprenoid metabolic pathway. The monoterpenoids usually play functions as allelochemicals in quinoa. The triterpenoids are present in the seed coats (also called bran or hull), and have a characteristic bitter or astringent taste to protect it from birds and insects, and possess detergent properties [2]. The saponins are also of interest as valuable adjuvants and the first saponin-based vaccines have been introduced commercially [203].

Sesquiterpenoids and Their Biological Activities or Functions
Only one sesquiterpene namely caryophyllene (81) was identified in quinoa [204]. Its structure is shown in Figure 9.

Triterpenoids and Their Biological Activities or Functions
Triterpenoids, including their aglycones (sapogenins) and glycosides (saponins), are mainly present in the bran to protect quinoa from pests and herbivores (i.e., birds and insects) and pathogenic microorganisms [206]. Quinoa saponins are characterized as the bitter metabolites. The quinoa could be classified into bitter and sweet varieties according to the triterpenoid saponin content, which is much lower in the sweet varieties and higher in the bitter ones [138,207].
The crude saponin fraction inhibited the growth of Candida albicans at 50 µg/mL [208]. The alkali-transformed saponin from quinoa bran showed inhibition against halitosis-related bacterium Fusobacterium nucleatum, with a minimum inhibitory concentration (MIC) of 31.3 µg/mL. It could be used as an antibacterial agent to treat halitosis [209]. When the fungal pathogen Botrytis cinerea was treated with the saponin extracts, mycelial growth and conidial germination were significantly inhibited [210].
When golden apple snails (Pomacea canaliculata, GAS) were treated with the crude saponin under laboratory conditions in 24 h at approximately 33 µg/mL, they were completely killed [211]. Similarly, when giant apple snails (Pomacea maculata) were treated with saponins above 7 µg/mL after 72 h, they were also 100% killed. Quinoa saponin could be a viable product to safely control P. maculata in rice fields [212]. Therefore, quinoa saponins could be developed into molluscicide. In addition, this molluscicide was found to be non-toxic to other non-target species such as goldfish (Carassius auratus) and tilapia (Oreochromis mossambicus), while providing adequate protection from Pomacea snails to newly sprouted rice seeds under laboratory conditions [211,213].

Spergulagenic Acid Derivatives and Their Biological Activities or Functions
Spergulagenic acid (84), a pentacyclic triterpene used in medicine, was found in diverse plant families [258]. Until now, three spergulagenic acid glycosides (Table 11) were identified in quinoa [217,256], though spergulagenic acid as the aglycone has not been isolated from quinoa. Their structures are shown in Figure 13. Table 11. Spergulagenic acid derivatives and their biological activities or functions.

Name Quinoa Part Used for Isolation
Biological Activity or Function Ref.

Name Quinoa Part Used for Isolation
Biological Activity or Function Ref.

Phytolaccagenic Acid Derivatives and Their Biological Activities or Functions
Phytolaccagenic acid (86) might be originated from serjanic acid (85) by subsequent oxidative enzymatic steps involving the formation of the corresponding alcohol substituted at C-23 in planta [11]. It is one of the main structures of quinoa sapogenins. About 10 phytolaccagenic acid analoques have been identified in quinoa. They are listed in Table 13, and the structures are shown in Figure 15.
Phytolaccagenic acid saponins are highly concentrated in the bran (seed coats), which are more exposed to water during germination compared to oleanolic acid saponins [259]. It was suggested that a short saccharide chain (1 or 2 glycosyl residues) requires the presence of an additional longer one to make the saponin water-soluble [260]. Phytolaccagenic acid was employed as the anti-inflammatory drug of oral administration [234].

Name Quinoa Part Used for Isolation
Biological Activity or Function Ref.

Name Quinoa Part Used for Isolation
Biological Activity or Function Ref.

Other Triterpenoids and Their Biological Activities or Functions
Other triterpenoids include tetracyclic and pentacyclic triterpenoids. Their biological activities are shown in Table 17, and the structures are shown in Figures 19 and 20.

Meroterpenoids and Their Biological Activities or Functions
Mertoterpenoids are natural products of mixed biosynthetic origin which are partially derived from terpenoids. Meroterpenoids were also found in quinoa that include tocopherols (141)(142)(143)(144) and tocotrienols (145,146). Their biological activities are listed in Table 18, and the structures are shown in Figure 21.
Both α-tocotrienol (145) and β-tocotrienol (146) were also identified in quinoa seeds [275]. They were the members of the vitamin E family to show antioxidant and anti-inflammatory properties [282].

C 27 -Steroids and Their Biological Activities or Functions
Eleven C 27 -steroids were identified in quinoa seeds which are listed in Table 19. Their structures are shown in Figure 22. Among them, ecdysteroids are main steroids which are insect moulting hormones and protect plants against non-adapted insects and nematodes [21]. Ecdysteroids are mainly present in the bran, the major component is 20-hydroxyecdysone (148) possessing a 14α-hydroxy-7-en-6-one chromophore and A/B-cis ring fusion (5β-H) [21].
Eating quinoa seeds or quinoa-derived products provides significant amounts of ecdysteroids that may be beneficial to animal or human health [22]. Quinoa extract enriched in 20-hydroxyecdysone has an antiobesity activity in vivo and could be used as a nutritional supplement for the prevention and treatment of obesity and obesity-associated disorders. The findings indicated that the extract acted by reducing both fatty acid uptake and esterification in adipocyte [286]. It was found that 8 isolated ecdysteroids showed a stronger free-radical-scavenging activity, which was almost 3 to 8 times higher than that of the well-known antioxidant compound, BHA, and also possessed a strong ability to sequester ferrous ions. This observation supported that if the number of hydroxyl and methyl groups bearing the carbon skeleton of ecdysteroids is higher, the antioxidant activity becomes stronger. The ability of ecdysteroids to sequester ferrous ions is thought to be due to their carbonyl conjugated to a double bond attached to the C-7. Ecdysteroids are also able to inhibit skin collagenase, and could therefore also prevent skin ageing [287]. In addition, ecdysteroids have been reported to occur in Chenopodiaceae to show their possible chemotaxonomic and ecological implications [21].

C 28 -Steroids and Their Biological Activities or Functions
About 14 C 28 -steroids such as campesterol (160), makisterone A (166), and their derivatives have been identified from quinoa seeds [34]. Their biological activities are listed in Table 20, and the structures are shown in Figure 23. The main biological activities include antioxidant activity [289], antiangiogenic activity [291], and inhibitory activity on collagenase [287].

C 29 -Steroids and Their Biological Activities or Functions
The main C 29 -steroids in quinoa included avenasterol (172/173), sitosterol (176), stigmasterol (181), and their derivatives. They were all identified in the lipid extract of quinoa seeds [261]. Their biological activities are listed in Table 21, and the structures are shown in Figure 24.

Nitrogen-Containing Metabolites and Their Biological Activities or Functions
About 12 nitrogen-containing metabolites have been identified in quinoa seeds. They belong to the derivatives of glycine and tyrosine. Their biological activities are listed in Table 22, and the structures are shown in Figure 25.
Betalains showed promising bioactive potential, such as high antioxidant and free radical scavenging activities [30]. Betacyanins and betaxanthins showed the highest antioxidant activity by comparing the white and black quinoa varieties [35]. These two varieties are characterized by a high content of dopaxanthin (188), whose dihydroxylated substructure demonstrated high antioxidant capacity [36].

Conclusions and Future Perspectives
This review focuses on the structures, isolation parts, biological activities or functions of quinoa secondary metabolites during the past 40 years. Flavonoids and phenolic acids were mostly derived from quinoa seeds. Steroids were mostly separated from quinoa bran. Triterpenoids were also mainly located in the bran. Their biological activities or functions have been reported but not comprehensive, and are needed to be systematically evaluated in the future.
The bitter taste associated with saponins (triterpenoids) greatly limits the use of quinoa as food [18]. Approximately 34% of quinoa saponins are present in the bran, indicating that dehulling could remove almost one half of the saponins. The seeds should be milled to remove the bran (seed coats) to make them edible [239]. Another method to remove saponins from the seeds is washing due to the high water solubility of saponins although this method can lead to the loss of some nutrients such as vitamins and minerals [18].
With the increased demand for quinoa, the problem that comes with it is that the bran is discarded as an industrial production waste. In order to increase the added value of quinoa, the bran (seed coat) should be fully exploited and utilized [321]. Quinoa saponins have shown their great potential applications. They can be used in the pharmaceutical industry as the saponins can induce changes in intestinal permeability which can be useful for the absorption of specific medicines and in hypocholesterolemia [15,[322][323][324]. Quinoa saponins are also of interest as valuable adjuvants and the first saponin-based vaccines have been introduced commercially [203]. In addition, the saponins can be used as bitters, antibiotics to control pathogenic fungi and bacteria, or to protect crop against attack by birds and other pests [325]. Quinoa saponins have been successfully developed as a bioinsecticide in Bolivia [326]. They can also be used as emulsifiers and detergents due to surface active characteristics which saponins have [327]. Quinoa saponins might be developed into products like soaps, shampoos, and bitters in the future. As phenolic acids, flavonoids, and steroids are also abundant in the bran, they can be developed into antimicrobials, antioxidants, and insect moulting hormones, respectively [5,21]. It is worth mentioning that 20-hydroxyecdysone (148), mainly present in the bran, has potential for development as an insect moulting hormone [21]. After the above secondary metabolites are extracted from the bran, the remaining residues, which mainly contain cellulose, could be either used as feed, or femented into biofuels and biofertilizer.
Biosynthesis research on quinoa secondary metabolites has rarely been reported. Methyl jasmonate was reported to induce accumulation of saponins in quinoa leaves and induce the expression of saponin biosynthetic genes in quinoa [328]. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars. Genome sequencing of quinoa revealed a diversity of biosynthetic core genes of secondary metabolites [329], indicating the great potential of this plant to produce various secondary metabolites with biological activities or functions which merit further investigation.