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Table of Contents
Abstract
1. Introduction
2. Review Strategy
3. Chemodiversity Of Calendula Genus
3.1. Monoterpenes
3.2. Sesquiterpenes
3.3. Diterpenes
3.4. Triterpenes
3.5. Carotenoids
3.6. Phenols
3.7. Benzoic Acid Derivatives
3.8. Hydroxycinnamates
3.9. Coumarins
3.10. Flavonoids And Anthocyanins
3.11. Other Compounds
3.12. Polysaccharides
4. Separation Of Calendula Metabolites By GC And LC
4.1. Sterols
4.2. Triterpenes And Glycosides
4.3. Carotenoids
4.4. Fatty Acids
4.5. Phenolic Compounds
5. Concluding Remarks And Future Perspectives Of Calendula Metabolites Research
Footnotes
Supplementary Materials
Author Contributions
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts Of Interest
Abstract
Marigold ( Calendula ), an important asteraceous genus, has a history of many centuries of therapeutic use in traditional and officinal medicines all over the world. The scientific study of Calendula metabolites was initiated at the end of the 18th century and has been successfully performed for more than a century. The result is an investigation of five species (i.e., C. officinalis , C. arvensis , C. suffruticosa , C. stellata , and C. tripterocarpa ) and the discovery of 656 metabolites (i.e., mono-, sesqui-, di-, and triterpenes, phenols, coumarins, hydroxycinnamates, flavonoids, fatty acids, carbohydrates, etc.), which are discussed in this review. The identified compounds were analyzed by various separation techniques as gas chromatography and liquid chromatography which are summarized here. Thus, the genus Calendula is still a high-demand plant-based medicine and a valuable bioactive agent, and research on it will continue for a long time.
1. Introduction
Calendula (marigold; Caléndula L.) is a genus of herbaceous plants from the Asteraceae family, whose members are widely used for medicinal and decorative purposes. Genus Calendula includes 12 species of which Calendula officinalis L. is the most famous plant and the oldest medical remedy [ 1 ]. To date, experimental science has accumulated a considerable amount of scientific information about this genus; therefore, we performed a scientometric study of the available information. There are more than 2200 articles related to the study of the Calendula species for the period of 1891–2022 ( Figure 1 ). Statistical studies indicate an exponential growth in scientific interest in Calendula ; the value of the determination coefficient (r 2 ) for the ‘curve of interest’ (Y = 0.6718·e 0.0726·X ) is 0.9435, which indicates the reliability of these statements. Thus far, the greatest scientific impact on the total number of studies on Calendula was made during 2010–2019 (44% of publications); however, because during 2020–2022, approximately 19% of studies on this topic were completed, the picture may change in the near future. Among the scientific areas in which Calendula research is performed, the agricultural and biological (approximately 38% of publications), medical (approximately 28%), and pharmacology/toxicology sciences (approximately 25%) occupy a predominant position ( Table S1 ). The largest number of works published by authors are from India (208), USA (200), Iran (189), Brazil (158), and Italy (148), and the authors with the largest number of articles are Kasprzyl Z. (35), Janiszowska W. (33), Szakiel A. (24), and Bransard G. (10). The top 10 most-cited articles with more than 100 citations include studies on chemical composition (triterpenoids, lipids), biological activity (anti-inflammatory, antioxidant, hypoglycemic), as well as clinical trials and allergic properties [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ] ( Table S2 ). As expected, this level of scientific interest has led to the fact that review papers on various Calendula aspects are published in the scientific literature with varying frequency. In total, twelve reviews have been published from 2006 to 2022 ( Table 1 ). All identified review articles had an important goal of generalizing data on the pharmacological activity of Calendula extracts to the detriment of information on the chemical composition. As a result, the total number of compounds mentioned in these works was 0–155. The work that cites the largest number of compounds (155) was published in 2009; therefore, this information needs to be updated. None of the reviews summarized data on the methods of analysis and/or separation of Calendula metabolites, which is a very important aspect of practical research of plant samples. Therefore, the aim of this work is to summarize the scientific information about the Calendula genus regarding the metabolite’s diversity as well as methods of analysis and separation.
2. Review Strategy
The resources of international databases (e.g., Scopus, Web of Science, PubMed, and Google Scholar) were used, and only original papers written in English and published in journals prior to October 2022 were considered. The search keywords used included plant names (e.g., “Calendula”, “Calendula officinalis”, etc.) and metabolite names. Metabolites with tentative structure (e.g., “quercetin- O -desoxyhexosyl- O -hexoside”, etc.) were excluded from the study. The structures of well-known metabolites (e.g., monoterpenes, sesquiterpenes, fatty acids, amino acids, etc.) are not discussed in this paper.
3. Chemodiversity of Calendula Genus
Some of the earliest chemical studies of the Calendula genus are the reports of F.A. Wirth (1891) [ 24 ], A. Kirchner (1892) [ 25 ], and A. Hilger (1894) [ 26 ] on the coloring pigments of C. officinalis flowers, which indicated the presence of phytosterols and some esters. Later, H. Kylin (1926) determined that the color of marigold flowers was primarily due to the carotenoid pigment calendulin, which differs from carotene; in 1932, L. Zechmeister and L. von Cholnoky characterized calendulin as a mixture of lycopene and violaxanthin [ 27 ]. Research on C. officinalis carotenoids was continued only in 1951 [ 28 ], after which investigations of the metabolites of this species and the genus became regular and have continued to this day for more than 70 years. The chemical studies of Calendula genus metabolites include five species: C. officinalis or pot marigold (common marigold) is the most famous and widely distributed medicinal plant; C. arvensis or field marigold and C. suffruticosa or bush marigold are native to Central and Southern Europe; C. stellata or star marigold is grown in Northwestern Africa, Malta, and Sicily; and small tripterous marigold C. tripterocarpum occurs in Spain, Iran, and Africa. During 1892–2022, more than 650 compounds ( 1 – 656 ) have been identified for the genus Calendula , including monoterpenes ( 1 – 44 ), sesquiterpenes ( 45 – 173 ) and sesquiterpene glycosides ( 174 – 207 ), diterpenes ( 208 , 209 ), triterpenes ( 210 – 342 ), carotenoids ( 343 – 437 ), phenols ( 438 – 443 ), benzoic acid derivatives ( 444 – 456 ), hydroxycinnamates ( 457 – 478 ), coumarins ( 479 – 488 ), flavonols ( 489 – 516 ), anthocyanins ( 517 – 524 ), alkanes ( 525 – 550 ), aliphatic alcohols ( 551 – 559 ), aliphatic aldehydes and ketones ( 560 – 565 ), fatty acids and esters ( 566 – 602 ), chromanols ( 603 – 613 ), organic acids ( 614 – 616 ), carbohydrates ( 617 – 630 ), amino acids ( 631 – 646 ), and other groups ( 647 – 656 ) ( Table 2 ). In addition, several polysaccharides have been isolated and characterized. Among the species mentioned, the most studied is C. officinalis for which 529 compounds are known, followed by C. arvensis (187 comp.), C. suffruticosa (68 comp.), C. stellata (27 comp.), and C. tripterocarpa (5 comp.). In terms of the organ-specific distribution of known metabolites of C. officinalis , the flowers are the best-studied part and are known to contain 403 compounds, while the leaves, roots, and seeds are known to contain 138 compounds. Studies on other species have been performed mainly on samples of the aerial part.
3.1. Monoterpenes
Monoterpenes 1 – 44 were found in the essential oils of C. officinalis , C. arvensis , and C. stellata herb, flowers, and leaves [ 16 , 29 , 30 , 31 , 32 , 33 , 34 ]. The typical compounds of the Calendula genus are linalool ( 15 ), limonene ( 17 ), β-myrcene ( 21 ), α/β-pinene ( 27 / 29 ), sabinene ( 33 ), γ-terpinene ( 40 ), terpinene-4-ol ( 42 ), α-terpinolene ( 43 ), and α-tujene ( 44 ) because these are routinely identified in essential oil samples using gas chromatography–mass spectrometry (GC-MS). These compounds are likely responsible for the characteristic odor of marigold flowers, although this has not been confirmed by olfactory analysis.
3.2. Sesquiterpenes
A total of 163 compounds of sesquiterpene nature were detected or isolated from four calendulas, i.e., 129 non-glycosidic compounds ( 45 – 173 ) and 34 glycosides ( 174 – 207 ) ( Figure 2 ). All non-glycosides were detected in the essential oils of C. arvensis , C. officinalis , and C. suffruticosa [ 30 , 31 , 37 ]. Structurally, derivatives of cadinane, carotane, caryophyllane, cubebane, eromophyllane, eudesmane, muurolane, and selinane dominated in all samples studied. The sesquiterpene glycosides of the Calendula genus (a rare group of natural terpenoids) have attracted much greater interest. The first compound, arvoside A ( 174 ), isolated from C. arvensis , is a very rare 4- epi -cubebol glycoside [ 44 ]. Later, viridiflorol derivatives ( 175 – 189 ) were found in C. arvensis (as C. persica ) and C. officinalis . This is the largest group of sesquiterpene glycosides in which hydroxyl can be substituted by fucose or chinovose acylated by acetic [ 44 ], isobutyric [ 45 ], isovaleric [ 44 ], methylpentenoic [ 44 , 46 ], methylpropanoic [ 47 , 48 ], methylbutenoic [ 46 , 47 ], senecic [ 45 , 46 ], 4-methylsenecic [ 46 ], angelic [ 45 ], and tiglic acids [ 45 ]. Similar to viridiflorol fucosides and chinovosides of β-eudesmol, 190 – 196 were identified in C. arvensis [ 46 ] and C. officinalis [ 45 ]. Rare angeloyl fucosides of 4α-hydroxygermacra-1(10) E ,5 E -diene ( 197 ) [ 46 ], α-elemol ( 207 ) [ 45 ], and 3α,7β-dihydroxy-5β,6β-epoxyeudesm-4(15)-ene ( 203 – 208 ) [ 48 ], as well as megastigmane glucosides officinoside A ( 199 ) and B ( 200 ) [ 50 ], icariside C 3 ( 198 ) [ 9 ], and glucosyl fucosides officinoside C ( 201 ) and D ( 202 ) [ 50 ] showed the unique sesquiterpene profile of Calendula plants.
3.3. Diterpenes
Two diterpenes, neophytadiene ( 176 ) and phytol ( 177 ), were identified in the essential oils of C. arvensis , C. officinalis , and C. suffruticosa [ 40 , 41 ].
3.4. Triterpenes
Triterpenes of the genus Calendula are present in plants both in the free state and as esters with fatty acids (lauric, myristic, palmitic) or alcohols (methanol, n -butanol), as well as in the glycosidic form. Isolated and characterized compounds were derived from eleven parent structures, including stigmastane ( 211 – 220 ), ergostane ( 221 – 224 ), cholestane ( 225 – 229 ), lanostane ( 230 , 231 ), dammarane ( 232 ), cycloartane ( 233 , 234 ), fridelane ( 235 , 236 ), lupane ( 237 – 246 ; Figure 3 ), ursane ( 247 – 270 ; Figure 3 ), oleanane ( 271 – 340 ; Figure 4 ) and tirucallane ( 341 , 342 ). The only aliphatic triterpene squalene ( 210 ) was found in C. suffruticosa [ 42 ]. Stigmastanes, ergostanes, cholestanes, and lanostanes represent sterol derivatives of the Calendula genus that are most abundant in C. officinalis [ 51 , 58 , 59 ]. Cycloartanes, fridelanes, lupanes, and ursanes are non-glycosidic compounds that exist in the form of alcohols, aldehydes, and ketones. Selected lupanes (lupane-3β,16β,20-triol, calenduladiol) and ursanes (α-amyrin, faradiol, arnidiol, arnitriol) are esterified by lauric, myristic, and palmitic acids [ 53 , 56 , 60 ]. In the oleanane group, oleanolic acid ( 286 ) and derivatives ( 287 – 322 ) have shown the largest diversity. The structural features of oleanolic acid glycosides that distinguish Calendula from other Compositae species are the ability to form mono- and oligoglycosides with one and/or two points of attachment of carbohydrate fragments at the C-3 and C-28 positions. Two types of glycosides have been identified in Calendula plants, i.e., acidic and neutral. Acidic glycosides contain a glucuronic acid fragment at C-3, which can be linked to glucose and galactose at C-2′, galactose at C-3′, and/or esterified at C-6′ with methanol or butanol. Neutral glycosides are characterized by some differences; after the addition of glucose to C-3, a complication of the structure has been observed as a result of the introduction of additional glucose fragments at C-2′, galactose, glucose, di- and tri-glucosyl fragments at C-3′, and also glucose, galactose, and a di-galactosyl moiety at C-4′. At position C-28 of oleanolic acid, only glucose can exist. Glycosides of other triterpene acids (e.g., morolic acid ( 323 ), moronic acid ( 325 ), echinocystic acid ( 327 ), cochalic acid ( 332 ), machaerinic acid ( 335 ), and mesembryanthemoidigenic acid ( 338 )) are both neutral and/or acidic derivatives. In C. officinalis , two compounds related to rare 3,4- seco -terpene alcohols, which are derivatives of tirucallan (3,4- seco -cucurbitane or 3,4- seco -19(10→9) abeo -euphane), have been identified as helianol ( 341 ) and thirucalla-7,24-dienol ( 342 ) [ 3 ]. Previously, both compounds were found in tubular flowers of Helianthus annus L. [ 110 ]. The most distributed triterpene glycoside is glucoside D ( 295 ), which has been found in four species: C. arvensis , C. officinalis , C. stellata , and C. suffruticosa . Three species ( C. arvensis , C. officinalis , C. stellata ) contain glucoside C ( 300 ), calenduloside C ( 312 ), and calenduloside D ( 320 ), and seven glycosides ( 293 , 299 , 303 , 309 , 318 , 319 , 333 ) were identified in two species. Triterpenoids are quantitatively the main group of Calendula metabolites, which reaches up to 3–4% of the total level of fatty esters of faradiol, arnidiol, and calenduladiol [ 58 ], and up to 9% of triterpenoid glycosides [ 111 ]. Scientometric studies have shown a number of mismatches in the names of some triterpenoid glycosides; specifically, for individual compounds, several trivial names are used. For the first time, six glycosides of oleanolic acid (containing a glucuronic acid residue at the C-3 position of the aglycone) were isolated from the flowers of C. officinalis and characterized by Kasprzyk Z. and Wojciechowski Z. in 1967, giving them the names glucosides A ( 291 ), B ( 293 ), C ( 295 ), D ( 296 ), E ( 300 ), and F ( 303 ) [ 67 ]. Later, Wojciechowski Z. et al. (1971) established the existence of a second group of oleanolic acid glycosides in C. officinalis containing a glucose residue at the C-3 position of the aglycone, named glucosides I ( 308 ), II ( 310 ), III ( 311 ), IV ( 313 ), V ( 314 ), VI ( 315 ), VII ( 316 ), and VIII ( 321 ) [ 71 ]. The latter research group used other names for glycosides A–F, such as glucuronides A–F, which are still relevant [ 112 ]. Therefore, the question of the priority of names for compounds 291 , 293 , 295 , 296 , 300 , and 303 remains open; the use of both variants is legitimate. Of note, the variants of names for glucosides C ( 295 ), D ( 296 ), and F ( 303 ), such as calendulosides H, G, and E, respectively, proposed by Vecherko L.P. et al., who isolated these compounds from C. officinalis in 1975–1976 [ 69 , 74 , 76 , 79 , 80 , 81 ], can be considered as synonyms. Calenduloside F ( 299 ) was isolated and characterized by Vecherko L.P. et al. (1975) [ 76 ]; however, the final identification of this compound under the name glucoside D 2 was performed by Vidal-Oliver E. (1989) [ 70 ]. Later, compound 299 was also named glucuronide D 2 [ 9 ].
3.5. Carotenoids
Since the discovery of carotene, lycopene, and violaxanthin in pigmented marigold petals [ 27 ], approximately a hundred carotenoids ( 343 – 437 ) have been found and identified in C. officinalis . Only this species was studied for this group of compounds. Carotenoids have been found in free and esterified forms, including myristic, palmitic, and stearic acid mono- and di-esters [ 85 ]. The most diverse carotenoid aglycone is lutein, which forms 32 compounds ( 376 – 407 ), followed by violaxanthin ( 418 – 428 ), cryptoxanthin ( 362 – 369 ), and zeaxanthin ( 429 – 434 ). Owing to the wide variety of colors of calendula flowers (ranging from white to burgundy and maroon), different varieties have different levels of carotenoids, ranging from trace amounts to 200 mg per 100 g of dry flower petals [ 113 , 114 ].
3.6. Phenols
Six simple phenols (i.e., p -cymene ( 438 ), p -cymenene ( 439 ), carvacrol ( 440 ), thymol ( 441 ), p -anethole ( 442 ), and estragole ( 443 )) are the minor constituents of the essential oil of C. officinalis [ 30 , 35 , 36 ] and C. arvensis [ 16 ] ( Figure 5 ).
3.7. Benzoic Acid Derivatives
Seven simple benzoic acids were identified as minor components of methanolic and ethanolic extracts of C. officinalis flowers, including salicylic acid ( 444 ), o -anisic acid ( 445 ), p -hydroxybenzoic acid ( 446 ), protocatechuic acid ( 447 ), vanillic acid ( 448 ), gentisic acid ( 449 ), and syringic acid ( 450 ) [ 35 , 87 , 89 ] ( Figure 5 ). Later, six glucosides of p -hydroxybenzoic acid ( 451 , 452 ), protocatechuic acid ( 453 , 454 ), and vanillic acid ( 455 , 456 ) were identified in leaves and pollen of C. officinalis [ 90 , 91 ].
3.8. Hydroxycinnamates
Twenty two derivatives of cinnamic acid of Calendula genus (i.e., cinnamic acid ( 457 ), coumaric acids ( 458 , 459 ), caffeic acid ( 460 ), ferulic acid ( 461 ), isoferulic acid ( 462 ), mono- O -caffeoyl quinic acids ( 464 – 467 ), di- O -caffeoyl quinic acids ( 468 – 472 ), tri- O -caffeoyl quinic acids ( 473 , 474 ), 5- O -feruloylquinic acid ( 475 ), 1,5-di- O -feruloylquinic acid ( 476 ), 1,5-di- O -isoferuloylquinic acid ( 477 ), and 1- O -caffeoyl glucose ( 478 )) were identified in the herb, roots, and pollen of C. arvensis , C. officinalis , C. suffruticosa , and C. tripterocarpa [ 75 , 89 , 92 ] ( Figure 6 ). Hydroxycinnamates are typical metabolites of asteraceous plants [ 115 ]; therefore, it is not surprising that they have been identified in calendulas. The dominant hydroxycinnamates in the flowers (3- O -caffeoylquinic acid ( 465 ) and 3,5-di- O -caffeoyl quinic acid ( 471 )) amounted to 1–7 mg/g for 465 and 0.5–2 mg/g for 471 ; while in the leaves, the content of 465 can reach 9 mg/g [ 89 ].
3.9. Coumarins
A small group of α-pyrone compounds or coumarins (ten compounds ( 479 – 488 )) has been identified in small amounts in the flowers, leaves, and herb of C. officinalis [ 90 , 94 , 95 ] and C. tripterocarpa [ 92 ], including umbelliferone ( 479 ), esculetin ( 480 ) and glycosides ( 481 – 484 ), scopoletin ( 485 ), and glycosides ( 486 – 488 ) ( Figure 7 ). The carbohydrate moieties of glycosides contain a glycose in esculin ( 481 ), cichoriin ( 482 ), and scopolin ( 486 ), neohesperidose in neoisobaisseoside ( 483 ) and haploperoside D ( 487 ), and rutinose in haploperoside ( 484 ) and isobaisseoside ( 488 ).
3.10. Flavonoids and Anthocyanins
Since the discovery of isorhamnetin ( 505 ), isorhamnetin-3- O -glucoside ( 507 ), and narcissin ( 514 ) in C. officinalis flowers in 1962 [ 116 , 117 ], twenty-eight flavonoids of C. arvensis , C. officinalis , C. stellata , C. suffruticosa , and C. tripterocarpa were also identified; the glycosyl derivatives of kaempferol ( 489 – 491 ), quercetin ( 492 – 504 ), and isorhamnetin ( 505 – 516 ) are the predominant forms of flavonoids ( Figure 8 ). Carbohydrate fragments may exist as monosaccharides (incorporate one moiety of rhamnose, galactose, and glucose), disaccharides (including neohesperidose (2- O -ramnosylglucose), such as calendoflavobioside ( 500 ) and calendoflavoside ( 511 ) [ 97 ]; rungiose (3- O -ramnosylglucose) such as calendoside II ( 501 ) and IV ( 512 ) [ 91 ]; 4- O -ramnosylglucose, such as calendoside I ( 502 ) and III ( 513 ) [ 91 ]; rutinose (6- O -ramnosylglucose), such as nicotiflorin ( 490 ), kaempferol-7- O -rutinoside ( 491 ), rutin ( 503 ), and narcissin ( 514 ) [ 93 , 97 , 98 ]; and 2- O -ramnosylrhamnose, such as quercetin-3-O-(2″- O -ramnosyl)-rhamnoside ( 499 ) and calendoflaside ( 515 ) [ 97 ]), and trisaccharides (2,6-di- O -ramnosylglucose in manghaslin ( 504 ) and thyphaneoside ( 516 ) [ 98 , 100 ]). Monoglucosides of quercetin and isorhamnetin may sometimes be acylated by acetic acid giving mono- ( 495 , 496 , 508 , 509 ) or diacetates ( 497 , 510 ) [ 89 , 91 ]. The content of flavonoids in different parts varies from trace amounts in the roots and seeds to 2–4% in the tubular and ligular flowers; isorhamnetin derivatives are typically the major components [ 89 , 114 ]. Anthocyanins 517 – 524 , as components of red colored marigold ray florets, are glycosides of cyanidin, delphinidin, malvidin, paeonidin, pelargonidin, and petunidin with a total content of 0.6–1.2% [ 89 ].
3.11. Other Compounds
Highly lipophilic compounds found in essential oils and hexane fractions of C. arvensis , C. officinalis , and C. suffruticosa include alkanes ( 525 – 550 ), aliphatic alcohols ( 551 – 559 ), aliphatic aldehydes and ketones ( 560 – 565 ), fatty acids and esters ( 566 – 602 ), and chromanols ( 603 – 613 ) [ 33 , 34 , 36 , 39 ]. In methanolic and water extracts of Calendula species, various hydrophilic compounds have been identified, including organic acids ( 614 – 616 ), carbohydrates ( 617 – 630 ), and amino acids ( 631 – 646 ) [ 41 , 108 ]. In essential oils of C. officinalis , 3-cyclohexene-1-ol, 3-cyclohexene-1-ol 4-methyl ester, loliolide, 1,2,3,5,8,8α-hexahydronaphthalene 6,7-dimethyl ester, 4-methylacethophenone, and 1-methyl ethyl hexadecanoate were identified [ 30 , 31 , 35 , 109 ]; tricyclene, 1H-benzocyclohepten-9-ol, and 2-pentyl furane were identified in C. arvensis [ 16 , 33 , 41 ]; naphthalene was detected in C. suffruticosa [ 42 ].
3.12. Polysaccharides
The study of Calendula polysaccharides started in the mid-1980s [ 118 ] and refers only to C. officinalis flowers; none of the other species have been studied ( Table 3 ). A group of German researchers conducted a systematic study of plant polysaccharides and their immunostimulating properties [ 8 ]. After the 0.5 M NaOH extraction of C. officinalis flowers, three neutral polysaccharides were isolated and characterized as rhamnoarabino-3,6-galactan and two arabino-3,6-galactans [ 119 ]. Later, five water-soluble polymers with 24.1–57.2 mol% of uronic acids were identified and demonstrated a wide variation of arabinose (4.0–12.5 mol%) and galactose (14.1–40.8 mol%) levels [ 120 ]. Polysaccharide fractions were also isolated from the industrial C. officinalis flower wastes; a high uronic content was typical for them (58.3–64.0 mol%) as well as variation in the level of neutral monosaccharides [ 121 , 122 ]. The exact structure of the acidic polysaccharides of C. officinalis is still unknown.
4. Separation of Calendula Metabolites by GC and LC
The chemical characteristics and chromatographic properties of the Calendula metabolites determine which technique is used to achieve satisfactory separation of target compounds. Some differences exist between gas chromatography and liquid chromatography (LC) methods designed for analyzing sterols, triterpenes, carotenoids, fatty acids, and phenolic compounds that are found in Calendula plants ( Table 4 ).
4.1. Sterols
Both GC and LC techniques were designed to separate sterols with various structures. Various 30 m columns (e.g., ZB-1 [ 123 ], HP-5MS UI [ 124 ], DB 17 [ 3 ], and RTX ® -1 MS [ 56 ]) were used to analyze sterol alcohols and esters by GC with flame ionization detection (GC-FID) and mass spectrometric detection (GC-MS). Fatty acid esters of arnitriol, faradiol, arnidiol, and maniladiol demonstrated appropriate LC separation on 250 mm reversed-phase (RP) columns (e.g., LiChrosphere RP-8 [ 125 ] and RP-18e [ 126 ], Hypersil ODS [ 60 ], Nucleosil 100-5 C18 [ 58 ], and Superiorex ODS C18 [ 3 ]) using isocratic elution with methanol [ 3 , 60 , 125 ], a water–methanol mixture [ 58 ], as well as gradient elution with trifluoroacetic acid–methanol mixtures [ 126 , 127 , 128 ] and ultraviolet (UV) or diode array (DAD) detection at 210 nm. Shorter columns (e.g., Kinetex C18 (100 mm) and Kromasil 100Å (50 mm)) showed good separation of 10 sterol esters by LC with atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometric detection (LC-APCI-QTOF-MS) [ 56 ].
4.2. Triterpenes and Glycosides
Aglycones (oleanolic acids) and glycosides were analyzed using high-performance liquid chromatography with UV (HPLC-UV) and mass-spectrometric detection (HPLC-UV-MS) assays using 250 mm (KromaPhase C18 [ 129 ], Eurospher 100 C18 [ 130 ]) and 150 mm RP columns (Waters Sunfire RP C 18 [ 111 ], C18 Luna [ 131 ]) with isocratic [ 129 ] or gradient elution in mixtures of acetic acid and acetonitrile [ 111 , 130 , 131 ]. Detection at 205–215 nm and MS detection in negative ionization mode allowed the analysis of two to six components [ 111 , 129 , 130 , 131 ].
4.3. Carotenoids
The chromatographic separation of Calendula carotenoids was realized using HPLC with diode-array detection (DAD) and HPLC-UV-MS techniques. To qualitatively and quantitatively analyze carotenes, lutein, lycopene, and other pigments, the RP sorbents are traditionally used in 250 mm and in 300 mm columns (e.g., C30 YMC [ 85 ], Nucleosil ODS C18 [ 86 ], YMC [ 132 ], Bondclone C18 [ 133 ], Nucleodur C18 [ 134 ], and Inertsil ODS-3 C18 [ 135 ]). Isocratic elution (with methanol–acetonitrile–methylene chloride–cyclohexene [ 133 ], acetone–water [ 134 ], methanol–tetrahydrofuran–water [ 135 ], and acetonitrile–methanol [ 136 ] mixtures) and gradient elution (with acetonitrile–water–ethyl acetate [ 86 ] and methanol–methyl tert -butyl ester–water [ 85 , 132 ] mixtures) were successfully performed. The strong absorption of carotenoids in the visible spectral region allowed their detection at 450–474 nm wavelengths [ 132 , 133 , 134 , 135 , 136 ] as well as by MS detection using atmospheric pressure chemical ionization (APCI) [ 85 ].
4.4. Fatty Acids
The fatty acid composition of C. officinalis seeds was extensively studied by GC assays on BPx-70 (60 m) [ 105 ], DB-23 (30 m) [ 137 ], HP-88 (100 m) [ 138 ], and Supelco SP-2560 (100 m) [ 139 ] columns and resulted in the quantification of 7–17 compounds with electron impact [ 105 , 137 , 138 ] and chemical ionization [ 139 ] MS detection.
4.5. Phenolic Compounds
Evaluation of phenolic compounds in Calendula plants is an important task, as indicated by the known HPLC protocols found in the scientific literature. To separate target compounds, only RP C18 columns with varying lengths were used, such as 75 mm ProntoSIL-120-5-C18 [ 90 , 114 , 144 ]; 100 mm Phenomenex C18 [ 142 ], Zorbax SB-C18 [ 143 ], and LiChrosorb RP18 [ 100 , 153 ]; 150 mm Luna C18 [ 131 ], SiliaChrom C-18 [ 140 ], Eclipse XDB-C18 [ 146 ], Spherisorb S3 ODS-2 C18 [ 149 ], Zorbax Eclipse Plus C18 [ 151 ], and Aquity UPLC HSS T3 [ 154 ]; 220 mm Aquapore RP-300 [ 152 ]; 250 mm Shim-pack C-18 [ 96 ], Hypersil C18 [ 141 , 147 ], X-Bridge C18 [ 145 ], and Phenomenex C18 [ 148 ]; 300 mm Bondclone C18 [ 133 ]; and 1000 mm Hypersil Gold [ 75 ]. The presence of various eluents requires the frequent use of formic acid [ 75 , 96 , 142 , 150 , 151 ], acetic acid [ 133 , 141 , 148 ], phosphoric acid [ 140 , 146 ] as the polar eluent and methanol [ 133 , 140 ] and acetonitrile [ 96 , 114 , 141 , 142 ] as the non-polar eluent. The addition of lithium perchlorate [ 90 , 114 , 144 ] and tetrahydrofuran [ 152 ] resulted in better resolution and improved peak shapes. Detection in the region at 254–280 nm and/or 330–370 nm corresponds to the maximum absorption of most phenolic compounds. The optimized LC conditions resulted in the separation of basic flavonoids and hydroxycinnamates of Calendula .
5. Concluding Remarks and Future Perspectives of Calendula Metabolites Research
Based on the results of previous studies, for the genus Calendula , a situation has been observed that is typical for industrial plant species that are widely used in human life. For such species, knowledge is skewed in favor of a single plant that is a commercial product, such as C. officinalis , which is the only species from the genus that is widely used. An incomparably smaller amount of information is available for C. arvensis , C. stellata , C. suffruticosa , and C. tripterocarpum , and seven other species ( C. eckerleinii , C. karakalensis , C. lanzae , C. maroccana , C. meuselii , C. pachysperma , C. palaestina ) are still unstudied. Of note, C. officinalis is an example of the use of only one part of the plant (flowers) to the detriment of the rest of the biomass (leaves, stems, roots), which has been understudied and is typically wasted. Table 5 presents a synopsis of known knowledge and clearly demonstrates the current situation regarding the Calendula genus. The actual situation in the field of studying Calendula chemodiversity indicates that essential oils of this genus are most often subjected to research. This occurs owing to the greater availability of instruments for this type of analysis, which is usually performed using the GC-MS technique, as well as the simplicity of sample preparation, which requires hydrodistillation (as the most common method of isolation). The same applies to the analysis of lipophilic extracts (hexane, dichloroethane, chloroform), which contain sterols, alkanes, aliphatic alcohols, aldehydes, ketones, and fatty acids. That is why there is an abundance of information on non-polar compounds. Of note, the lipophilic components of Calendula are currently of no practical importance; thus, excessive attention to them is not justified, at least until further studies are performed. Sesquiterpene glycosides, unlike the sesquiterpene components of essential oils, have proven antiviral activity against a vesicular stomatitis virus (VSV) and rhinovirus (HRV type 1B) [ 47 ], antiprotozoal activity against Leishmania donovani [ 49 ], and anti-inflammatory activity [ 155 ]. However, the study of these valuable compounds is limited to only three species; in C. officinalis , only flowers have been studied; although, given the discovery of these compounds in the herb of C. arvensis , it would be worth paying attention to other parts of C. officinalis . Researchers have made considerable progress in the study of triterpene alcohols, esters, and glycosides of Calendula . However, these studies refer primarily to C. officinalis from which 91 compounds have been isolated out of 109 known compounds. Compared to other compounds, for triterpenoid esters and glycosides, more in-depth pharmacological studies have been performed. Pharmacological studies demonstrated the anti-ulcer effect of calenduloside B ( 319 ) [ 156 ], antimutagenic activity of glycosides 291 , 295 , 296 , 299 , 300 , 303 , 309 , 312 , 318 , 320 [ 157 ], the anti-inflammatory activity of faradiol ( 197 ), lupeol ( 189 ) [ 6 ], and other triterpene alcohols [ 3 ] and some esters [ 125 ], hypoglycemic and gastroprotective potential of glucoside A ( 303 ), B ( 296 ), C ( 300 ), D ( 295 ), and F ( 291 ) [ 9 ], as well as their antibacterial, antiparasitic [ 158 ], and other activities. Owing to the clear potential of using triterpenoids as biologically active agents, it is necessary to expand the search for new compounds and new sources within the Calendula genus. Phenolic compounds of the Calendula genus have been extensively studied; however, most of the scientific information related to C. officinalis does not allow global conclusions about the features of the phenolic distribution within the genus. The question of domination of only two flavonol aglycones (quercetin and isorhamnetin) in Calendula plants remains interesting and unexplored. The studies of carotenoids, anthocyanins, and polysaccharides are limited to a single object, C. officinalis flowers, and these studies require more attention because of the availability and wide spectrum of bioactivity of these phytochemicals. Moreover, a detailed study of the fine stricture of polysaccharides of C. officinalis flowers is needed owing to the lack of information. Because C. officinalis is an industrial plant, it is necessary to expand research on non-floral parts of the plant, such as leaves, stems, roots, and seeds. The volume of production of these parts of the plant must be gigantic, but there are currently no examples of their rational practical application. In terms of marigold pharmaceutic production, the waste from the industrial processing of C. officinalis flowers is not used as a resource for obtaining valuable products. Moreover, there are few examples of recycling waste from the pharmaceutical processing of plants. Currently, this wasteful approach can be regarded as irrational and requires more attention and reasonable proposals for processing plant waste. In general, after almost a century of studying the genus Calendula , despite its widespread use, it is still the subject of numerous studies. Scientists are trying to expand the horizons of knowledge about its metabolites, application, and analysis because there are still many areas that need to be clarified. Taking into account the identified trends in the study of Calendula , we will still require scientific progress in the field of genus chemistry for a long period of time.
Footnotes
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Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27238626/s1 , Table S1: Distribution of Calendula publications between research areas; Table S2: Top 10 cited articles aimed to Calendula research.
Author Contributions
Conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing—original draft preparation, writing—review and editing, visualization, funding acquisition, D.N.O. and N.I.K.; supervision, project administration, D.N.O. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Not applicable.
Conflicts of Interest
The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.
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