Fatty acid composition of the seed oil revealed the presence of lauric (3.90 %), myristic (3.58 %), palmitic (14.96 %), stearic (10.13 %), palmitoleic (4.55 %), oleic (16.26 %), linoleic (39.45 %) and linolenic (7.15 %) acids (Saleem et al. 1986). In the seed oil, conjugated acid was present to the extent of 4.5 % whereas the percentage of non-conjugated acid (linolenic acid) was only 2.65 %. The residual meal after the extraction of oil was also studied for its proteins (18 %) and amino acids composition.

The amounts (%) of lipids in C. officinalis seeds were: neutral lipids, 15.7 %; glycolipids (GLs), 0.9 %; and phospholipids (PhLs), 0.6 % (Ul’chenko et al. 1998). The lipid yield of extracts from the flowers (EF) was 17.1 % and from the leaves (EL) 9.3 %. The following classes of lipids were found (% by weight) hydrocarbons, 0.9 %; esters of sterols and triterpenols with fatty acids, 0.5 %; triacylglycerols (TAGs), 20.0 % comprising TAGs-I, 59.0; TAGs, 2–11.8 %; TAGs, 3–4.3 %; free fatty acids, trace; hydroxy-TAGs, 0.8 %; free sterols and free triterpenols, 0.4 %; diacylglycerols and monoacylglycerols, 1.2 %; and unidentified components, 1.1 %. The phospholipid complex of Marigold seeds consisted of eight classes, which may be arranged in order of content by weight as follows: PCs (phosphatidylcholines) > PIs (phosphatidylinositols) > N-acyl-PEs (N-acyl phosphatidylethanolamines) > N-acyl-lyso-PEs (N-acyl-lyso-phosphatidylethanolamines) > lyso-PIs (lyso-phosphatidylinositols) > PSs (phosphatidylserines) > lyso-PCs (lyso-phosphatidylcholines) > PEs (phosphatidylethanolamines). The glycolipids of the seeds were represented by four components forming the following sequence by mass content: SGs (sterylglycosides) > ESGs (ester sterylglycosides) > MGDGs (monogalactosyldiacylglycerols) > DGDGs (digalactosyldiacylglycerols).

The lipid content of seed of 11 genotypes varied between 13.6 and 21.7 g oil/100 g seeds (Dulf et al. 2013). PUFA contents varied between 60.4 and 66.4 %, while saturates comprising mainly palmitic and long chain saturated fatty acids were found in higher amounts in sterol esters (49.3–55.7 % of total fatty acids). Calendic acid [18:3 (8 t, 10 t, 12c) (n−6)] with contents of 51.47–57.63 % of total fatty acids was the predominant polyunsaturated fatty acid (PUFA) followed by linoleic acid [18:2 (n−6)] (28.50–31.86 %), oleic acid [18:1 (n−9)] (4.44–6.25 %) and palmitic acid (16:0) (3.86–4.55 %). Small and very small (or trace) amounts (<2 %) of stearic (18:0), β-calendic [18:3 (8 t, 10 t, 12 t) (n−6)], elaidic [18:1 (9 t) (n−9)], arachidic (20:0), behenic (22:0), gondoic [20:1 (n−9)], α-linolenic [18:3 (n−3)], linoelaidic [18:2 (9 t,12 t) (n−6)], cis-7-hexadecenoic [16:1 (n−9)], palmitoleic [16:1 (n−7)], lauric (12:0), myristic (14:0), pentadecanoic (15:0) and margaric (17:0) acids were also present. Cromack and Smith (1998) reported the seed oil content of around 20 %, of which up to 60 % was calendic acid, a useful industrial feedstock. Chisholm and Hopkins (1967) reported that Calendula could accumulate more than 40 % of calendic acid. Ozgul-Yucel found that Turkish calendula seed oil was characterized by high concentration of linoleic acid (43.5 %) and low content of CLNAs (calendic acid (18.3 %) + β-calendic (11.2 %). Angelini et al. (1997) reported 16–46 % levels of calendic acid in the Italian Pot Marigold seed oils.

Crombie and Holloway (1985) found that linoleic and oleic acids were precursors in the biosynthesis of calendic acid in Marigold (C. officinalis) seeds but not linolenic acid. (9S)-Hydroxyoctadeca-(10E,12Z)-dienoic acid (α-dimorphecolic acid) was isolated and converted into (R/S)-hydroxy- and -hydroperoxy-[9-3H]octadeca-(10E,l2Z)-dienoic acids but neither labelled specimen was converted into calendic acid by Marigold seed homogenate. Also α-dimorphecolic acid, a minor component of Marigold seed oil, was found to be a terminus rather than an intermediate for calendic acid.

In the flowers, stem and leaves of C. officinalis, 15 amino acids were detected in the free state: alanine, arginine, aspartic acid, asparagine, valine, histidine, glutamic acid, leucine, lysine, proline, serine, tyrosine, threonine, methionine and phenylalanine (Absavoa et al. 1994). Among them, six predominated: arginine, proline, glutamic acid, phenylalanine, lysine and leucine. The leaves contained about 5 % of amino acids, the stems 3.5 % and the flowers 4.5 %. Szakiel et al. (2005) found that Marigold (C. officinalis) synthesized significant amounts of oleanane saponins, found not only in flowers but also in all organs of plant. These glycosides comprised two series of structurally related compounds, that is, derivatives of 3-O-monoglucoside of oleanolic acid (hence named ‘glucosides’) and derivatives of 3-O-monoglucuronide (‘glucuronides’), depending on the first sugar moiety linked to the C-3 hydroxyl group of oleanolic acid, which is either glucose or glucuronic acid. The occurrence of both series of oleanolic acid glycosides occurring in C. officinalis was earlier reported by Kasprzyk and Wojciechowski (1967) and Wojciechowski et al. (1971). ‘Glucuronides’, known as the series I, were designated with letters (F, D, D2, C, B, A) and ‘glucosides’, forming the series II—with Roman numerals from I to VIII. ‘Glucuronides’ were found in relatively large amounts (up to 2 % of the dry mass) in flowers and in considerably lower quantity in green organs of the plant. Ruszkowski et al. (2003) found ‘glucuronides’ in roots of young Marigold plants, while ‘glucosides’ accumulated mainly in roots of grown and senescing plants and also found in green organs of the plant. However, only glycosides I, II, III, VI and VII were found in Marigold shoots. Oleanolic acid and its 3-O-glucuronide derivatives and 3-O-glucoside derivatives were found in vacuoles prepared from protoplasts and cell walls obtained from leaf cells of Calendula officinalis (Szakiel and Kasprzyk 1989). In both cell compartments 37 % of total cellular oleanolic acid accumulated, 0.6 % occurring as free oleanolic acid (only in vacuoles). Glucuronides accounted for 31.1 % (20.7 % in vacuoles and 10.4 % in cell walls) and glucosides for 5.3 % (2.6 % in vacuoles and 2.7 % in cell walls). Szakiel et al. (1995) found that [3-3H]oleanolic acid glycosides formed in the cytosol of C. officinalis leaf cells were transported to the extracellular space in the form of pentaglucoside VI (44 %), whereas glucuronides derived from [3-3H]oleanolic acid 3-O-monoglucuronide (29 %) as well as a part of glucosides (24 %) were transported into the cell walls.

Studies confirmed that plastoquinone occurred only in the chloroplasts, ubiquinone only in the mitochondria and α-tocopherol in both these subfractions of C. officinalis leaves (Janiszowska et al. 1976). In Calendula officinalis leaves the cyclization of squalene to β-amyrin and its further oxidation to oleanolic acid as well as the biosynthesis of all derivatives of oleanolic acid 3-glucoside and some derivatives of oleanolic acid 3-glucuronoside were found to occur in the microsomal fraction (Janiszowska and Kasprzyk 1977). The final metabolites of oleanolic acid 3-glucoside series, that is, pentaglycosides, were translocated from this fraction, one to the cell wall and plasmalemma fraction and the other to the cytosol. The derivatives of oleanolic acid 3-glucoronoside were synthesized partially in other fractions and accumulated in the different membranous structures of the cell.

Polyphenolic compounds (g/kg dry matter) in the aerial Marigold plant parts were determined as follows: chlorogenic acid 0.55 g, 3,5-DCQA (dicaffeoylquinic acid) 0.78, total caffeoyl derivatives 1.33 g, total dihydroxycinnamic acid derivatives 7.54 g, total flavonoids 5.12 g, total dihydroxycinnamic acid derivatives + flavonoids 12.66 g and total polyphenolic compounds 24.97 g (Fraisse et al. 2011).

Fatty acids C12–C22 were found to be components of acylated steryl glucosides in Calendula officinalis (Zdzislaw et al. 1975). As a source of acyl groups for the synthesis of steryl acylglucosides, various phospholipids obtained from the same plant were utilized in the following sequence: phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylcholine. It does not utilize triacylglycerols and monogalactosyldiacylglycerols.

In 3-day and 14-day-old seedlings and leaves of Calendula officinalis, the following sterols were identified: cholestanol, campestanol, stigmastanol, cholest-7-en-3-β-ol, 24-methylcholest-7-en-3β-ol, stigmast-7-en-3β-ol, cholesterol, campesterol, sitosterol, 24-methylcholesta-5,22-dien-3β-ol, 24-methylenecholesterol, stigmasterol and clerosterol (Adler and Kasprzyk 1975). Sitosterol was predominant in young and stigmasterol in old tissues. Young tissues contained relatively more campesterol, but in old tissues a C₂₈Δ^5,22 diene was present suggesting transformation of campesterol to its Δ^5,22 analog, similar to that of sitosterol to stigmasterol. All the identified sterols were present as free compounds and also in the steryl esters, glucosides, acylated glucosides and water-soluble complexes.

Petroleum ether extract of Calendula officinalis leaf showed the presence of fatty acids, and chloroform extracts showed the presence of triterpenes and sterols (Chakraborthy 2010). Flavonoids, carbohydrates, amino acids, and saponins were present in methanol extract, and saponins, phenolic substances, and tannins were present in the water extract of Calendula officinalis.

Thirty and twenty-one compounds were identified in the fresh leaf and dry leaf oils of C. officinalis and the yield was 0.06 and 0.03 %, respectively (Okoh et al. 2008). Sesquiterpenoids dominated the fresh leaves (59.5 %) while the monoterpenes dominated the oil in the dry leaves (70.3 %). The fresh leaf oil was dominated by T-muurolol (40.9 %), α-thujene (19.2 %) and δ-cadinene (11.4 %), while the dry leaves oil was found to be rich in 1,8-cineole (29.4 %), γ-terpinene (11.6 %), δ-cadinene (9.0 %), β-pinene (6.9 %) and α-thujene (6.3 %).

The yield in Calendula officinalis leaf essential oil showed a maximum at the full flowering stage (0.97 %) and a minimum during the pre-flowering stage (0.13 %) (Okoh et al. 2007). The compositions also showed different patterns at different phases of the vegetative cycle. Sesquiterpenes (α-cadinene, α-cadinol, T-muurolol and epi-bicyclosesquiphellandrene) and monoterpenes (limonene, 1,8-cineole and trans-β-ocimene) showed the highest correlations with the age of the plant. Aiming the use of essential oil as a food ingredient, the most interesting stage is the post-flowering period, the essential oil at this time being rich in α-cadinene, α-cadinol, t-muurolol, limonene and 1,8-cineole, with p-cymene present at lower levels. The total yields of the essential oils at the different stages of the vegetative cycle increased with the age of the plant and ranged between 0.13 % (3rd week) and 0.97 % (12th week, flowering); α-cadinene is an important flavouring agent in baked foods, candy and chewing gum and also used as a fragrance in cosmetics and detergents. T-muurolol and α-cadinol are important antimicrobial agents. The essential oil at 12 week was dominated by geraniol (44.5 %), α-cadinol (24.20 %), δ-cadinene (23.8 %), T-muurolol (22.50 %), 1,8-cineole (22.10 %), cadina-1,4-diene (12.20 %), germacrene D (11.50 %), α-cadinene (10.7 %) and calarene (5.70 %). Other minor components included α-pinene (2.90 %), limonene (2.6 %), α-cubebene (1.7 %), α-humulene (1.7 %), β-pinene (1.4 %), nerolidol (1.3 %), sabinene (0.9 %), endobourbonene (1.0 %), β-caryophyllene (0.9 %), α-ylangene (0.8 %), palustron (0.7 %), epi-bicyclosesquiphellandrene (0.5 %), β-selinene (0.3 %), alloaromadendrene (0.2 %), α-bourbonene (0.2 %), muurolene (0.10 %), terpene-4-ol (0.10 %), p-cymene (0.10 %), carvacrol (0.10 %), α-thujene (0.10 %) and α-gurjunene (0.10 %). These compounds occurred in trace amounts: δ-3-carene, nonanal, 3-cyclohexene-1-ol, α-phellandrene, bornyl acetate, sabinyl acetate, α-copaene, β-cubebene, aromadendrene and oplopenone.

A new series of glycosides of oleanolic acid was found in the roots of old Calendula officinalis plants (Wojciechowski et al. 1971). These compounds were derivatives of 3-glucoside of oleanolic acid and were different, from those previously isolated from the flowers, derivatives of 3-glucuronoside of oleanolic acid. The sugar components of eight representatives of the new series are glucose and galactose in following ratios: in glucoside I, 1:0; in II, 1:1; in III, 1:2; in IV, 2:1; in V, 3:1; in VI, 3:2; in VII, 4:1; and in VIII, 4:1. The sugars in I–VII were attached only in position 3 of oleanolic acid, but in VIII one glucose molecule was joined to 28-carboxyl of oleanolic acid. Glucoside I was identified as 3-monoglucoside and II as 3-(4′-galactosyl)-glucoside of oleanolic acid. Besides these compounds, 6′-methyl ester of 3-glucuronoside of oleanolic acid was also found in the roots.

A monoside 3-O-β-d-glucopyranoside of oleanic acid and a bioside (calenduloside A) with the structure 3-galactosylglucosyloleanolic acid (Vecherko et al. 1969, 1971b) and calenduloside B with the structure O-β-d-galactopyranosyl-(1 → 4)-O-β-d-glucopyranosyl-(1 → 3)-oleanoloyl-(28 → 1)-α-d-glucopyranoside (Vecherko et al. 1971a); oleanolic acid 3-{[galactopyranosido-(1 → 3)] [glucopyranosido–(1 → 2)]-β-d-glucopyranoside} (calenduloside C) and 28-acyl-β-d-glucopyranoside of calenduloside C (calendulososide D) (Vecherko et al. 1975); glucopyranosyl oleanolate 3-O-β-d-glucuronopyranoside (calenduloside F) (Vecherko et al. 1973), oleanolic acid 3-O-β-d-galactopyranosyl-(1 → 3)-β-d-glucuronoside (calenduloside G) and 28-acyl-β-d-glucopyranoside of calenduloside G (calenduloside H) (Vecherko et al. 1974) were isolated from the roots of C. officinalis. Calenduloside B, trioside of oleanolic acid, was isolated from the roots (Iatsyno et al. 1978).

Chemical studies have revealed the presence of various classes of compounds, the main being triterpenoids, flavonoids, coumarins, quinones, volatile oil, carotenoids and amino acids in C. officinalis plant parts (Muley et al. 2009; Khalid and da Silva 2012). The extract of this plant as well as pure compounds isolated from it had been demonstrated to possess multifold pharmacological activities: antioxidant, antiinflammatory, antiedematous, immunostimulant, anticancer, lymphocyte and wound healing, hepatoprotective, antibacterial and antifungal, anti-HIV, spasmolytic and spasmogenic, cytotoxic, genotoxic and antigenotoxic, inhibition of heart rate and antiviral, among others.

Superoxide radicals and hydroxyl radicals were observed in decreasing concentrations in the presence of increasing concentrations of C. officinalis butanolic fraction with IC₅₀ values of 1 and 0.5 mg/ml, respectively, suggesting a possible free radical scavenging effect (Cordova et al. 2002). Lipid peroxidation in liver microsomes induced by Fe²⁺/ascorbate was 100 % inhibited by 0.5 mg/ml of the fraction (IC₅₀ = 0.15 mg/ml). Its total reactive antioxidant potential (TRAP) (in μM Trolox equivalents) was 368.14 and its total antioxidant reactivity (TAR) was calculated to be 249.19 μM. The results suggested the butanolic fraction of C. officinalis to have significant free radical scavenging and antioxidant activity.

The methanol and water extracts of wild Marigold, Calendula arvensis (GWM) and cultivated Marigold, Calendula officinalis (CM), in a concentration range of 0.10–0.90 mg/ml, scavenged all types of investigated radicals in dependence on their applied concentrations (Ćetković et al. 2004). Generally, CM extracts possessed higher scavenging and antioxidant activity than GMW extracts, while methanol extracts exhibited lower activities than water extracts. Water extracts of CM had the best antioxidant properties; 0.75 mg/ml extracts completely eliminated hydroxyl radical, which was generated in the Fenton system. The same concentration of this extract scavenged 92 % DPPH and 95 % peroxyl radical during lipid peroxidation. Antioxidant properties were in correlation with the contents of total phenolic compounds (14.49–57.47 mg/g) and flavonoids (5.26–18.62 mg/g) in extracts. The electron spin resonance (ESR) spectroscopy data demonstrated that methanol and water extracts of CM possessed similar free radicals scavenging and antioxidative activity as synthetic antioxidants BHA. Total antioxidant capacity (%) (DPPH scavenging activity) of C. officinalis aerial plant parts was 1.52 % and contribution from the main caffeoyl derivatives was as follows: chlorogenic acid 3.15 %, 3,5-DCQA (dicaffeoylquinic acid) 6.90 % and total caffeoyl derivatives 10.05 % (Fraisse et al. 2011).

Calendula officinalis flower top extract was found to scavenge superoxide radicals generated by photoreduction of riboflavin and hydroxyl radicals generated by Fenton reaction and inhibited in-vitro lipid peroxidation (Preethi et al. 2006). Concentrations needed for 50 % inhibition (IC₅₀) were 500, 480 and 2,000 mg/ml, respectively. The extract scavenged ABTS radicals and DPPH radicals with IC₅₀ of 6.5 and 100 mg/ml, respectively. The extract also scavenged nitric oxide (IC₅₀ = 575 mg/ml). Oral administration of Calendula extract inhibited superoxide generation in macrophages in vivo by 12.6 and 38.7 % at doses of 100 and 250 mg/kg body weight. Oral administration of the extract to mice for 1 month significantly increased catalase activity and produced significant increase in glutathione levels in the blood and liver. Glutathione reductase was found to be increased, whereas glutathione peroxidase was found to be decreased after extract administration. The results indicated Calendula officinalis possessed significant antioxidant activity in-vitro and in-vivo.

The propylene glycol extract of Calendula officinalis exerted its anti-ROS (reactive oxygen species) and anti-RNS (reactive nitrogen species) activity in a concentration-dependent manner during polymorphonuclear leukocytes burst, with significant effects being observed at even very low concentrations: 0.20 μg/ml without l-arginine, 0.10 μg/ml when l-arginine was added to the test with phorbol 12-myristate 13-acetate and 0.05 μg/ml when it was added to the test with N-formyl-methionyl-leucyl-phenylalanine (Braga et al. 2009). Electron paramagnetic resonance (EPR) spectroscopy confirmed these findings, 0.20 μg/ml being the lowest concentration of C. officinalis extract that significantly reduced 2,2-diphenyl-1-picrylhydrazyl (DPPH). Calendula officinalis propylene glycol extracts were found to have protective effect against oxidative DNA damage and lipid peroxidation induced by high polyunsaturated fatty acid (PUFA) intake in young growing pigs (Frankic et al. 2009). It elicited a numerical trend towards the reduction of plasma malondialdehyde and urinary isoprostane (iPF2α-VI) excretion. Its effect was comparable with that of vitamin E. The extract from flower tops showed less antioxidant potential than the extract from petals. They concluded that the amount of C. officinalis extracts proposed for internal use by traditional medicine protected the organism against DNA damage induced by high PUFA intake.

Methanol enabled more efficient extraction of flavonoids from C. officinalis flowers than the other solvents isopranol and ethanol tested (Butnariu and Coradini 2012). From cv Petran and cv Plamen flowers, methanol extracted, respectively, 2.04 and 2.142 mg/100 g FW of carotene pigments (lycopene, lutein), 10.358 and 5.222 mg/100 g FW of total photosynthetic pigments (chlorophyll a, b), total flavonoid content 96.17 and 90.37 mg quercetin equivalent (QE)/100 ml and total phenolic content of 134 and 153.23 mg GAE/100 ml. The highest antioxidant activity correlated to the polyphenol content was obtained for extracts prepared using methanol. For the methanol extract of cv Petran and cv Plamen, the DPPH radical scavenging activity was2.64 and 2.97 mmol Trolox/g and the ferric reducing antioxidant power (FRAP) was 0.29 and 1.55 mmol Fe²⁺/g.

Studies by Ozkol et al. (2012) showed that administration of Calendula officinalis extract protected rats against subacute cigarette smoke-induced cell injury. Marigold extract decreased the elevated levels of malondialdehyde, reduced glutathione and protein carbonyl caused by cigarette smoke. Further, Marigold extract increased the diminished levels of glutathione peroxidase (GPx), superoxide dismutase activities and β-carotene and vitamins A and C caused by cigarette smoke.

Bernatoniene et al. (2011) confirmed dry Calendula officinalis extract to be an effective scavenger of H₂O₂ radicals in in-vitro studies with the mitochondria of rat cardiac muscles. Calendula extract incorporated into hydrophilic cream containing complex emulsifier provided significant antioxidant effect due to the content of carotenoids, polyphenols and flavonoids and good emulsion quality. Cream with the best properties (0.9 % of Calendula extract) contained 0.73 mg/100 g of total carotenoids expressed as β-carotene.

Laser Activated Calendula Extract (LACE) showed a potent in-vitro inhibition (70–100 %) of tumour cell proliferation when tested on a wide variety of human (leukaemias, melanomas, fibrosarcomas and cancers of the breast, prostate, cervix, lung, pancreas and colorectal) and murine tumour cell lines (Jiménez-Medina et al. 2006). Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and caspase-3-induced apoptosis. In contrast, the same extract showed an opposite effect when tested on human peripheral blood lymphocyte (PBL) and NKL (natural killer) cell line, in which in-vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE in nude mice inhibited in-vivo tumour growth of Ando-2 melanoma cells and prolonged the survival day of the mice. Two triterpene glycosides from the flowers, calenduloside F 6′-O–n-butyl ester (9) and calenduloside G 6′-O–n-methyl ester, exhibited potent cytotoxic effects against colon cancer, leukaemia and melanoma cells (Ukiya et al. 2006).

Simultaneous administration of C. officinalis flower extract to tumour-bearing male C57BL/6 mice reduced the lung B16F-10 melanoma tumour nodules by 74 % with 43.3 % increase in life span (Preethi et al. 2010). Elevated levels of hydroxyproline, uronic acid, hexosamine, serum sialic acid and γ-glutamyl transpeptidase in the metastatic controls were significantly lowered in the C. officinalis-treated animals. The extract also inhibited expression of MMP-2, MMP-9, prolyl hydroxylase and lysyl oxidase and activated TIMP-1 and TIMP-2 and downregulated proinflammatory cytokines. The results indicated antimetastatic effects of Calendula officinalis flowers through the inhibition of key enzymes were involved in processes of metastasis.

Studies demonstrated that chamomile and Marigold (Calendula officinalis) tea exerted selective dose-dependent cytotoxic action against target cancer cells; cytotoxicity of Marigold tea was higher than chamomile (Matić et al. 2013). However, the cytotoxic effect of chamomile tea was very weak to healthy peripheral blood mononuclear cells (PBMC), while the effect of Marigold tea on PBMC was more pronounced. Marigold tea exerted highly selective antitumor effect especially to melanoma Fem-x cells in comparison to the action to normal healthy PBMC. Chemical analyses showed that dominant phenolic compounds in examined infusions and decoctions were flavonoid glycosides and hydroxycinnamic acid derivatives. Ethanol extracts of C. officinalis and other Asteraceae species were found to have antileukemic properties and to induce J-45.01 human acute T leukaemia cell death via apoptosis. The correlation between antileukemic activity and total polyphenol content was determined (Wegiera et al. 2012).

Three major flavonoid fractions separated from C. officinalis flower methanol extract did not exhibit inhibitory effect on the parent and tamoxifen-resistant T47D human breast cancer (Ostad et al. 2004). Quercetin increased cell proliferation of the resistant T47D cells in the presence of tamoxifen but no effect was detected by using quercetin itself. Also it was found that isorhamnetin did not have any proliferative or antiproliferative activity on the both cell lines.

Studies on surgically induced skin wound in Wistar albino rats showed that application of % unguentum containing fractions C1 and C5, isolated from Calendula officinalis flowers of in combination with allantoin, significantly stimulated physiological skin regeneration and epithelialization (Klouchek-Popova et al. 1982).