Componential Profile and Amylase Inhibiting Activity of Phenolic Compounds from Calendula officinalis L. Leaves

An ethanolic extract and its ethyl acetate-soluble fraction from leaves of Calendula officinalis L. (Asteraceae) were found to show an inhibitory effect on amylase. From the crude extract fractions, one new phenolic acid glucoside, 6′-O-vanilloyl-β-D-glucopyranose, was isolated, together with twenty-four known compounds including five phenolic acid glucosides, five phenylpropanoids, five coumarins, and nine flavonoids. Their structures were elucidated based on chemical and spectral data. The main components, isoquercitrin, isorhamnetin-3-O-β-D-glucopyranoside, 3,5-di-O-caffeoylquinic acid, and quercetin-3-O-(6′′-acetyl)-β-D-glucopyranoside, exhibited potent inhibitory effects on amylase.


Introduction
The Compositae annual herbaceous plant, Calendula officinalis L., commonly called marigold, or pot marigold is widely cultivated as an ornamental, culinary and valuable medicinal herb due to its various pharmacological properties: antiinflammatory, antioxidant, antibacterial, antifungal, and so forth [1]. A number of chemical investigations have revealed the presence of several classes of compounds, with the main ones being terpenes, flavonoids, carotenoids, lipids, and carbohydrates.
Despite the wide use of C. officinalis flowers, leaves of this species have not currently found a practical application. The productivity of the vegetative foliage is much greater than that of the flowers, making it possible to consider the foliage as a new kind of useful plant material. The presence of various classes of compounds in C. officinalis leaves was determined as a result of chemical investigations. Isorhamnetin, isorhamnetin-3-O--d-glucopyranoside, and narcissin [2] belong to the phenolic compounds. Previously it has been shown that phenolic compounds are responsible for the presence of antioxidant and anticholinesterase activity in extracts of C. officinalis [3]. Carotenoids represent 12 compounds, dominated by lutein, -carotene, and violaxanthin; the total carotenoid content in the leaves of C. officinalis growing in Bulgaria reaches 0.85 mg/g [4]. The most investigated groups of compounds found in the leaves of C. officinalis are triterpene glycosides and sterols. In young leaves the presence of cholestanol, campestanol, stigmastanol, and clerosterol derivatives in free, esterified, and glycosylated forms was revealed [5]. Mono-, di-, tri-, and tetraglucosides of olenolic acid isolated from C. officinalis leaves growing in Poland demonstrated antibacterial and antiparasitic activity [6]. The essential oil of C. officinalis leaves contains a set of compounds, dominated by sesquiterpenes ( -muurolol, -cadinene) and monoterpenes ( -thujene) [7]. A number of neutral components, phospho-, and glycolipids have also been identified in the lipid complex [8].

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The Scientific World Journal Early, Yoshikawa et al. revealed a high hypoglycemic activity of the extracts and individual compounds from C. officinalis flowers that can be considered marigold as a forthcoming antidiabetic remedy [10]. In the course of our studies on the bioactivity of C. officinalis we found that an ethanolic extract from the leaves of this plant species showed inhibitory effect on the amylase. It is known that inhibitors of amylase, a carbohydrate hydrolyzing enzyme in the small intestine, are relevant to type II diabetes [11].
In this study, we present the results of phytochemical investigation of C. officinalis leaves from nine doubleflowered varieties growing in the Russian Federation. As a result, twenty-five compounds were isolated including a new glycoside, 6 -O-vanilloyl--d-glucopyranose. In addition, we describe the inhibitory effects of the isolated compounds on amylase.

Plant Material.
Plants of Calendula officinalis L. in nine double-flowered varieties ("Egypt Sun, " "Flame Dance, " "Geisha, " "Green Heart Orange, " "Indian Prince, " "Radio, " "Red Black Centered, " "Russian Size, " "Touch of Red") were grown from authenticated seeds obtained from Tsitsin's Main Botanical Garden of the Russian Academy of Science (Moscow, Russian Federation) by cultivation in the fields of the Botanical Garden of the Institute of General and Experimental Biology (IGEB, Ulan-Ude, Russian Federation). The leaves were collected in the middle of August, 2012, and then dried in vacuo at 40 ∘ C (12 h) and stored at 4 ∘ C in the IGEB Plant Repository.

Extraction and Isolation.
Air-dried, ground leaves of C. officinalis (1.65 kg) were extracted three times with 60% EtOH at 80 ∘ C and the extracts were concentrated under reduced pressure to yield 462.7 g of crude extract. The crude extract was resuspended in water (1 : 6, v/v) and successively partitioned with hexane, CHCl 3 , EtOAc, and n-BuOH.

Phenolic Compounds Content in Russian Varieties of C. officinalis Leaves.
Preliminary chemical research on the composition of C. officinalis leaves was carried out for nine varieties cultivated widely in the territory of Russia. These varieties are characterized by high productivity and simplicity of cultivation. As a result, it was found that flavonoid content in the leaves examined ranged from 8.49 ("Indian Prince") to 13.08 mg g −1 ("Radio"), while the total content of phenolic compounds varied from 29.21 ("Russian Size") to 50.24 mg g −1 ("Radio") ( Figure 1). The maximal content of phenolic compounds was observed for C. officinalis leaves of the "Radio" variety, which were further subjected to detailed chemical study.

Extraction and Isolation of Phenolic Compounds from C.
officinalis Leaves of "Radio" Variety. A 60% ethanolic extract of C. officinalis leaves of "Radio" variety was partitioned with CHCl 3 , EtOAc, and n-BuOH to yield three fractions. The crude extract was found to exhibit inhibitory activity on an amylase with an inhibitory value (IC 50 ) of 38.02 ± 1.29 g mL −1 (Table 1). In the same bioassay, the ethylacetatesoluble fraction still showed potent inhibitory activity with a lower value, 24.52 ± 0.88 g mL −1 .
Its 13 C-NMR spectrum exhibited a carbonyl group at 166.7 and a methoxyl group at 56.3. In its HMBC spectrum, the carbonyl group ( 166.7) showed a correlation with the aromatic proton of H-6 at 7.63 and the aromatic carbon at 147.5 (C-3) correlated with methoxyl protons at 3.97 (Figure 3).
Alkaline hydrolysis of 6 with 1 M KOH gave vanillic acid, which was identified by co-HPLC with an authentic sample and 13 C-NMR. These data suggest the presence of a vanilloyl moiety in the structure of 6. The presence of d-glucose was confirmed after acidic hydrolysis followed by HPLC analysis of the hydrolysate. In addition, the remaining 6 carbon signals in its 13 C-NMR spectrum are in good agreement with  those for -glucose. The linkage of the vanilloyl moiety and glucose was solved by an analysis of the HMBC spectrum. The carbonyl group ( 166.7) showed correlation with the proton at 4.05, suggesting that the vanilloyl moiety is connected to C-6 of the glucose moiety. Moreover, the results of the 13 C-NMR analysis showed a downfield shift in the resonance of the glucose C-6 , as well as an upfield shift of the C-5 resonance comparing with those of free glucose. Assignment of the protons and carbons signals was achieved by a combination of the HMBC and 1 H-1 H COSY spectral data. Since the anomeric hydroxyl group of the glucose moiety of 6 is unacylated, it may be obtained as anomers (and -forms) mixture. The 13 C-NMR data obtained showed a presence of a week signal at 92.1 that corresponded toform of 6. However, the ratio of the peak areas for C-1 atoms of -( 92.1) and -forms ( 98.3) is about 1 : 92. Thus, we can conclude that the predominant component of mixture is -forms (total content of -form is no more than 1.1%). These results enabled compound 6 to be identified as 6 -Ovanilloyl--d-glucopyranose.

HPLC-UV Analysis of the Main Phenolic Compounds in C. officinalis Leaves.
A quantitative analysis of the main phenolic compounds found in C. officinalis leaves was performed using microcolumn HPLC-UV method which allowed separating 15 components (Figure 4). This method was developed previously for analysis of C. officinalis flowers [9].

Amylase
Inhibiting Activity of C. officinalis Phenolic Compounds. All substances isolated were tested for their amylase inhibiting activity by the microplate method. Compounds 7, 13, 17, and 21 showed significantly higher inhibitory activity at concentration ranging 1.02-2.64 g mL −1 comparing with the reference compound acarbose (9.54 g mL −1 ) ( Table 4). It should be noted that concentrations of the mentioned compounds in C. officinalis leaves are insignificant; therefore, they cannot be considered as an active substances. Comparative analysis of the inhibitory activities of the dominant compounds showed that the actual antiamylase components in the crude extracts were 18 (isoquercitrin; 15.45 g mL −1 ), 22 (isorhamnetin-3-O--dglucopyranoside; 23.41 g mL −1 ), 9 (3,5-di-O-caffeoylquinic acid; 25.63 g mL −1 ), and 19 (quercetin-3-O-(6 -acetyl)--dglucopyranoside; 32.70 g mL −1 ). It is known that the phenolic phytosubstances are the perspective natural compounds which exhibited antidiabetic activity [33]. Flavonoids and phenylpropanoids demonstrate a high inhibitory activity on amylase and glucosidase, the key enzymes of carbohydrates digestion process [34]. Some caffeoylquinic acids (chlorogenic acid, isochlorogenic acid) and derivatives of quercetin and isorhamnetin, such as rutin (quercetin-3-O-rutinoside) and narcissin (isorhamnetin-3-O-rutinoside), have been previously reported as inhibitors of glucosidase [35]. The results of our experiments seemed to agree with this finding, probably due to the presence of large amounts of phenolic compounds in C. officinalis leaves.
The present investigation is the first extended study of the phenolic composition from C. officinalis leaves. As a result, twenty-five compounds were isolated, of which sixteen had not previously been reported in this plant source. The presence of undiscovered benzoic acid glucosides early in the Calendula genus, one of which was novel (6 -O-vanilloyl-d-glucopyranose), as well as the amylase inhibiting activity of the crude extract and individual compounds, illustrates the need for in-depth research even on very familiar species such as C. officinalis.