Bioactive compounds from marigold processing waste: Extraction, isolation, and antidiabetic activity

Diabetes is a chronic metabolic disorder characterized by excessive blood sugar levels. According to the International Diabetes Federation, 537 million adults are living with diabetes today and is projected to rise to 643 million by 2030 and 783 million by 2045. Marigold or Tagetes erecta Linn. is a common plant used in diverse cultures in India. Different parts of this plant are used in folk medicine to treat colic, diarrhea, vomiting, fever, skin diseases, and hepatic disorders. During marigold cultivation, the leaves are the most common agricultural waste. In the present study, we aim to evaluate the in-vitro antidiabetic activity of hydroalcoholic extract of leaves of marigold and the isolation and characterization of active constituents. The hydroalcoholic extract was fractionated by column chromatography using different stationary phases and solvents ranging from highly non-polar to polar as mobile phase by gradient elution method. In-vitro alpha-glucosidase inhibition of the hydroalcoholic extract and isolated compounds was performed and compared with the standard drug acarbose. The isolated compounds were tentatively characterized as kaempferol di-O-rhamnoside (compound 1), kaempferol O-rhamnoside-O-pentoside (compound 2), and kaempferol O-rhamnoside (compound 3). The isolated compounds were more active than the hydroalcoholic extract. Kaempferol O-rhamnoside was found to possess the highest antidiabetic activity with an IC 50 value of 61 μg/ml.

Diabetes is a chronic metabolic disorder characterized by excessive blood sugar levels.According to the International Diabetes Federation, 537 million adults are living with diabetes today and is projected to rise to 643 million by 2030 and 783 million by 2045(Elyamani et al., 2021;;Sun et al., 2022).The α-glucosidase is an enzyme located in the brush border of the enterocytes of the jejunum, and its function is to cleave oligosaccharides and disaccharides into monosaccharides for better absorption (Akmal and Wadhwa, 2022).The inhibition of this enzyme results in the reduction in postprandial blood glucose levels (Bu et al., 2010).However, there is no systematic study reported on the fractionation and isolation of antidiabetic principles from the leaves of T. erecta.Hence, in the present study, it was proposed to evaluate leaves of T. erecta to isolate active constituents and compare the activity of the hydroalcoholic extract, and isolated compounds by in-vitro α-glucosidase enzyme inhibitory model.

Identification and extraction
Leaves of T. erecta L. were obtained from Mysuru, Karnataka, authenticated, and the voucher specimen of the same was deposited at National Ayurveda Dietetics Research Institute, Bangalore (Voucher no: RRCBI-MUS-138).Extraction was carried out by the reflux method.500 g of the dried, powdered leaves were extracted with hydro alcohol (1:8 drug to solvent ratio), the extract was concentrated in a rotary evaporator and dried using a vacuum tray drier to obtain powder and was stored in polythene bags until analysis.

Chromatographic separation and isolation
The hydroalcoholic extract was subjected to fractionation by silica gel (60-120 mesh) column chromatography (column 1), and elution was started with hexane, ethyl acetate (EA), and methanol.Eleven fractions were collected, based on the thin layer chromatography (TLC) pattern [n-Butanol: Glacial acetic acid: Water (16:4:20)], five fractions (5%, 25%, 50%, 75% methanol in EA, and 100% methanol) were combined, subjected to further column chromatography (column 2), and performed the same abovementioned procedure.From column 2, 12 fractions were collected and 5% methanol in EA, 5 subfractions were collected.First and second subfractions yielded crude crystals with impurities, and TLC showed two bands, so these fractions were combined and subjected to further silica gel (300-400 mesh) column chromatography (column 3).Elution has started with hexane, chloroform, EA, and methanol.Twenty-one fractions were collected, from 20% EA in chloroform, four subfractions were collected, first and second fractions got purified spots in TLC, so these fractions were combined (compound 3).
From column 1, 25% of methanol in EA, the major part, was utilized for column 2, green to white specs were observed in the sides of the test tubes, and acetone was added to obtain precipitate (compound 1).From column 2, two subfractions 5% and 7.5% of methanol in EA were separately concentrated, acetone was added to each fraction, and a precipitate was obtained and showed a similar band pattern in TLC.Thus, these compounds were combined to obtain compound 2.

Characterization
The isolated compounds were characterized using ultra violet (UV), TLC, Fourier transform infrared (FTIR), and mass spectrophotometer.The MALDI-Mass spectrophotometer data were collected utilizing a 25 kV accelerating voltage, a 90 ns time delay, and a 50 Hz pulsed nitrogen laser (λ = 337 nm) on an Ultraflex TOF/TOF spectrometer (Bruker Daltonics, Bremen, Germany, and Billerica, Massachusetts, USA).TLC of all three compounds showed a single spot (Fig. 14); therefore, all three compounds were subjected to the high-performance liquid chromatography (HPLC) analysis for assessing their purity.A new HPLC method was developed and standardized in the laboratory using a Shimadzu HPLC instrument (Scientific Instruments, Kyoto, Japan), with the following conditions: • Column: Shim-pack C18 (250 × 4.6 mm, 5 microns), • Mobile phase: Acetonitrile in phosphate buffer, pH:3.5, • Elution: Gradient elution, • Flow rate: 1.5 ml/minute, • Detector: Photodiode array detector, and • Injection volume: 20 µl.

Sample preparation
2 mg of each sample was dissolved in 5 ml of methanol with the help of sonication for 2 minutes.The mobile phase was prepared by mixing HPLC grade acetonitrile with phosphate buffer of pH 3.5 by adding 0.136 g of anhydrous potassium dihydrogen orthophosphate (KH 2 PO 4 ) in 900 ml of HPLC grade water (obtained from Millipore, Milli-Q Water purification system) and 0.5 ml of orthophosphoric acid and volume was made up to 1,000 ml with water, filtered through 0.45 µ membrane and degassed in a sonicator for 3 minutes (Table 1).

Compound 1 (From column 1)
From column 1, 25% of methanol in EA, the major part, was utilized for column 2, green to white specs were observed in the sides of the test tubes, acetone was added, and a precipitate was obtained; light green, crystalline powder; the yield was 94.5 mg; melting point or mp 201°C-205°C; HPLC three peaks were observed.The retention time of one major peak was 11.432 minutes with 77% area and two minor peaks were 11.297 and 11.668 minutes with 13.3% and 8% areas, respectively.UV-Vis (λ max , ethanol): 263, 344 nm; FTIR (KBr, cm −1 ): 3,352.39(-OH), 2,980.12(-CH aromatic), 2,926.11(-CH aliphatic), 1,602.90,1,492.95(C=C Compound 2 (From column 2) From column 2, two subfractions 5% and 7.5% of methanol in EA were separately concentrated, acetone was added to each fraction, and a precipitate was obtained, which showed a similar band pattern in TLC and similar peaks in HPLC.Thus, these compounds were combined.Light green crystalline powder; the yield was 240 mg; mp 240°C-245°C; HPLC three peaks were observed.One major peak was seen at 16.109 minutes and two insignificant minor peaks.UV-Vis (λ max , ethanol): 265, 347 nm; FTIR (KBr, cm −1 ): 3,419.

Alpha-glucosidase inhibitory assay
The inhibition of α-glucosidase activity was assessed using the spectrophotometric microplate reader.10 μl of α-glucosidase enzyme (1 U/ml) was pre-incubated with different concentrations of the test material (100, 150, 200, and 400 μg/ ml) at 37°C and allowed to react with PNPG (20 μl, 5 mM).The reaction was terminated by adding sodium carbonate solution (50 μl, 0.1 M), and p-nitrophenol released in the reaction mixture was monitored at 410 nm compared with the control, and acarbose was used as a standard.Acarbose is the most widely prescribed α-glucosidase inhibitor.The experiment was conducted in triplicates and percentage inhibition was calculated (Telagari and Hullatti, 2015).

Isolation and characterization
The hydroalcoholic extract was fractionated by column chromatography using different stationary phases and solvents ranging from highly non-polar to polar as mobile phase by gradient elution method.The isolated kaempferol di-O-rhamnoside (1) was a light green, crystalline powder with mp 201°C-205°C.In HPLC, three peaks were observed.The retention time of one major peak was 11.432 minutes (77% area) and two minor peaks was 11.297 and 11.668 minutes (13.3% and 8% areas, respectively) (Fig. 2).From UV-Vis, major peaks at 263 and 344 nm (Fig. 5) indicate the 1 to be flavonol derivatives (Negri et al., 2013).TLC (n-butanol: glacial acetic acid: water-16:4:20) showed a single black to greenish-brown spot with ferric chloride reagent indicating a pure, single compound and polyphenolic in nature (Forsyth, 1955) (Fig. 14).FTIR showed prominent peaks at frequencies, 3,352.39cm −1 for the -OH stretching.The -CH stretching of aromatic nature was interpreted by a peak at 2,980.12 and 2,926.11cm −1 indicating the presence of -CH aliphatic stretching, 1,602.90 and 1,492.95cm −1 representing the C=C aromatic stretching.The additional peaks at 1,656.91 cm −1 correspond to an ethylenic double bond and C-O-C stretching (Fig. 8).Negative electrospray ionization mass spectrometry (ESI-MS) spectra showed molecular ion peak at m/z 577.27 that generated two MS2 fragment ions at m/z 431 (M-146), loss of a rhamnose moiety; and at m/z 283 (M-146-146-2H)-, loss of two rhamnose moieties (Barros et al., 2011).The position of substitution of the sugar residues could not be concluded and the fragmentation pattern was similar to flavonoid di-O-glycosides (Fig. 11).The result indicates that 1 could be kaempferol di-Orhamnoside (1) (Fig. 1).The isolated kaempferol O-rhamnoside-O-pentoside (2) was light green crystalline powder with mp 240°C-245°C.HPLC, one major peak was at 16.109 minutes and two insignificant minor peaks (Fig. 3).UV-Vis major peaks at 265 and 347 nm indicate 2 to be flavonol derivatives (Negri et al., 2013) (Fig. 6).TLC showed similar features to 1 and confirms it as polyphenolic (Forsyth, 1955) (Fig. 14).FTIR showed prominent peaks at frequency 3,419.90cm −1 representing -OH stretching.The peak at 3,976.26 cm −1 accounted for -CH aromatic stretching and 2,924.18cm −1 for -CH aliphatic stretching.1,597.11cm −1 corresponds to C=C aromatic stretching, 1,658.91 and 1,346.36cm −1 indicated C=C and C-O-C groups, respectively (Fig. 9).The   et al., 2011) (Fig. 12), and relative abundances of the two fragment ions suggested that they were located at different positions on the aglycone, although it was also not possible to conclude about the precise position.The compound was tentatively assigned as kaempferol O-rhamnoside-O-pentoside (2) (Fig. 1).The isolated kaempferol O-rhamnoside (3) was a pale-yellow semi solid with mp 150°C-155°C.HPLC chromatogram showed a single peak with 97% purity with a retention time of 16.559 minutes (Fig. 4).UV showed two peaks at 264 and 363 nm indicating the presence of flavonol derivatives (Negri et al., 2013) (Fig. 7).TLC showed similar features to 1 and confirms it as polyphenolic (Forsyth, 1955) (Fig. 14).FTIR showed prominent peaks at 3,473.91 cm −1 for -OH stretching and 3,093.92cm −1 for -CH aromatic -CH aliphatic represents 2,929.97cm −1 and C=C aromatic stretching at 1,554.68 cm −1 .Ethylenic double bond at 1,668.48 and 1,176.62cm −1 for C-O-C stretching (Fig. 10).Negative ESI-MS of 3 showed a molecular ion peak (M-H) -at m/z 431.Its MS2 gave a major fragment at m/z 285 (M-146)-, loss of a rhamnose moiety (Zhao et al., 2014) (Fig. 13).Relative abundances of the two fragment ions suggested that they were located at different positions on the aglycone, although it was also not possible to conclude the precise position.Thus, the compound was tentatively assigned as kaempferol O-rhamnoside (3) (Fig. 1)./ Journal of Applied Pharmaceutical Science 13 (Suppl 1); 2023: 021-027

Alpha-glucosidase enzyme inhibitory assay
The α-glucosidase enzyme inhibitory assay demonstrated that the hydroalcoholic extract is somewhat pharmacologically active and less powerful than isolated components in contrast to standard acarbose (Table 2).With complex combinations of natural products, less well-known phenomena termed antagonism; where the effects of active materials are hidden by those of other compounds.Removing these molecules is one of the key rationales stated for isolating active compounds in the conventional pharmaceutical development process (Caesar and Cech, 2019).The results showed α-glucosidase inhibitors from the leaves were kaempferol di-Orhamnoside (1), kaempferol O-rhamnoside-O-pentoside (2), and kaempferol O-rhamnoside (3).Three compounds showed potent anti-α-glucosidase activity, kaempferol O-rhamnoside (IC 50 value 61 μg/ml) was more active followed by kaempferol O-rhamnoside-Opentoside (IC 50 value 84.7 μg/ml) and kaempferol di-O-rhamnoside (IC 50 value 118.81 μg/ml) (Table 2).Our present study revealed the leaves extract and isolated compounds along with the previously reported articles on the plant make the plant a good source of active antidiabetic agents.
Quercetagetin, the primary flavonoid in marigold inflorescence residue, was isolated and purified.Quercetagetin's in-vitro anti-diabetic efficacy was tested and compared to conventional quercetin and rutin.The IC 50 values for quercetagetin on α-glucosidase and α-amylase were 180.11 ± 3.68 and     / Journal of Applied Pharmaceutical Science 13 (Suppl 1); 2023: 021-027 137.71 ± 3.55 µmol/l, respectively (Wang et al., 2016).Lutein extract from marigold flowers can reduce blood glucose levels and works as an antioxidant in mice, as indicated by a drop in malondialdehyde levels (Kusmiati et al., 2019).Esterified lutein from T. erecta flower petals exhibits considerable antidiabetic action in alloxan-induced diabetic mice (Saigun and Sasidhar, 2019).

CONCLUSION
The present study revealed that the hydroalcoholic extract of T. erecta leaves showed potent antidiabetic activity.On further fractionation of the hydroalcoholic extract, three active principal constituents were identified, namely kaempferol di-Orhamnoside (1), kaempferol O-rhamnoside-O-pentoside (2), and kaempferol O-rhamnoside (3) which were not previously reported from this plant.All the compounds were tentatively characterized using UV, FTIR, and MS analyses.On the other hand, their purity was evaluated by HPLC and mp.This appears to be the first report on the comparative study of hydroalcoholic extract, and three isolated compounds using the in-vitro α-glucosidase inhibitory assay.The pharmacological activity of T. erecta leaf extract and column fractions could be due to the presence of kaempferol glycosides.Furthermore, based on the in-vitro results, the in-vivo evaluation will be carried out using the streptozotocin-induced diabetic rat model.

LIST OF ABBREVIATIONS
EA: Ethyl acetate; ESI-MS: electrospray ionization mass spectrometry; FTIR: fourier transform infrared; HPLC: High-performance liquid chromatography; TLC: Thin layer chromatography; UV: ultra violet.The authors report no financial or any other conflicts of interest in this work.

Figure 1 .
Figure 1.Chemical structure of isolated compounds.

Table 1 .
The gradient elution conditions.