TY - JOUR
AU - Rai, Lalit, Kumar
AB - Abstract In the present study, a systematic validated method was developed for the determination of two key dietary dihydrochalcones (DHC) viz. phloridzin (PZ) and phloretin (PT) in the leaves of Sikkim crabapple (Malus sikkimensis) using HPLC-Photo Diode Array (PDA). Chromatographic separation was optimized on a C18 column using a gradient elution of water/acetonitrile with the flow rate of 1.0 mL/min at 25°C at 280 nm. Sample preparation approach is rapid and energy efficient, and it requires no pre-concentration before analysis. Validation showed a good analytical performance in terms of specificity, linearity (r2 > 0.999), precision (% RSD < 1.08), recovery (97–100.4%) and sensitivities (limits of detection: 12.48 and 14.95 ng/mL; limit of quantification: 41.61 and 49.85 ng/mL) of PZ and PT, respectively. Developed approach was employed for targeted phytochemical analysis in the bark and fruits of M. sikkimensis. The PZ content in the bark and leaves was highest (12–13 mg/100 mg), about 90-fold higher than fruits. PT was only present in the leaves (0.57 mg/100 mg). The comparative data on PZ and PT content in various wild apple species/cultivar from different countries have also been discussed. The reliability of the validated method was established by analyzing global and expanded uncertainties in two DHC determinations in wild apple. The present method fulfills the technical requirement of ISO 17025:2017 for quality control of M. sikkimensis. Introduction Apples are considered to be a significantly rich source of phytochemicals, particularly dietary flavonoids (1). Dietary flavonoids are reported to inhibit cancer cell proliferation, protect against lipid oxidation, regulate inflammatory and immune responses (2). Malus (family Rosaceae) is a genus of about 30–55 species of small shrubs including the domesticated orchard apple (Malus pumila). The other species are collectively known as crabapples or wild apples. Malus sikkimensis or Sikkim crabapple native to Arunachal Pradesh, India, is a good source of various polyphenols in juice and fruit tissues (3). The functional food value of M. sikkimensis fruits has long been recognized due to the presence of a significant number of polyphenols. The presence of a number of anti-oxidant compounds, e.g., catechin, epi-catechin (Flavan-3-ols), PT, phloridzin (PZ; dihydrochalcone and its glycoside), quercetin (flavonols), chlorogenic acid (phenolic acids) and procyanidin, has been reported in the wild apples (4). Dihydrochalcones (DHCs) belong to flavonoid class and have characteristic C6-C3-C6 structural backbone with no heterocyclic C ring. DHCs are important precursors and play a critical role in flavonoids biosynthesis in plants. Interestingly, most of the DHCs are sweet and considered as natural sweetening agents. Not only the fruits but also the leaves of the apple accumulate high amounts of PZ (5). In addition to PZ and PT, five DHCs—trilobatin, 3-hydroxyphloretin, phloretinrutinoside, 6′′-O-coumaroyl-4′-O-glucopyranosyl PT and 3′′′-methoxy-6′′-O-feruloy-4′-O-glucopyranosyl-phloretin—have been reported from M. crabapples cv. radiant leaves (6). Phytochemical variable in different crabapples is due to obvious reasons of genotype × environment (G × E) interaction. PZ (PT 2′-O-glucoside or phlorizoside), a prominent member of DHCs has been reported worthwhile in the treatment of several physiological disorders (7). The principal pharmacological action of PZ has been reported to produce renal glycosuria and block intestinal glucose absorption through inhibition of sodium–glucose cotransporter, strongly decreases high K+-induced contraction in phasic muscle such as tenia coli. (8). Similarly, its aglycone moiety, i.e., PT, reported to possess the number of biological activities viz. antioxidant, anti-inflammatory, antimicrobial and anti-diabetic activities (α-glucosidase IC50 = 31.26 μg/L) (9). Significant positive anticancer activities of PT and its derivatives were demonstrated against several human cancer cell lines (6). The clinical pharmacology and toxicology of PZ, including investigational uses of PZ and its derivatives in the treatment of diabetes, obesity and stress hyperglycemia, are well studied (10). The significant properties of DHCs pose a challenge to detect, analyze and separate their content rapidly with acceptable reproducibility and sensitivity. However, to date, the phytochemical investigation as well as the quality specification of M. sikkimensis is not established. Several methods (11–14) either based on high-performance liquid chromatography (HPLC)–PDA or Ultra High-Performance Liquid Chromatography (UHPLC)-Diode Array Detection/ Electron Spray Ionisation-Mass Spectrometry (DAD/ESI-MS) demonstrating the determination of varying number of phenolics including DHCs in a different cultivar of apple have been reported. But, till date no validated method has been reported for M. sikkimensis. These methods have a drawback such as lack of validation data, undisclosed sample extraction efficiency, longer run time and/or high cost of initial investment (accurate mass spectrometry). Because of the different sample matrix and mentioned drawbacks, the reported method could not be implemented as such for rapid and accurate screening of wild cultivars of M. sikkimensis. It is the first validated method that provides the simultaneous and accurate analysis of five bioactive phytoconstituents of Drosophila erecta in short time with defined traceability and accuracy profile for holistic quality analysis of raw medicinal herb and its preparation. In the present paper, we have the first time reported the isolation and characterization of medicinally important bioactive compound, i.e., PZ and PT, from the leaves of M. sikkimensis as well as the quality specification of M. sikkimensis. Additionally, the consistency of the validated method was also studied the first time by calculating the global and expanded uncertainties. Experimental Plant material The leaves of M. sikkimensis were collected from Lachung of Gangtok, Sikkim. The taxonomical authentication was done by a botanist. A specimen voucher (gbp-sikkim/46a) was deposited to the herbarium of G. B. Pant Himalayan Institute, Gangtok, Sikkim. The shade-dried plant material was stored at room temperature following good storage practices. Dried and finely powdered plant material (100 mg) was kept for a different extraction process. Chemicals and reagents HPLC-grade trifluoroacetic acid, methanol, water and acetonitrile solvents were purchased from Merck, India. Before use, all solvents were filtered through a 0.22-micron membrane filter (Millipore, Billerica, MA, USA). Marker compounds, PZ and PT (purity > 99%), were isolated from the leaves of M. sikkimensis and characterized with the help of spectral data. Instrumentation The 1H and 13C spectra were recorded with tetramethylsilane as an internal standard on a Bruker Advance instrument (300-MHz Nuclear Magnetic Resonance Spectroscopy (NMR)). The DEPT experiments were used to resolve the multiplicities of carbon atoms. Chemical shifts were given in parts per million. Correlation Spectroscopy (COSY), Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC) and Heteronuclear Multiple-Bond Correlation Spectroscopy (HMBC) were performed using standard pulse programs. The ESI–MS was obtained on an MS-2010EV (Shimadzu, Japan) at 70 eV by flow injection into the electrospray source. Microwave-assisted extraction (MAE) was performed with a convection microwave oven (Magicook 22C, 900 W, Whirlpool, New Delhi, India) with programmable heating power and 3D wave distribution. Ultrasonic-assisted extraction (UAE) was carried out in ultrasonication bath (Microclean-109, Oscar Ultrasonics, Mumbai, India; 30.0 × 25.0 × 12.5 cm, 34 ± 3 kHz, PZT sandwich-type six transducer, 250 W), respectively. HPLC equipment (Shimadzu, Japan) consists of pumps (Liquid Chromatography (LC)-10AT), autoinjector (SIL-10 AD), PDA (SPD-M10A) and analytical column (Waters, Spherisorb ODS2, 250 mm × 4.6 mm). PT and PZ were purified on a semi-preparative LC-8A (Shimadzu) using a reverse phase column (Supelco C18, 10 × 100 mm, 10 μm). Extraction and isolation of bioactive DHCs Dry leaves (45 g) were finely crushed using a grinder and then extracted using methanol in a Soxhlet for 8 hrs. The combined extract was concentrated under vacuum to give a methanolic extract. The dried extract (4.5 g) was subjected to fractionation on a silica gel (60–120 mesh) column eluting with a mixture of solvents with gradually increasing polarity starting from hexane followed by ethyl acetate and methanol. Fractions 15–20 eluted with hexane:ethyl acetate (30:70, v/v) were further purified using semi-preparative HPLC (mobile phase acetonitrile: water: 25:75, v/v; flow rate: 3 mL/min, detection 280 nm), which resulted into purified marker compound PT (Rt 11.28 min). Fractions 22–32 eluted with hexane:ethyl acetate (10, 90, v/v) were further purified through semi-preparative HPLC, which resulted into purified compound PZ (Rt 3.58 min). The structure elucidation has established the identity as PT and PZ, respectively (Figure 1). Figure 1 Open in new tabDownload slide Schematic representation of isolation and HPLC chromatogram of (a) mix standards (PZ and PT 0.5 mg/mL each) and (b) methanolic extract of M. sikkimensis (100 mg/mL) at 280 nm; characteristic UV spectra of respective presented in the inset. Figure 1 Open in new tabDownload slide Schematic representation of isolation and HPLC chromatogram of (a) mix standards (PZ and PT 0.5 mg/mL each) and (b) methanolic extract of M. sikkimensis (100 mg/mL) at 280 nm; characteristic UV spectra of respective presented in the inset. Chromatographic parameters The separation of PT and PZ was achieved using binary solvent composition of the mobile phase of water (containing 0.1% of trifluoroacetic acid; solvent A) and acetonitrile (solvent B). The optimum resolution was obtained by using different gradients as follows: 0–8 min, 25% B; 8–11 min, 25–45% B; 11–20 min, 45% B; and 20–25 min, 25% B. The equilibration time between two consecutive runs in series was set at 10 min. The flow rate was 1.0 mL/min throughout the analysis. Injection volume was 10 μL. All analyses were performed maintaining column temperature at 30°C. The data acquisition was performed in the range of 190–400 nm to monitor any possible co-elution in the plant sample solution. Both targeted compounds (PZ and PT) have UV maxima at 282 and 283 nm, respectively (15–17). Therefore, for simultaneous quantization 280 nm was selected considering optimum chromatographic signal response for simultaneous quantization. Additionally, interference due to possible co-elution of sample matrix impurity was also checked by peak purity at the start, apex and end of the peak in post-processing of chromatogram. Statistical analysis Results were presented as the mean ± SD/SE of triplicate observations. One-way analysis of variance followed by Turkey’s test was used to determine statistical significance for multiple comparisons. The P-value of <0.05 was taken as statistically significant. MS Excel and Graph Pad Prism version 4.0 were used to perform statistical calculations. The uncertainties of measurement have been using the validation parameters following EURACHEM/CITAC CG 4 statistical procedure (18). Results The robust functional potential is questionable until the chemical standardization of medicinal food is not defined. Till date, the standardization of crabapple (M. sikkimensis) is still lacking. The present work demonstrates the isolation of bioactive secondary metabolites, validated HPLC method for quality analysis with uncertainty measurement of determination and chemical standardization of M. sikkimensis based on two dietary DHCs. Isolation and identification of isolated phytochemicals from M. sikkimensis leaves Marker chemicals have been isolated by silica gel column chromatography, purified through preparative LC as described above and finally characterized as PT and PZ (Figure 1). The authenticity of marker compounds was confirmed by its spectral characteristics. All data were found in good coherence with previously reported in the literature (19–22). Compound 1: (C15H14O5; 274) was identified as PT, amorphous powder, ESI–MS m/z 297 [M + Na]+in positive ion mode and 273 [M-H]− in negative ion mode was recorded. 13C-NMR (MeOD, 75 MHz): δ 104.5(C-1), 164.7(C-2), 95.0(C-3), 155.4(C-4), 95.0(C-5), 155.4(C-6), 205.4(C-7), 46.2(C-8), 30.4(C-9), 107.4(C-1′), 123.9(C-2′), 128.4(C-3′), 115.0(C-4′), 163.1(C-5′) and 115.0(C-6′). 1H-NMR (MeOD, 300 MHz) analysis yielded chemical shift at 2.84 (2H, t, J = 9.6 Hz H-9), 3.27 (2H, t, J = 9.6, H-8), 5.94 (1H, s, H-3 and 5), 6.74 (1H, d, J = 8.4, H-3′,5′) and 7.05 (1H, d, J = 8.4, H-2′,6′). Compound 2: (C21H24O10, 436) was identified as PZ, crystalline, ESI–MS m/z 459 [M + Na]+, negative ESI–MS m/z 435 [M-H]−. The UV/Visible spectrum of the compound showed λmax at 226 and 283 nm characteristic of PZ as found in literature (23). The chemical shift values observed for PZ at δ 13C-NMR (MeOD, 75 MHz) are as follows: δ 105.3(C-1), 164.7(C-2), 100.8(C-3), 165.4(C-4), 96.9(C-5), 160.9(C-6), 204.8(C-7), 45.1(C-8), 29.1(C-9), 131.7(C-1′), 129.3(C-2′), 115.1(C-3′), 155.3(C-4′), 115.1(C-5′), 129.3(C-6′),105.3(C-1″), 73.3(C-2″), 77.3(C-3″), 69.6(C-4″), 76.8(C-5″) and 60.7(C-6″); 1H-NMR (MeOD, 300 MHz): 2.88 (2H, t, J = 7.5, H-9), 3.32 (1H, m, H-3″), 3.42 (1H, m, H-2″), 3.45 (1H, m, H-4″, H-5″), 3.68 (1H, m, H-6″), 3.71 (CHC(O)), 3.92 (2H, m, H-8), 5.96 (1H,d, J = 2.1, H-5), 6.18 (1H, d, J = 2.1, H-3,5′), 6.68 (1H, d, J = 8.4, H-3′) and 7.06 (1H, d, J = 8.1, H-2′, 6′). Table I Extraction efficiency of different solvents and techniques for bioactive compounds PZ and PT from the aerial part of M. sikkimensis Solvents Content of DHCs (mg/100 mg, dry weight basis) CP HE UAE MAE PZ PT PZ PT PZ PT PZ PT Hexane 1.40 ± 0.13 0.47 ± 0.02 1.37 ± 0.09 0.57 ± 0.07 1.17 ± 0.06 0.52 ± 0.06 SNP SNP EtOAc 7.97 ± 0.28 0.17 ± 0.01 7.27 ± 0.21 0.23 ± 0.01 8.63 ± 0.11 0.21 ± 0.01 7.57 ± 0.17 0.11 ± 0.01 Methanol 11.97 ± 0.18 0.53 ± 0.02 12.13 ± 0.32 0.53 ± 0.03 12.53 ± 0.30 0.57 ± 0.04 10.71 ± 0.27 0.27 ± 0.02 Water 4.67 ± 0.03 0.36 ± 0.03 4.87 ± 0.17 0.32 ± 0.02 4.40 ± 0.04 0.80 ± 0.06 1.75 ± 0.09 0.42 ± 0.03 Solvents Content of DHCs (mg/100 mg, dry weight basis) CP HE UAE MAE PZ PT PZ PT PZ PT PZ PT Hexane 1.40 ± 0.13 0.47 ± 0.02 1.37 ± 0.09 0.57 ± 0.07 1.17 ± 0.06 0.52 ± 0.06 SNP SNP EtOAc 7.97 ± 0.28 0.17 ± 0.01 7.27 ± 0.21 0.23 ± 0.01 8.63 ± 0.11 0.21 ± 0.01 7.57 ± 0.17 0.11 ± 0.01 Methanol 11.97 ± 0.18 0.53 ± 0.02 12.13 ± 0.32 0.53 ± 0.03 12.53 ± 0.30 0.57 ± 0.04 10.71 ± 0.27 0.27 ± 0.02 Water 4.67 ± 0.03 0.36 ± 0.03 4.87 ± 0.17 0.32 ± 0.02 4.40 ± 0.04 0.80 ± 0.06 1.75 ± 0.09 0.42 ± 0.03 Parameters for optimum extraction in each technique were described in the experimental section. Results are mean ± SD of three replicate analyses. Open in new tab Table I Extraction efficiency of different solvents and techniques for bioactive compounds PZ and PT from the aerial part of M. sikkimensis Solvents Content of DHCs (mg/100 mg, dry weight basis) CP HE UAE MAE PZ PT PZ PT PZ PT PZ PT Hexane 1.40 ± 0.13 0.47 ± 0.02 1.37 ± 0.09 0.57 ± 0.07 1.17 ± 0.06 0.52 ± 0.06 SNP SNP EtOAc 7.97 ± 0.28 0.17 ± 0.01 7.27 ± 0.21 0.23 ± 0.01 8.63 ± 0.11 0.21 ± 0.01 7.57 ± 0.17 0.11 ± 0.01 Methanol 11.97 ± 0.18 0.53 ± 0.02 12.13 ± 0.32 0.53 ± 0.03 12.53 ± 0.30 0.57 ± 0.04 10.71 ± 0.27 0.27 ± 0.02 Water 4.67 ± 0.03 0.36 ± 0.03 4.87 ± 0.17 0.32 ± 0.02 4.40 ± 0.04 0.80 ± 0.06 1.75 ± 0.09 0.42 ± 0.03 Solvents Content of DHCs (mg/100 mg, dry weight basis) CP HE UAE MAE PZ PT PZ PT PZ PT PZ PT Hexane 1.40 ± 0.13 0.47 ± 0.02 1.37 ± 0.09 0.57 ± 0.07 1.17 ± 0.06 0.52 ± 0.06 SNP SNP EtOAc 7.97 ± 0.28 0.17 ± 0.01 7.27 ± 0.21 0.23 ± 0.01 8.63 ± 0.11 0.21 ± 0.01 7.57 ± 0.17 0.11 ± 0.01 Methanol 11.97 ± 0.18 0.53 ± 0.02 12.13 ± 0.32 0.53 ± 0.03 12.53 ± 0.30 0.57 ± 0.04 10.71 ± 0.27 0.27 ± 0.02 Water 4.67 ± 0.03 0.36 ± 0.03 4.87 ± 0.17 0.32 ± 0.02 4.40 ± 0.04 0.80 ± 0.06 1.75 ± 0.09 0.42 ± 0.03 Parameters for optimum extraction in each technique were described in the experimental section. Results are mean ± SD of three replicate analyses. Open in new tab Table II Overview of method development for the quantitation of PZ and PT in M. sikkimensis Parameters PZ PT  Retention time (min) 7.50 16.51  Linearity range (ng/mL) 100–500 100–500 Linearity best-fit values   Slope 6213 ± 81.75 5833 ± 91.96   Y-intercept −69880 ± 27110 −54030 ± 30500  Goodness of fit   Correlation coefficient 0.9995 0.9993   Sy.x 25850 29080   P-value <0.0001 <0.0001  Sensitivities   LOD (ng/mL) 12.48 14.95   LOQ (ng/mL) 41.61 49.85  Precision and accuracy   Intraday–reproducibility (% RSD of percent content; n = 10) 0.87 0.58   Interday-repeatability (% RSD of percent content): Day 1/Day 2/Day 3 (n = 5; P > 0.05) 0.71 /0.56/1.08 0.85/0.75/0.97  Recovery   Target concentration in sample matrix (mg/100 mg) 12.13 0.57   Spiked amount (mg/100 mg), i.e., (±50% of respective compound in plant; n = 3) 6.06 0.28   Recovery range (%) 90.6–100.4 97.0–98.1   Mean ± RSD% 98.8 ± 0.42 97.4 ± 0.63 Parameters PZ PT  Retention time (min) 7.50 16.51  Linearity range (ng/mL) 100–500 100–500 Linearity best-fit values   Slope 6213 ± 81.75 5833 ± 91.96   Y-intercept −69880 ± 27110 −54030 ± 30500  Goodness of fit   Correlation coefficient 0.9995 0.9993   Sy.x 25850 29080   P-value <0.0001 <0.0001  Sensitivities   LOD (ng/mL) 12.48 14.95   LOQ (ng/mL) 41.61 49.85  Precision and accuracy   Intraday–reproducibility (% RSD of percent content; n = 10) 0.87 0.58   Interday-repeatability (% RSD of percent content): Day 1/Day 2/Day 3 (n = 5; P > 0.05) 0.71 /0.56/1.08 0.85/0.75/0.97  Recovery   Target concentration in sample matrix (mg/100 mg) 12.13 0.57   Spiked amount (mg/100 mg), i.e., (±50% of respective compound in plant; n = 3) 6.06 0.28   Recovery range (%) 90.6–100.4 97.0–98.1   Mean ± RSD% 98.8 ± 0.42 97.4 ± 0.63 Open in new tab Table II Overview of method development for the quantitation of PZ and PT in M. sikkimensis Parameters PZ PT  Retention time (min) 7.50 16.51  Linearity range (ng/mL) 100–500 100–500 Linearity best-fit values   Slope 6213 ± 81.75 5833 ± 91.96   Y-intercept −69880 ± 27110 −54030 ± 30500  Goodness of fit   Correlation coefficient 0.9995 0.9993   Sy.x 25850 29080   P-value <0.0001 <0.0001  Sensitivities   LOD (ng/mL) 12.48 14.95   LOQ (ng/mL) 41.61 49.85  Precision and accuracy   Intraday–reproducibility (% RSD of percent content; n = 10) 0.87 0.58   Interday-repeatability (% RSD of percent content): Day 1/Day 2/Day 3 (n = 5; P > 0.05) 0.71 /0.56/1.08 0.85/0.75/0.97  Recovery   Target concentration in sample matrix (mg/100 mg) 12.13 0.57   Spiked amount (mg/100 mg), i.e., (±50% of respective compound in plant; n = 3) 6.06 0.28   Recovery range (%) 90.6–100.4 97.0–98.1   Mean ± RSD% 98.8 ± 0.42 97.4 ± 0.63 Parameters PZ PT  Retention time (min) 7.50 16.51  Linearity range (ng/mL) 100–500 100–500 Linearity best-fit values   Slope 6213 ± 81.75 5833 ± 91.96   Y-intercept −69880 ± 27110 −54030 ± 30500  Goodness of fit   Correlation coefficient 0.9995 0.9993   Sy.x 25850 29080   P-value <0.0001 <0.0001  Sensitivities   LOD (ng/mL) 12.48 14.95   LOQ (ng/mL) 41.61 49.85  Precision and accuracy   Intraday–reproducibility (% RSD of percent content; n = 10) 0.87 0.58   Interday-repeatability (% RSD of percent content): Day 1/Day 2/Day 3 (n = 5; P > 0.05) 0.71 /0.56/1.08 0.85/0.75/0.97  Recovery   Target concentration in sample matrix (mg/100 mg) 12.13 0.57   Spiked amount (mg/100 mg), i.e., (±50% of respective compound in plant; n = 3) 6.06 0.28   Recovery range (%) 90.6–100.4 97.0–98.1   Mean ± RSD% 98.8 ± 0.42 97.4 ± 0.63 Open in new tab Optimization of sample preparation Before analysis, the sample preparation has been optimized for extraction time, solvent and techniques. The extraction efficiency of different solvents was evaluated using four solvents (hexane, ethyl acetate, methanol and water). The extraction efficiency was estimated using both classical procedure—cold percolation (CP) and hot extraction (HE) by refluxing—as well as advanced—UAE and MAE—techniques. Dried and finely powdered plant material (100 mg) was used to optimize extraction parameters of CP (3 × 15 mL, 24 hrs, room temperature), HE (3 × 15 mL, 30 min, 100°C) by refluxing over water bath (15 mL, 8 hrs), MAE (3 × 5 mL, 2 min at 50°C, 350 W) and UAE (3 × 5.0 mL, 10 min, 60°C) using different solvents by determination of targeted DHCs. Extracted samples were made up to 1.0 mL and centrifuged at 10,000 rpm for 5 min before HPLC analysis. Different parameters have been for solvents, techniques, time and temperature in order to achieve the optimum extraction efficiency of (Table I). Method validation The method validation was achieved by the evaluation of linearity, sensitivities (limit of detection [LOD] and quantification), precision, accuracy, specificity and recovery data (24, 25). For the simultaneous determination of PZ and PT, optimum separation was found at 7.50 and 16.51 min, respectively. No closely related component eluted was evident and thus caused no interferences in the quantitation of both the compounds. Further, it was also confirmed by peak purity determination in sample analysis of M. sikkimensis (Figure 1). Linearity The capability of the method for the linear relationship of the detector’s response at 280 nm with the varying concentration of the both PZ and PT was studied in the selected range (100–500 ng/mL). For both DHCs, excellent linearity (correlation coefficient, >0.999) was obtained in the specified concentration range. The slope and intercept of the linear equations of the calibration lines are presented in Table II. LOD and limit of quantitation The sensitivities of the method calculated as the LOD and limit of quantification (LOQ) of PZ and PT from the goodness of fit data of linear regression equations (25): $$ LOD=\frac{\mathrm{S} yx}{\mathrm{slope}}\times 3;\quad LOQ=\frac{\mathrm{S} yx}{\mathrm{slope}}\times 10 $$ The LOQ and the LOD of PZ and PT were found to be 41.61, 12.48, 49.85 and 14.95 ng/mL, respectively (refer to Table II). Specificity The specificity of the determination obtained by retention time as well as UV–Vis spectral matching of the respective of PZ and PT in the sample run with standard peak was stored in the library. In addition to spiking the known quantity of the reference in the sample, interference due to possible co-elution of sample matrix component was assessed using peak purity. The peak purity was checked at the start, apex and end of the peak of corresponding compound; the purity index was higher than the threshold value, which further confirmed the peak purity of both PZ and PT in post-processing of chromatogram. Precision The result of intra-day precision and inter-day accuracy (Table II) experiments demonstrate the suitability of the method (% RSD, <1.08 and 0.97, respectively). Recovery The extraction recovery of PZ and PT was calculated by spike method. The ratio of the areas obtained from the spiked samples and the areas obtained from the standard solutions used for recovery calculation. The excellent extraction efficiency (99% and 97%, respectively) is illustrated in Table II. Assessment of global uncertainty Five separate sources of uncertainty viz—uncertainty associated with calibration curve (U1), uncertainty linked to precision (due to different analysts [U2] and change in day of analysis [U3]) and uncertainty associated with accuracy (due to analyst change [U4] and change in day of analysis [U5])—were taken into account (26, 27) and were calculated. The values of individual factors of uncertainty as well as global uncertainty of measurement in the determination of PZ and PTM. sikkimensis leaves are summarized (Table III). Table III Result of individual and global uncertainties of compounds at their content level* S. No Compound Calibration curve Precision Accuracy Global uncertainty Expanded uncertainty U1 U2 U3 U4 U5 U 2 U 1 PZ 0.038 0.271 0.156 0.242 0.225 0.932 1.864 2 PT 0.002 0.181 0.172 0.363 0.294 1.012 2.024 S. No Compound Calibration curve Precision Accuracy Global uncertainty Expanded uncertainty U1 U2 U3 U4 U5 U 2 U 1 PZ 0.038 0.271 0.156 0.242 0.225 0.932 1.864 2 PT 0.002 0.181 0.172 0.363 0.294 1.012 2.024 *Method uncertainty values calculated following EURACHEM/CITAC CG 4 statistical procedure. U1 indicates uncertainty associated with calibration curve; U2, uncertainty linked to precision due to different analysts; U3, uncertainty linked to precision due to change in day of analysis; U4, uncertainty associated to accuracy due to analyst change; U5, uncertainty associated to accuracy change in day of analysis. Open in new tab Table III Result of individual and global uncertainties of compounds at their content level* S. No Compound Calibration curve Precision Accuracy Global uncertainty Expanded uncertainty U1 U2 U3 U4 U5 U 2 U 1 PZ 0.038 0.271 0.156 0.242 0.225 0.932 1.864 2 PT 0.002 0.181 0.172 0.363 0.294 1.012 2.024 S. No Compound Calibration curve Precision Accuracy Global uncertainty Expanded uncertainty U1 U2 U3 U4 U5 U 2 U 1 PZ 0.038 0.271 0.156 0.242 0.225 0.932 1.864 2 PT 0.002 0.181 0.172 0.363 0.294 1.012 2.024 *Method uncertainty values calculated following EURACHEM/CITAC CG 4 statistical procedure. U1 indicates uncertainty associated with calibration curve; U2, uncertainty linked to precision due to different analysts; U3, uncertainty linked to precision due to change in day of analysis; U4, uncertainty associated to accuracy due to analyst change; U5, uncertainty associated to accuracy change in day of analysis. Open in new tab Table IV Status of distribution of dietary DHCs contents (mg/100 mg dry weight basis) in different plant parts of wild Malus species (crabapples) of different region/country Crabapple species (cultivar) Origin/country Plant part Content (mg/100 mg on dry weight) except† Reference PZ PT Malus sylveslris Mill. Saint Nolff/France Fruits 0.20 - (28) Malus hupehensis Xianyang/China Fruits 1.55 - (29) Malus hupehensis var. pinyiensis Xianyang/China Fruits 1.47 - Malus toringoides Xianyang/China Fruits 0.21 - Malus rockii Rehder (Lijiangshandingzi) Xingcheng/China Fruits 0.01 - (30) Malus xiaojinensis Cheng et Jiang (Xiaojinhaitang) Xingcheng/China Fruits <0.01 - Malus coronaria (L.) Mill (Huaguanhaitang) Xingcheng/China Fruits 0.01 - Malus prunifolia (Wild) Borkh (Donghongguo) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd. (Sankuaishihaitang01) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Sankuaishihaitang02) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Xiaofanshanhaitang) Xingcheng/China Fruits <0.01 - Malus micromalus (Carr.) Rehd (Balenghaitang) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Pingdinghaitang) Xingcheng/China Fruits <0.01 - Malus prunifolia Mill. (Honghaitang) Xingcheng/China Fruits <0.01 - Malus toringoides Hughes Aba Tibetan/China Leaves 10.52 - (31, 32) Fruits 0.21 - Malus transitoria Schneid Aba Tibetan/China Leaves 12.90 - Fruits 0.27 - Malus kansuensis Schneid Aba Tibetan/China Leaves 5.36 - Fruits 0.06 - Malus maerkangensis Aba Tibetan/China Leaves 4.95 - Fruits 0.04 - Malus setokVassilcz Aba Tibetan/China Leaves 9.47 - Fruits 0.38 - Malus xiaojinensis Aba Tibetan/China Leaves 9.10 - Fruits 0.38 - Crabapple (Kerr) Valmiera/Latvia Leaves 0.04 - (33) Stem 4.05 - Crabapple (Riku) Valmiera/Latvia Leaves 0.01 - Stem 0.57 Crabapple (Quaker Beautu) Valmiera/Latvia Leaves 0.01 - Stem 0.30 Crabapple (Ritika) Valmiera/Latvia Leaves 0.01 - Stem 0.53 Crabapple (K-8/9–24) Valmiera/Latvia Leaves 0.01 - Stem 1.09 Crabapple (Kuku) Valmiera/Latvia Leaves 0.01 - Stem 0.55 Crabapple (Ruti) Valmiera/Latvia Leaves 0.01 - Stem 0.29 Siberian crabapple Irkutsk/Russia Flesh 0.70 0.01 (32) Peel 3.24 0.02 Palmetta Irkutsk/Russia Flesh 0.19 0.02 Peel 0.28 0.01 Siberian souvenir Irkutsk/Russia Flesh 0.31 0.01 Peel 0.47 0.02 Altayskoerumyanoe Irkutsk/Russia Flesh 0.19 <0.01 Peel 0.39 0.01 Podruga Irkutsk/Russia Flesh 0.16 0.06 Peel 0.32 0.01 Nejenka Irkutsk/Russia Flesh 0.09 0.03 Peel 0.23 <0.01 Podaroksadovodam Irkutsk/Russia Flesh 0.83 0.08 Peel 0.99 0.01 Phoenix altayski Irkutsk/Russia Flesh 0.33 0.03 Peel 0.97 0.01 Yellow-green peel Croatia Flesh <0.01 <0.01 (34) Peel <0.01 - Malus sikkimensis Sikkim/India Leaves 12.53† 0.57 Present study Fruits 0.14† ND Bark 13.08† ND Crabapple species (cultivar) Origin/country Plant part Content (mg/100 mg on dry weight) except† Reference PZ PT Malus sylveslris Mill. Saint Nolff/France Fruits 0.20 - (28) Malus hupehensis Xianyang/China Fruits 1.55 - (29) Malus hupehensis var. pinyiensis Xianyang/China Fruits 1.47 - Malus toringoides Xianyang/China Fruits 0.21 - Malus rockii Rehder (Lijiangshandingzi) Xingcheng/China Fruits 0.01 - (30) Malus xiaojinensis Cheng et Jiang (Xiaojinhaitang) Xingcheng/China Fruits <0.01 - Malus coronaria (L.) Mill (Huaguanhaitang) Xingcheng/China Fruits 0.01 - Malus prunifolia (Wild) Borkh (Donghongguo) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd. (Sankuaishihaitang01) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Sankuaishihaitang02) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Xiaofanshanhaitang) Xingcheng/China Fruits <0.01 - Malus micromalus (Carr.) Rehd (Balenghaitang) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Pingdinghaitang) Xingcheng/China Fruits <0.01 - Malus prunifolia Mill. (Honghaitang) Xingcheng/China Fruits <0.01 - Malus toringoides Hughes Aba Tibetan/China Leaves 10.52 - (31, 32) Fruits 0.21 - Malus transitoria Schneid Aba Tibetan/China Leaves 12.90 - Fruits 0.27 - Malus kansuensis Schneid Aba Tibetan/China Leaves 5.36 - Fruits 0.06 - Malus maerkangensis Aba Tibetan/China Leaves 4.95 - Fruits 0.04 - Malus setokVassilcz Aba Tibetan/China Leaves 9.47 - Fruits 0.38 - Malus xiaojinensis Aba Tibetan/China Leaves 9.10 - Fruits 0.38 - Crabapple (Kerr) Valmiera/Latvia Leaves 0.04 - (33) Stem 4.05 - Crabapple (Riku) Valmiera/Latvia Leaves 0.01 - Stem 0.57 Crabapple (Quaker Beautu) Valmiera/Latvia Leaves 0.01 - Stem 0.30 Crabapple (Ritika) Valmiera/Latvia Leaves 0.01 - Stem 0.53 Crabapple (K-8/9–24) Valmiera/Latvia Leaves 0.01 - Stem 1.09 Crabapple (Kuku) Valmiera/Latvia Leaves 0.01 - Stem 0.55 Crabapple (Ruti) Valmiera/Latvia Leaves 0.01 - Stem 0.29 Siberian crabapple Irkutsk/Russia Flesh 0.70 0.01 (32) Peel 3.24 0.02 Palmetta Irkutsk/Russia Flesh 0.19 0.02 Peel 0.28 0.01 Siberian souvenir Irkutsk/Russia Flesh 0.31 0.01 Peel 0.47 0.02 Altayskoerumyanoe Irkutsk/Russia Flesh 0.19 <0.01 Peel 0.39 0.01 Podruga Irkutsk/Russia Flesh 0.16 0.06 Peel 0.32 0.01 Nejenka Irkutsk/Russia Flesh 0.09 0.03 Peel 0.23 <0.01 Podaroksadovodam Irkutsk/Russia Flesh 0.83 0.08 Peel 0.99 0.01 Phoenix altayski Irkutsk/Russia Flesh 0.33 0.03 Peel 0.97 0.01 Yellow-green peel Croatia Flesh <0.01 <0.01 (34) Peel <0.01 - Malus sikkimensis Sikkim/India Leaves 12.53† 0.57 Present study Fruits 0.14† ND Bark 13.08† ND †Content of PZ and PT in the crabapple cultivar from Irkutsk/Russia, Xingcheng/China (reported as μg/g in cited paper converted to mg/100 mg) and Croatia (reported as mg/Kg on fresh weight basis converted to mg/100 mg for uniform presentation) are based on fresh weight basis. ND: not detected. Open in new tab Table IV Status of distribution of dietary DHCs contents (mg/100 mg dry weight basis) in different plant parts of wild Malus species (crabapples) of different region/country Crabapple species (cultivar) Origin/country Plant part Content (mg/100 mg on dry weight) except† Reference PZ PT Malus sylveslris Mill. Saint Nolff/France Fruits 0.20 - (28) Malus hupehensis Xianyang/China Fruits 1.55 - (29) Malus hupehensis var. pinyiensis Xianyang/China Fruits 1.47 - Malus toringoides Xianyang/China Fruits 0.21 - Malus rockii Rehder (Lijiangshandingzi) Xingcheng/China Fruits 0.01 - (30) Malus xiaojinensis Cheng et Jiang (Xiaojinhaitang) Xingcheng/China Fruits <0.01 - Malus coronaria (L.) Mill (Huaguanhaitang) Xingcheng/China Fruits 0.01 - Malus prunifolia (Wild) Borkh (Donghongguo) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd. (Sankuaishihaitang01) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Sankuaishihaitang02) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Xiaofanshanhaitang) Xingcheng/China Fruits <0.01 - Malus micromalus (Carr.) Rehd (Balenghaitang) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Pingdinghaitang) Xingcheng/China Fruits <0.01 - Malus prunifolia Mill. (Honghaitang) Xingcheng/China Fruits <0.01 - Malus toringoides Hughes Aba Tibetan/China Leaves 10.52 - (31, 32) Fruits 0.21 - Malus transitoria Schneid Aba Tibetan/China Leaves 12.90 - Fruits 0.27 - Malus kansuensis Schneid Aba Tibetan/China Leaves 5.36 - Fruits 0.06 - Malus maerkangensis Aba Tibetan/China Leaves 4.95 - Fruits 0.04 - Malus setokVassilcz Aba Tibetan/China Leaves 9.47 - Fruits 0.38 - Malus xiaojinensis Aba Tibetan/China Leaves 9.10 - Fruits 0.38 - Crabapple (Kerr) Valmiera/Latvia Leaves 0.04 - (33) Stem 4.05 - Crabapple (Riku) Valmiera/Latvia Leaves 0.01 - Stem 0.57 Crabapple (Quaker Beautu) Valmiera/Latvia Leaves 0.01 - Stem 0.30 Crabapple (Ritika) Valmiera/Latvia Leaves 0.01 - Stem 0.53 Crabapple (K-8/9–24) Valmiera/Latvia Leaves 0.01 - Stem 1.09 Crabapple (Kuku) Valmiera/Latvia Leaves 0.01 - Stem 0.55 Crabapple (Ruti) Valmiera/Latvia Leaves 0.01 - Stem 0.29 Siberian crabapple Irkutsk/Russia Flesh 0.70 0.01 (32) Peel 3.24 0.02 Palmetta Irkutsk/Russia Flesh 0.19 0.02 Peel 0.28 0.01 Siberian souvenir Irkutsk/Russia Flesh 0.31 0.01 Peel 0.47 0.02 Altayskoerumyanoe Irkutsk/Russia Flesh 0.19 <0.01 Peel 0.39 0.01 Podruga Irkutsk/Russia Flesh 0.16 0.06 Peel 0.32 0.01 Nejenka Irkutsk/Russia Flesh 0.09 0.03 Peel 0.23 <0.01 Podaroksadovodam Irkutsk/Russia Flesh 0.83 0.08 Peel 0.99 0.01 Phoenix altayski Irkutsk/Russia Flesh 0.33 0.03 Peel 0.97 0.01 Yellow-green peel Croatia Flesh <0.01 <0.01 (34) Peel <0.01 - Malus sikkimensis Sikkim/India Leaves 12.53† 0.57 Present study Fruits 0.14† ND Bark 13.08† ND Crabapple species (cultivar) Origin/country Plant part Content (mg/100 mg on dry weight) except† Reference PZ PT Malus sylveslris Mill. Saint Nolff/France Fruits 0.20 - (28) Malus hupehensis Xianyang/China Fruits 1.55 - (29) Malus hupehensis var. pinyiensis Xianyang/China Fruits 1.47 - Malus toringoides Xianyang/China Fruits 0.21 - Malus rockii Rehder (Lijiangshandingzi) Xingcheng/China Fruits 0.01 - (30) Malus xiaojinensis Cheng et Jiang (Xiaojinhaitang) Xingcheng/China Fruits <0.01 - Malus coronaria (L.) Mill (Huaguanhaitang) Xingcheng/China Fruits 0.01 - Malus prunifolia (Wild) Borkh (Donghongguo) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd. (Sankuaishihaitang01) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Sankuaishihaitang02) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Xiaofanshanhaitang) Xingcheng/China Fruits <0.01 - Malus micromalus (Carr.) Rehd (Balenghaitang) Xingcheng/China Fruits <0.01 - Malus robusta (Carr.) Rehd (Pingdinghaitang) Xingcheng/China Fruits <0.01 - Malus prunifolia Mill. (Honghaitang) Xingcheng/China Fruits <0.01 - Malus toringoides Hughes Aba Tibetan/China Leaves 10.52 - (31, 32) Fruits 0.21 - Malus transitoria Schneid Aba Tibetan/China Leaves 12.90 - Fruits 0.27 - Malus kansuensis Schneid Aba Tibetan/China Leaves 5.36 - Fruits 0.06 - Malus maerkangensis Aba Tibetan/China Leaves 4.95 - Fruits 0.04 - Malus setokVassilcz Aba Tibetan/China Leaves 9.47 - Fruits 0.38 - Malus xiaojinensis Aba Tibetan/China Leaves 9.10 - Fruits 0.38 - Crabapple (Kerr) Valmiera/Latvia Leaves 0.04 - (33) Stem 4.05 - Crabapple (Riku) Valmiera/Latvia Leaves 0.01 - Stem 0.57 Crabapple (Quaker Beautu) Valmiera/Latvia Leaves 0.01 - Stem 0.30 Crabapple (Ritika) Valmiera/Latvia Leaves 0.01 - Stem 0.53 Crabapple (K-8/9–24) Valmiera/Latvia Leaves 0.01 - Stem 1.09 Crabapple (Kuku) Valmiera/Latvia Leaves 0.01 - Stem 0.55 Crabapple (Ruti) Valmiera/Latvia Leaves 0.01 - Stem 0.29 Siberian crabapple Irkutsk/Russia Flesh 0.70 0.01 (32) Peel 3.24 0.02 Palmetta Irkutsk/Russia Flesh 0.19 0.02 Peel 0.28 0.01 Siberian souvenir Irkutsk/Russia Flesh 0.31 0.01 Peel 0.47 0.02 Altayskoerumyanoe Irkutsk/Russia Flesh 0.19 <0.01 Peel 0.39 0.01 Podruga Irkutsk/Russia Flesh 0.16 0.06 Peel 0.32 0.01 Nejenka Irkutsk/Russia Flesh 0.09 0.03 Peel 0.23 <0.01 Podaroksadovodam Irkutsk/Russia Flesh 0.83 0.08 Peel 0.99 0.01 Phoenix altayski Irkutsk/Russia Flesh 0.33 0.03 Peel 0.97 0.01 Yellow-green peel Croatia Flesh <0.01 <0.01 (34) Peel <0.01 - Malus sikkimensis Sikkim/India Leaves 12.53† 0.57 Present study Fruits 0.14† ND Bark 13.08† ND †Content of PZ and PT in the crabapple cultivar from Irkutsk/Russia, Xingcheng/China (reported as μg/g in cited paper converted to mg/100 mg) and Croatia (reported as mg/Kg on fresh weight basis converted to mg/100 mg for uniform presentation) are based on fresh weight basis. ND: not detected. Open in new tab Discussion Optimization of extraction efficiency for chromatographic analysis The extraction efficiency of different solvents and procedures revealed that the amount of both PZ and PT extracted using methanol was significantly higher (P < 0.05, Student t-test) than other solvents in all techniques except UAE, whereas water proved to be the better choice for PT, not PZ. The varying concentration of methanol in water could improve the PZ extraction. Considering the optimum extraction efficiency of both DHCs, UAE using methanol was selected for sample preparation for PZ and PT analysis in M. sikkimensis leaves (Table I). Table V Status of method validation in different apple cultivars Apple variety and part used Analytical method Extraction method/solvent PZ PT Chromatographic condition Reference Red delicious, Shandong Fuji and Xinjiang Fuji (peel and pulp) HPLC DAD Ultrasonic bath (30 min in MeOH with 1% acetic acid 2.56 μg/g - C18 column (5 μm, 150 × 4.6 mm); column temperature, 40°C; flow rate, 1.00 mL/min; injection volume, 20 μL; wavelength, 190–600 nm; solvent system: gradients elution of 1% acetic acid in water and acetonitrile with run time of 12.30 min (11) Red delicious, Shandong Fuji and Xinjiang Fuji (peel and pulp) Classic HPLC Ultrasonic bath (30 min in MeOH with 1% acetic acid 3.58 μg/g - C18 column (5 μm,150 mm × 4.6 mm); column temperature, 30°C; flow rate, 0.80 mL/min; injection volume, 20 μL; wavelength 280 nm; solvent system: gradient elution 1% acetic acid in water, methanol and acetonitrile with run time of 70 min (11) Maluspumila Mill (fruit of different cultivars) HPLC–DAD Ultrasonic bath (60 min in EtOH: Water) 0.92–1257.30 μg/g 0.54–0.62 μg/g C18 column (5 mm, 4.6 mm); column temperature, 20°C; flow rate, 1.0 mL/min; wavelength, 280 nm; solvent system: gradient elution of 1.0% (v/v) acetic acid water solution and methanol with run time of 40 min (12) Malus domestica Barkh cv. Kanzi peel (UHPLC) (DAD/ESI-am-MS) Ultrasonic sonicator (60 min in MeOH:water - - C18 column (1.7 μm,150 mm × 3 mm); column temperature, 40°C; flow rate, 1.0 mL/min; injection volume, 5 μL; wavelength 280–320 nm; solvent system: gradient elution of water 0.1% formic acid and acetonitrile with 0.1% formic acid with runtime of 23 min (13) Eight apple cultivars viz. golden delicious, red delicious, McIntosh, empire, Ida red, northern spy, Mutsu, and Cortland HPLC–PDA/MS CP with methanol Peel (37.6–172.0 μg/g)*; pulp (8–24.6μg/g)* - C18 column (5 μm, 250 × 4.6 mm); injection volume, 10 μL/min; wavelength, 280–520 nm; solvent system: gradient elution of 6% acetic acid in 2 mM sodium acetate buffer (pH 2.55, v/v) and acetonitrile with run time of 70 min (14) Apple variety and part used Analytical method Extraction method/solvent PZ PT Chromatographic condition Reference Red delicious, Shandong Fuji and Xinjiang Fuji (peel and pulp) HPLC DAD Ultrasonic bath (30 min in MeOH with 1% acetic acid 2.56 μg/g - C18 column (5 μm, 150 × 4.6 mm); column temperature, 40°C; flow rate, 1.00 mL/min; injection volume, 20 μL; wavelength, 190–600 nm; solvent system: gradients elution of 1% acetic acid in water and acetonitrile with run time of 12.30 min (11) Red delicious, Shandong Fuji and Xinjiang Fuji (peel and pulp) Classic HPLC Ultrasonic bath (30 min in MeOH with 1% acetic acid 3.58 μg/g - C18 column (5 μm,150 mm × 4.6 mm); column temperature, 30°C; flow rate, 0.80 mL/min; injection volume, 20 μL; wavelength 280 nm; solvent system: gradient elution 1% acetic acid in water, methanol and acetonitrile with run time of 70 min (11) Maluspumila Mill (fruit of different cultivars) HPLC–DAD Ultrasonic bath (60 min in EtOH: Water) 0.92–1257.30 μg/g 0.54–0.62 μg/g C18 column (5 mm, 4.6 mm); column temperature, 20°C; flow rate, 1.0 mL/min; wavelength, 280 nm; solvent system: gradient elution of 1.0% (v/v) acetic acid water solution and methanol with run time of 40 min (12) Malus domestica Barkh cv. Kanzi peel (UHPLC) (DAD/ESI-am-MS) Ultrasonic sonicator (60 min in MeOH:water - - C18 column (1.7 μm,150 mm × 3 mm); column temperature, 40°C; flow rate, 1.0 mL/min; injection volume, 5 μL; wavelength 280–320 nm; solvent system: gradient elution of water 0.1% formic acid and acetonitrile with 0.1% formic acid with runtime of 23 min (13) Eight apple cultivars viz. golden delicious, red delicious, McIntosh, empire, Ida red, northern spy, Mutsu, and Cortland HPLC–PDA/MS CP with methanol Peel (37.6–172.0 μg/g)*; pulp (8–24.6μg/g)* - C18 column (5 μm, 250 × 4.6 mm); injection volume, 10 μL/min; wavelength, 280–520 nm; solvent system: gradient elution of 6% acetic acid in 2 mM sodium acetate buffer (pH 2.55, v/v) and acetonitrile with run time of 70 min (14) *Fresh weight. Open in new tab Table V Status of method validation in different apple cultivars Apple variety and part used Analytical method Extraction method/solvent PZ PT Chromatographic condition Reference Red delicious, Shandong Fuji and Xinjiang Fuji (peel and pulp) HPLC DAD Ultrasonic bath (30 min in MeOH with 1% acetic acid 2.56 μg/g - C18 column (5 μm, 150 × 4.6 mm); column temperature, 40°C; flow rate, 1.00 mL/min; injection volume, 20 μL; wavelength, 190–600 nm; solvent system: gradients elution of 1% acetic acid in water and acetonitrile with run time of 12.30 min (11) Red delicious, Shandong Fuji and Xinjiang Fuji (peel and pulp) Classic HPLC Ultrasonic bath (30 min in MeOH with 1% acetic acid 3.58 μg/g - C18 column (5 μm,150 mm × 4.6 mm); column temperature, 30°C; flow rate, 0.80 mL/min; injection volume, 20 μL; wavelength 280 nm; solvent system: gradient elution 1% acetic acid in water, methanol and acetonitrile with run time of 70 min (11) Maluspumila Mill (fruit of different cultivars) HPLC–DAD Ultrasonic bath (60 min in EtOH: Water) 0.92–1257.30 μg/g 0.54–0.62 μg/g C18 column (5 mm, 4.6 mm); column temperature, 20°C; flow rate, 1.0 mL/min; wavelength, 280 nm; solvent system: gradient elution of 1.0% (v/v) acetic acid water solution and methanol with run time of 40 min (12) Malus domestica Barkh cv. Kanzi peel (UHPLC) (DAD/ESI-am-MS) Ultrasonic sonicator (60 min in MeOH:water - - C18 column (1.7 μm,150 mm × 3 mm); column temperature, 40°C; flow rate, 1.0 mL/min; injection volume, 5 μL; wavelength 280–320 nm; solvent system: gradient elution of water 0.1% formic acid and acetonitrile with 0.1% formic acid with runtime of 23 min (13) Eight apple cultivars viz. golden delicious, red delicious, McIntosh, empire, Ida red, northern spy, Mutsu, and Cortland HPLC–PDA/MS CP with methanol Peel (37.6–172.0 μg/g)*; pulp (8–24.6μg/g)* - C18 column (5 μm, 250 × 4.6 mm); injection volume, 10 μL/min; wavelength, 280–520 nm; solvent system: gradient elution of 6% acetic acid in 2 mM sodium acetate buffer (pH 2.55, v/v) and acetonitrile with run time of 70 min (14) Apple variety and part used Analytical method Extraction method/solvent PZ PT Chromatographic condition Reference Red delicious, Shandong Fuji and Xinjiang Fuji (peel and pulp) HPLC DAD Ultrasonic bath (30 min in MeOH with 1% acetic acid 2.56 μg/g - C18 column (5 μm, 150 × 4.6 mm); column temperature, 40°C; flow rate, 1.00 mL/min; injection volume, 20 μL; wavelength, 190–600 nm; solvent system: gradients elution of 1% acetic acid in water and acetonitrile with run time of 12.30 min (11) Red delicious, Shandong Fuji and Xinjiang Fuji (peel and pulp) Classic HPLC Ultrasonic bath (30 min in MeOH with 1% acetic acid 3.58 μg/g - C18 column (5 μm,150 mm × 4.6 mm); column temperature, 30°C; flow rate, 0.80 mL/min; injection volume, 20 μL; wavelength 280 nm; solvent system: gradient elution 1% acetic acid in water, methanol and acetonitrile with run time of 70 min (11) Maluspumila Mill (fruit of different cultivars) HPLC–DAD Ultrasonic bath (60 min in EtOH: Water) 0.92–1257.30 μg/g 0.54–0.62 μg/g C18 column (5 mm, 4.6 mm); column temperature, 20°C; flow rate, 1.0 mL/min; wavelength, 280 nm; solvent system: gradient elution of 1.0% (v/v) acetic acid water solution and methanol with run time of 40 min (12) Malus domestica Barkh cv. Kanzi peel (UHPLC) (DAD/ESI-am-MS) Ultrasonic sonicator (60 min in MeOH:water - - C18 column (1.7 μm,150 mm × 3 mm); column temperature, 40°C; flow rate, 1.0 mL/min; injection volume, 5 μL; wavelength 280–320 nm; solvent system: gradient elution of water 0.1% formic acid and acetonitrile with 0.1% formic acid with runtime of 23 min (13) Eight apple cultivars viz. golden delicious, red delicious, McIntosh, empire, Ida red, northern spy, Mutsu, and Cortland HPLC–PDA/MS CP with methanol Peel (37.6–172.0 μg/g)*; pulp (8–24.6μg/g)* - C18 column (5 μm, 250 × 4.6 mm); injection volume, 10 μL/min; wavelength, 280–520 nm; solvent system: gradient elution of 6% acetic acid in 2 mM sodium acetate buffer (pH 2.55, v/v) and acetonitrile with run time of 70 min (14) *Fresh weight. Open in new tab Optimization of chromatographic separation Chromatographic conditions were optimized for separation and quantification of targeted compounds, i.e., PZ and PT, without any sample matrix interference due to co-elution of neighboring components. For optimum separation, various parameters viz. columns, the organic component of the mobile phase and compositions were attempted. A satisfactory separation of PZ and PT without any interference of co-elution of any peak from sample matrix was obtained. The optimum separation was achieved with a reverse phase column and acetonitrile-water (trifluoroacetic acid) gradient elution (as mentioned in the experimental section). The acidic composition of mobile phase (solvent A) was found to be essential for improving the sharpness of targeted DHCs. For simultaneous quantitation, chromatogram was set at 280 nm. The specificity of the determination has been ensured by retention matching, spiking the standard UV–Vis spectral matching and peak purity. Representative chromatograms of pure marker compounds and M. sikkimensis extract are given (Figure 1). Quantitative evaluation of PZ and PT in M. sikkimensis The developed method was applied to quantify the PZ and PT in the different parts of wild apple (M. sikkimensis) collected from the north east state of India. Samples were prepared using the UAE of plant material for quantitative analysis. Results showed that the PZ content in the leaves and the bark was highest (12–13 mg/100 mg), about 90-fold higher than fruits. Further, PT only quantified in the leaves (0.57 mg/100 mg). The comparative data on PZ and PT content in various wild apple species/cultivar from China, Croatia, France, Latvia, Russia, and Tibet are presented in Table IV (28–34). By compiled reports, it could be summarized that PZ content of the leaves of Indian wild apple is comparable to the other wild apple species of Aba Tibetan, China. Before the present study, the PT content was reported in the crabapples of Russia. The variation in the crabapple species from different location is due to G × E interactions. The fitness of the method Previously reported chromatographic methods (11–14) deal with the PZ determination in different wild apple cultivar but not Indian wildapple species, i.e., M. sikkimensis (Table V), suggesting the reverse phase LC with gradient elution with acetonitrile as a universal choice. However, the reported chromatographic conditions could not apply as such due to longer runtime, a high concentration of acid in the mobile phase, lack of validation data and uncertainty in measurement. Therefore, chromatographic condition was optimized, and the method was validated following International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The fitness of the developed method was determined by satisfying the requirement of traceability, reliability criteria of analysis and generating several uncertainties measurement data (Table III). The uncertainty of bias at method validation level was taken into account for global uncertainty measurement. While expanded uncertainty was calculated by global uncertainty multiplied by a coverage factor k = 2 to define the results of unknown true value with a confidence level of 95%. The expanded uncertainties of PZ and PT compounds in M. sikkimensis leaves are 1.82% and 2.02%, respectively, which confirms that at a confidence level of 95%, the unknown true value is within ±2% range of the measured value. Green aspects of the method Nowadays, a strong opinion is emerging to develop a generic LC method to fit for the purpose, which not only demonstrates the analytical performance but also addresses the environmental concerns using green chemistry approaches (35–37). In comparison to the previous methods (Table V), the current validated method completes one analytical cycle in a very short time for the simultaneous determination of PZ and PT in crabapple without any interference. The present analytical procedure is environmentally benign and considered as green procedure due to minimal organic solvent usage, less sample time and energy efficiency with no pre-concentration step. The acoustic cavitation ruptures the plant cell, thus enhancing the extraction efficiency in short run time, i.e., 20 min without any negative influence on the environment as compared to other classical procedure (11). Conclusion The ethnobotanical study on M. sikkimensis reveals that the therapeutic potential of the plant needs to be explored as the plant contains some biologically crucial phytomolecules. The protective action crude extract of M. sikkimensis leaves was mainly due to the presence of PZ as demonstrated in pilot in vitro experimentation (data not shown). Therefore, propagation and cultivation of M. sikkimensis can be utilized as a rich source of dietary DHC, which has a beneficial action on various health ailments due to their antioxidant and anti-inflammatory properties. It is the first study that describes the isolation and characterization of PZ and PT as well as the quality assurance of Indian wild apple, i.e., M. sikkimensis. The study also demonstrated that the PZ content is highest in the Indian crabapple leaves. Comparative analysis revealed that the PZ content in M. sikkimensis leaves were even higher than other wild species of Russia, France and Latvia even North Central China origin. The crabapples of Aba Tibetan region of China have comparable PZ content to Indian species. Developed HPLC–PDA method is selective, sensitive and reliable for simultaneous quantification of two DHCs viz. PZ and PT in Indian crabapple. For the first time, global and expanded uncertainty measurements are reported to meet the requirement of traceability and reliability criteria of analysis of DHCs in apples. Acknowledgments This research is supported by Department of Science and Technology, New Delhi, through fellowship [DST-WoS(A)-SR/WOSA/C131/2012 to S.D.]. The authors are grateful to the Director, CSIR-CIMAP for providing the necessary facilities and infrastructure (MLP-Quality) for carrying out the work. References 1. 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TI - Quantitation of dietary dihydrochalcones in Indian crabapple (Malus sikkimensis) using validated high-performance liquid chromatography
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmz040
DA - 2019-08-16
UR - https://www.deepdyve.com/lp/oxford-university-press/quantitation-of-dietary-dihydrochalcones-in-indian-crabapple-malus-FstlaV5ygE
SP - 679
VL - 57
IS - 8
DP - DeepDyve
ER -