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Identification and quantification of secondary metabolites of Pterocarpus marsupium by LC–MS techniques and its in-vitro lipid lowering activity
Industrial Crops & Products 127 (2019) 26–35
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Identification and quantification of secondary metabolites of Pterocarpus marsupium by LC–MS techniques and its in-vitro lipid lowering activity
T
Pratibha Singha, Vikas Bajpaia, Abhishek Guptab, Anil N. Gaikwadb, Rakesh Mauryac, ⁎ Brijesh Kumara, a
Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow 226001, India Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India c Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India b
ARTICLE INFO
ABSTRACT
Keywords: HPLC-ESI-QTOF-MS/MS Lipid accumulation Phenolic compounds Pterocarpus marsupium Quantitation UPLC-ESI-QqQLIT-MS/MS 3T3-L1
Pterocarpus marsupium (Vijaysar) is a deciduous tree, considered to have high insulinogenic properties. Since ancient times Ayurveda practitioners believed that heartwood of vijaysar significantly reduced blood sugar levels in diabetes. Marsupsin, pterosupin and pterostilbene are the three important phenolic constituents of the heartwood of this plant reported to significantly lower the blood glucose level. For the dereplication of bioactive phytochemicals present in heartwood of Pterocarpus marsupium a method was developed to identify and elucidate the main fragmentation pathways of phytoconstituents in positive electrospray ionization mode using HPLC-ESIQTOF-MS/MS. Further, an UPLC-ESI-QqQLIT-MS/MS method was developed to determine the contents of bioactive compounds, namely 2,6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside, pterocarposide, epicatechin, vanillic acid, quercetin, formononetin and naringenin in heartwood of this plant. Twenty seven compounds were tentatively identified based on their retention time, accurate mass and MS/MS patterns. Invitro assessment of compounds in 3T3-L1 adipocytes revealed significant attenuation of lipid accumulation in extract from Pterocarpus marsupium, hence it may have certain phytochemicals which are responsible for reducing obesity. The analytical method for quantitation was validated and successfully applied for the simultaneous determination of eleven bioactive compounds. In conclusion, rapid and accurate HPLC-ESI-QTOF-MS/MS and UPLCESI-QqQLIT-MS/MS methods developed in positive ionization mode were successfully applied for identification and quantitation of bioactive phytochemicals present in heartwood of Pterocarpus marsupium.
1. Introduction Pterocarpus is a genus of pan tropical trees in the Fabaceae family and it contains about 35 species. Pterocarpus marsupium (Roxb.), one of the most well-known members of this genus, is a large tree common in central, western and southern parts of India and Sri Lanka (Therrell et al., 2007). P. marsupium (PM), is one of the most valuable multipurpose and important commercial forest trees which yields excellent timber for the international trade market. It is also a valuable medicinal plant, used mainly in Ayurveda, for the treatment of diabetes (Chatterjee and Pakrashi, 1991; Satyavati et al., 1987, 1989). Due to the high commercial significance in timber industry and its use in the preparation of anti-diabetic herbal drugs, the plant has been overexploited (Anis, 2005; Kapai et al., 2010). P. marsupium bark is very effective in preventing cataract formation and reducing hyperglycemia in alloxanized diabetic rats (Therrell et al., 2007). and heartwood
⁎
(PMH) is reported potentially useful as hypoglycemic agents (Sheehan et al., 1983). The heartwood of P. marsupium is more commonly used in the treatment of diabetes by Ayurvedic physicians (Chatterjee and Pakrashi, 1991). Pterostilbene is one of the most active ingredients of PMH extract along with other significant components like epicatechin, marsupin and pterosupin reported for their anti-diabetic potential. The active hypoglycemic principal of the bark of this plant is (-)-epicatechin (Sheehan et al., 1983). Phenolic compounds namely marsupsin, pterosupin, pterostilbene and liquiritigenin have shown anti-hyperlipidemic and anti-hyperglycemic activities (Jahromi et al., 1993a, 1993b; Manickam et al., 1997). Laboratory studies showed that gum resin of Pterocarpus helps to regenerate the beta cells in the pancreas (Badkhane et al., 2010; Khan et al., 2014). P. marsupium is the only pure herb ever found to regenerate beta cells in the pancreas (Khan et al., 2014). Other uses of Pterocarpus as herbal supplement include antioxidant support and skin health (Modak et al., 2007). It is a very rich source of bioactive
Corresponding author. E-mail address: [email protected] (B. Kumar).
https://doi.org/10.1016/j.indcrop.2018.10.047 Received 23 June 2018; Received in revised form 26 September 2018; Accepted 15 October 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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Table 1 Compound dependent parameters (MRM) of analytes. S. No.
Compound. No.
Compound Name
Retention time (min)
Precursor (Q1) mass (Da)
Product (Q3) mass (Da)
Dwell time (ms)
DP (eV)
EP (eV)
CE (eV)
CXP (eV)
1. 2. 3. 4. 5. 6. 7. 8. 9.
8 4 13 14 21 18 2 23 1
2.15 2.37 2.41 2.43 3.09 3.65 4.29 6.06 6.20
433.2 431.1 289.0 167.0 415.0 401.0 243.2 301.0 271.2
313.0 311.2 203.0 108.0 295.0 281.0 135.0 151.0 150.0
200 200 200 200 200 200 200 200 200
−15.0 −21.0 −12.0 −55.0 −21.0 −89.0 −86.0 −107.0 −42.0
−6.0 −8.0 −9.0 −6.0 −5.0 −6.0 −8.0 −9.0 −8.0
−21.0 −29.0 −29.0 −22.0 −32.0 −26.0 −28.0 −31.0 −20.0
−7.0 −35.0 −6.0 −9.0 −16.0 −7.0 −9.0 −12.0 −17.0
10. 11.
25 24
Marsuposide Vijyoside Epicatechin Vanillic acid Pterocarposide Pteroside Pterosupol Quercetin 2,6 dihydroxyphenyl glucopyranoside Naringenin Formononetin
6.22 6.35
271.3 267.0
119.0 251.9
200 200
−58.0 −108
−9.0 −9.3
−38.0 −29.0
−6.0 −23.0
DP-declustering potential; EP-entrance potential; CE-collision energy; CXP-cell exit potential.
Fig. 1. Base peak chromatogram of Pterocarpus marsupium heart woods (PMH) collected from different geographical locations.
phenolic compounds such as pterosupin, pterostilbene, marsupin and epicatechin (Tiwari et al., 2015). P. marsupium also exhibits strong astringent property which is used in the treatment of inflammation and non-insulin dependent diabetes mellitus (Rizvi and Mishra, 2013). The composition and quantity of phytochemicals present in medicinal plants may vary due to different localities, (Bajpai et al., 2017a, b) growing conditions, (chandra et al., 2016), age (Pandey et al., 2014) and gender (Bajpai et al., 2012, 2016). Thus, the phytochemical investigation of raw plants/parts and their finished products is crucial in order to ensure efficacy, safety, authenticity and quality (Kumar et al., 2017). The rapid qualitative and quantitative analysis of known compounds and identification of unknown compounds in trace amounts of extracts and formulations has been effectively and successfully
completed by LC–MS (Singh et al., 2014; Tolonen and Uusitalo, 2004). There is no LC–MS report available for the identification of phytochemicals in P. marsupium except for identification of pterostilbene metabolite in mouse urine (Shao et al., 2010). Therefore, HPLC-ESIQTOF-MS/MS and UPLC-QqQLIT-MS/MS methods are required to study qualitative and quantitative chemical profiles of secondary metabolites of the heartwood of P. marsupium. Hence, a rapid, sensitive, and efficient HPLC-ESI-QTOF-MS/MS method was developed for the qualitative analysis followed by an UPLC-QqQLIT-MS/MS method in MRM (multiple reaction monitoring) mode for the simultaneous determination of selected bioactive phytochemicals. Phytochemical variation was also investigated to see the variations among the selected samples according to different locations. 27
28
4.5 4.6
5.9
5.10
5.11
6.7
8.5
8.9 9.4
9.5
9.7 11.7 10.5
10.7 11.0 12.4
14.7 17.8
11.3 22.2 24.4
12.3
12.8 12.9
13.7 24.0
2 3
4
5
6
7
8
9 10
11
12 17 13
14 15 19
23 24
16 25 26
18
20 21
22 27
C16H14O6 C16H16O3
C21H20O9 C21H20O9
C21H20O8
C15H12O3 C15H12O5 C15H12O4
C15H10O7 C16H12O4
C8H8O4 C15H10O4 C15H10O5
C15H10O5 C15H10O5 C15H14O6
C21H24O10
C14H18N2O2 C21H24O10
C21H22O10
C21H22O11
C21H20O10
C21H20O10
C21H20O10
C15H16O3 C21H20O11
C12H16O7
Molecular formula
303.0863 257.1172
417.1180 417.1180
403.1387
241.0859 273.0757 257.0808
303.0858 269.0808
169.0459 255.0652 271.0601
271.0601 271.0601 291.0853
437.1442
247.1441 437.1442
435.1286
451.1235
433.1135
433.1135
433.1135
245.1172 449.1078
273.0969
Calculated mass [M+H]+
matched with authentic standards and quantified also.
4.2
1
a
tR (min)
S. No.
303.0865 257.1170
417.1181 417.1181
403.1388
241.0858 273.0752 257.0809
303.0856 269.0809
169.0459 255.0653 271.0604
271.0606 271.0601 291.0856
437.1441
247.1442 437.1441
435.1281
451.1235
433.1138
433.1138
433.1138
245.1173 449.1078
273.0960
Measured mass [M+H]+ 255.0762 (1.8), 237.0723 (3), 153.0540 (51.3), 147.0432 (10.4), 135.0439 (100), 123.0442(43.7), 226.0994 (17.2), 137.0603 (100), 108.0875 (98) 431.0943 (29), 413.0849 (34), 329.0648 (100), 299.0549 (27) 415.1030 (25.7), 397.0922 (39.2), 379.0767 (23), 313.0700 (100) 415.1030 (25.7), 397.0922 (39.2), 379.0767 (23), 313.0700 (100) 415.1030 (25.7), 397.0922 (39.2), 379.0767 (23), 329.0700 (25.1), 313.0700 (100) 433.1094 (61), 405.1151 (100), 331.0818 (8.8), 313.0689 (93), 285.0736 (79) 417.1147 (18), 389.1196 (100), 297.0738 (42), 269.0787 (51) 189.0795 (100) 419.1326 (39), 317.1011 (100), 299.0913 (35), 281.0779 (33.8) 419.1326 (39), 317.1011 (100), 299.0913 (35), 281.0779 (33.8) 242.0642 (7), 225.0646 (8), 137.0211(100) 242.0642 (7), 225.0646 (8),169.0131 (8), 153.0182 (12), 273.0725 (8), 165.0527 (23), 139. 0383 (100), 123.0440 (45) 151.0395 (14), 125.0591 (13), 93.0336 (100) 137.0218 (100), 127.0679 (7), 121.0284 (8) 242.0642 (7), 225.0646 (8), 151.0401 (15), 137.0211(100), 121.0279 (82) 285.0718 (30), 257.0777 (100), 194.0215 (15) 253.0477 (89), 237.0513 (62), 226.0602 (69), 197.0588 (100), 137.0229 (23) 223.0743 (7), 137.0220 (100), 195.0791 (13) 153.0174 (100), 147.0437 (55), 119.0490 (7) 239.0701 (8), 211.0749 (9), 147.0439 (40), 239.0701 (8), 211.0749 (9), 119.0491 (7) 386.1366 (22), 368.1260 (58), 313.1071 (100), 295.0965 (16), 283.0965 (9) 399.0931 (12), 371.1180 (10), 353.0977 (100), 279.0763 (8) 399.0931 (21), 371.1180 (14), 353.0977 (100), 279.0763 (11) 285.0747 (49.5), 257.0800 (100), 229.0850 (17.7) 226.0988 (62), 137.0640 (92), 107.0497 (100)
MS/MS Fragments
Table 2 List of identified compounds of Pterocarpus marsupium heart wood (PMH) samples.
−0.64 −0.61
−0.26 −0.26
0.19
0.56 0.39 0.08
0.26 0.33
0.05 −0.57 0.50
−1.01 0 −0.17
0.21
−0.54 0.21
−0.16
−0.08
0.12
0.12
0.12
0.32 −0.06
0.21
Error (Δppm)
1-benzofurans stilbenoid
Flavonoid Flavonoid
Flavonoid
Flavonoid Flavonoid Flavonoid
Flavonoid Flavonoid
phenolic acid Flavonoid Flavonoid
xanthone isoflavone Flavonoid
indole alkaloids 1-benzofurans
Flavonoid
Flavonoid
Flavonoid
Flavonoid
Flavonoid
Flavonoid Flavonoid
Flavonoid
Class of compounds
Carpusin Pterostillbene (Devgun et al., 2009)
Retusin-8-O-arabinoside Pterocarposidea
Pterosidea
7-Hydroxyflavanone Naringenina Liquirtigenin (Jahromi et al., 1993a)
Quercetina Formononetina
Vanillic acida 7,4'-Dihydroxyflavone Resokaempferol
1,3,7-trihydroxyxanthone 5,6,7-trihydroxyisoflavone Epicatechina(Jahromi et al., 1993a)
Coatlin A
Hypaphorin Pterosupin (Jahromi et al., 1993a)
Marsuposidea(Mishra et al., 2013)
Macrocarposide
Aureusidin-6-rhamnoside
8-C-glucosyl-5-deoxykaempferol
Vijyosidea
2,6-dihydroxyphenyl glucopyranosidea (Mishra et al., 2013) Pterosupola(Mishra et al., 2013) Fisetin-8-C-glucoside
Identifications
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Fig. 2. Proposed fragmentation pathway of Liquirtigenin.
Fig. 3. Proposed fragmentation pathway of Pterosupin.
Fig. 4. CID MS/MS spectra of [M+H]+ ions of antidiabetic compounds reported from Pterocarpus marsupium heart wood (PMH).
2. Materials and methods
purchased from the Sigma Aldrich (St. Louis, MO, USA) were used in sample preparation and LC–MS studies. Ultra-pure water was produced by a Direct-Q system (Millipore, Milford, MA, USA). The standard reference (purity≥95%) samples of 2, 6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside and pterocarposide were isolated from the heart wood of P. marsupium and their structures
2.1. Reagents, chemicals and materials Ethanol used for extraction was AR grade and purchased from Merck (Darmstadt, Germany). LC–MS grade acetonitrile and methanol 29
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Fig. 5. Proposed fragmentation pathway of Pterostillbene.
were unambiguously characterized by direct comparison of their 1H and 13C NMR spectral data with reported literature (Maurya et al., 2004; Handa et al., 2000). The standard reference samples of epicatechin, vanillic acid, quercetin, formononetin, genistein and naringenin were purchased from Sigma Aldrich (St. Louis, MO, USA). One sample of heart woods of Pterocarpus marsupium was purchased from district Allahabad (PMAL), one from district Nanpara (PMNp), two samples from district Bhinga (PMB1&B2) and two samples from district Baharaich (PMBH1&BH2) of Uttar Pradesh State, India in March 2017.
autosampler (G1329 A), thermostated column compartment (G1316C) and diode array detector (G1315D). The outlet of the diode array detector was introduced into the electrospray (ESI) interface of the mass spectrometer through a flow splitter (1:1). The HPLC separation was carried out on an Agilent Poroshell 120 EC C18 column (150 mm × 4.6 mm, 2.7 μm) operated at 25 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile with 0.1% formic acid (B) with flow rate of 0.5 mL/min under the gradient program of 10 to 30% (B) for initial 12 min, 30–45% (B) from 12 to 20 min, 45–90% (B) from 20 to 28 min, 90–90% (B) from 28 to 32 min, 90–10% (B) from 32 to 35 min followed by initial 10% (B) in 35–38 min. The sample injection volume was 2 μL. Mass spectrometric analysis was performed on Agilent 6520 QTOF mass spectrometer in positive ESI mode. The resolving power of QTOF analyser was above 10,000 (FWHM) and spectra were acquired within the mass range m/z 50–2000. Nitrogen was used as nebulizing, drying and collision gas. Capillary temperature was set to 350 °C and nebulizer pressure to 40 psi and the drying gas flow rate was 10 L/min. Ion source parameters such as VCap, fragmentor, skimmer and octapole rf peak voltage were set to 3500 V, 150 V, 65 V and 750 V, respectively. Collision energy for various MS/MS experiments ranged from 10 to 40 eV for all the selected masses. Accurate mass measurements were carried out by the auto mass calibration method using external mass calibration solution (ESI-L Low Concentration Tuning Mix; Agilent calibration solution B). The chromatographic and mass spectrometric analysis and prediction of chemical formula including the exact mass calculation were performed by Mass Hunter software version B.04.00 build 4.0.479.0 (Agilent Technology).
2.2. Extraction and sample preparation Hundreded g powder of the dried heart wood of P. marsupium sample was suspended with 20 mL ethanol (100%) and sonicated for 30 min at 25 °C in an ultrasonic water bath (Bandelin SONOREX, Berlin), left for 24 h at room temperature. The extract was collected and filtered through filter paper (Whatman No. 1) and the residue was reextracted three times with fresh solvent following the same procedure. The combined filtrates of each sample was concentrated using a Buchi rotary evaporator (Flawil, Switzerland) under reduced pressure at 20–50 kPa at 40 °C yielding dark yellow-brown semi-solid extract of P. marsupium heartwood (PMH). Yield of samples PM (AL), PM (Np), PM (B1&B2), PM (BH1 and BH2) were 6.336 g, 6.408 g, 6.300 g, 6.225 g 6.378 g and 6.4103 g respectively. All the extracts were prepared in same way from same method. One mg/mL solution of the dried ethanolic extract was prepared in methanol and filtered through a 0.22 μm polyvinylidene difluoride (PVDF) membrane (MILLEX GV filter unit, Merck Millipore, Darmstadt, Germany) prior to LCeMS analysis.
2.4.2. UPLC-MS/MS analysis for quantitative study All sample analysis for quantitative study was accomplished in multiple reaction monitoring mode. AB Sciex Analyst 1.6.2 software (Applied Biosystems) was used for data collection. The UPLC-ESIQqQLIT-MS/MS analysis was performed on a Waters Acquity UPLC™ system (Waters, Milford, MA, USA) interfaced with hybrid linear ion trap triple quadrupole mass spectrometer (API 4000 QTRAP™ MS/MS system from AB Sciex, Concord, ON, Canada) equipped with electrospray (Turbo V™) ion source. The Waters Acquity UPLC™system was equipped with a binary solvent manager, sample manager, column oven and photodiode array detector (PDA). The compound dependent MRM parameter optimization was performed by direct infusion using Harvard apparatus (USA) and flow injection analysis (FIA).
2.3. Preparation of standard and stock solutions Stock solutions (1.0 mg/mL) of eleven reference standards namely 2, 6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside, pterocarposide, epicatechin, vanillic acid, quercetin, formononetin and naringenin were prepared separately in LC–MS grade methanol. Then, methanol stock solution containing the mixture of eleven analytes was prepared and diluted in appropriate concentration to yield a series of concentrations from 0.1 to 1000 ng/mL. All stock solutions were stored in the refrigerator at −20 °C until further use. 2.4. Instrumentation and analytical conditions 2.4.1. HPLC-MS/MS analysis for qualitative study Qualitative analysis was performed with an Agilent 6520 quadrupole time-of-flight (QTOF) mass spectrometer connected with Agilent 1200 HPLC system via Dual electrospray ionization (ESI) interface (Agilent technologies, USA). The Agilent 1200 HPLC system consisted of quaternary pump (G1311 A), online vacuum degasser (G1322 A),
2.4.2.1. Ultra-performance liquid chromatography conditions. Chromatographic separation of compounds was obtained with an ACQUITY UPLC BEH™ C18 column (100 mm × 2.1 mm, 1.7 μm) operated at 25 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B), at a flow rate of 0.3 mL/ min under a gradient program: 30
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Fig. 6. Extracted ion chromatograms (EIC’s) of analytes quantified from Pterocarpus marsupium heart wood (PMH) in MRM mode.
5-5% (B) initial to 1.0 min, 5–20% (B) from 1.0 min to 1.5 min, 20–24 (B) from 1.5 min to 4.5 min, maintained at 24% (B) from 4.5 min to 5.2 min, 24–90% (B) from 5.2 min to 6.0 min, maintained at 90% (B) from 6.0 min to 6.5 min back to initial condition from 6.5 min to 7.5 min and maintained at 5% (B) from 7.5 min to 8.0 min. The sample injection volume used was 2 μL.
spectra were recorded by scanning in the range of m/z 100–1000 at a cycle time of 9 s with a step size of 0.1 Da. Nitrogen was used as the nebulizer, heater, and curtain gas as well as the collision activated dissociation gas. The compound-dependent parameters such as declustering potential (DP), entrance potential (EP), collision energy (CE) and cell exit potential (CXP) were optimized for each compound by direct infusion of 20 ng/mL solutions of the each analyte using a Harvard 144 ‘22’ syringe pump (Harvard Apparatus, South Natick, MA, USA) in negative ionization mode. The optimized parameters are shown in Table 1. Quadrupole 1 and quadrupole 2 were maintained at unit resolution. Quantitative analysis was performed using multiple reactions monitoring (MRM) mode. The optimized source dependent
2.4.2.2. Mass spectrometric conditions. Electrospray ionization in negative mode (-ESI) was used for sample ionization. Precursor ion scan mode was used for screening and MRM acquisition mode for quantitation of eleven bioactive compounds in six PMH samples. All the analytes were detected in -ESI using precursor ion scan and mass 31
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RSD(%)
2.24 1.30 1.01 2.20 1.50 2.60 1.10 2.20 1.15 1.82 0.97
Mean
98.3 102.9 99.60 101.35 95.72 97.40 101.20 100.6 95.21 105.1 99.60
Stability %RSD(n = 5)
2.5. Validation of quantitative analytical method
1.20 0.89 1.25 1.40 2.25 1.22 2.32 0.77 0.98 1.02 0.96 1.80 3.92 3.01 8.36 8.53 1.10 2.48 1.15 5.30 2.10 1.86
1.22 1.71 0.86 1.63 1.25 0.76 0.77 2.13 1.17 0.83 0.94
Intraday(n = 6) LOQ(ng ml−1)
Interday(n = 6)
1.72 0.74 1.80 2.82 2.12 1.24 1.93 1.22 0.97 0.96 1.01
The developed MRM method was validated for linearity, limits of detection (LOD), limits of quantitation (LOQ), precisions, stability and recovery studies according to the International Conference on Harmonization (ICH, Q2R1) guidelines, 2005 (Guideline, I.H.T., 2005). The linearity of calibration curve was performed by the analyte peak area ratio versus the nominal concentration. The linearity calibration curves were made using at least five experiments of each reference compound and evaluated by squared linear correlation coefficient ( r2). The LOD and LOQ were defined as a signal-to-noise ratio (S/N) equal to 3 and 10, respectively. The intra- and inter-day precisions were determined by analyzing known concentrations of the eleven analytes in the nine replicates during a single day and by triplicating the experiments on five successive days. For the recovery test, three spike levels were set as 50%, 100% and 200% of each reference standard. For comparison an unspiked sample was concurrently prepared and analyzed. 2.6. 3T3-L1 cell culture and Oil Red O staining
0.9999 0.9999 0.9999 0.9999 1 0.9999 0.9999 1 1 0.9999 0.9999 10-1000 10-1000 20-1000 25-1000 10-1000 10-1000 10-1000 20-1000 10-1000 10-1000 10-500
Y = 815x + 147 Y = 362x + 142 Y = 388x + 117 Y = 122x + 102 Y = 630x + 538 Y = 4230x + 466 Y = 446x + 111 Y = 938x + 108 Y = 820x + 435 Y = 2520x + 530 Y = 3090x + 577
0.59 1.29 0.99 2.75 2.81 0.36 0.82 0.37 1.75 0.69 0.61
In order to evaluate the effect of extracts isolated form heartwood of P. marsupium of different years, Oil Red O staining was performed. 3T3L1 pre-adipocytes were cultured in DMEM media supplemented with 10% Fetal bovine serum as described earlier (Gupta et al., 2017a, b). 3T3-L1 pre-adipocytes were seeded in 24 well cell culture plates either in presence or absence of 50 μg/ml of each extracts. After two days, fully confluent cells were induced DMEM (Dulbecco's Modified Eagle's medium) media (+10% FBS) (Fetal Bovine Serum) supplemented with 0.5 mM IBMX, 250 nM Dexamethasone and 5 μg/ml insulin (MDI) (Gupta et al., 2018). After 72 h, media replaced with DMEM media (+10% FBS) supplemented with 5 μg/ml insulin. After 48 h, media was changed with DMEM media (+10% FBS) for next 48 h for differentiation into mature adipocytes. Now, cells were stained with 0.36% Oil Red O in 60% isopropanol for 30 min. Excess Oil Red O was washed with PBS and images were captured using Leica inverted microscope at 10X and 40 × . Thereafter, captured Oil Red O was extracted using isopropanol and absorbance was measured at 492 nm for bargraph. 2.7. Statistical analysis
Marsuposide Vijyoside Epicatechin Vanillic acid Pterocarposide Pteroside Pterosupol Quercetin 2,6-dihydroxyphenyl glucopyranoside Naringenin Formononetin
r2 Linear range(ng ml−1)
Regression equation
LOD(ng ml−1)
Precision (%RSD) Linearity Reference standards Compound. No.
Table 3 Validation parameters of the developed method for quantitation of eleven phytochemicals in Pterocarpus marsupium heart wood (PMH) samples.
parameters were as follows: the ion spray voltage (IS) was set to −4200 V; the turbo spray temperature (TEM), 450 °C; nebulizer gas (GS 1) at 50 psi; heater gas (GS 2) at 50 psi; the curtain gas (CUR) at 20 psi and the collision-activated dissociation gas (CAD) was set as medium with the interface heater on. High-purity nitrogen was used for all the processes. AB Sciex Analyst software version 1.6.2 was used to control the UPLC–QqQLIT MS/MS system and for data acquisition and processing.
For in vitro experiments, the experiment was performed in triplicate and statistical significance was compared with MDI + by using Oneway ANOVA (analysis of variance) analysis followed by Dunnet’s multiple comparison test using GraphPad Prism software. “*” signifies p < 0.05, “**” signifies p < 0.01, “***” signifies p < 0.001. 3. Results and discussion 3.1. Analysis of phytochemicals using HPLC-ESI-QTOF-MS/MS The ESI in positive ionization mode was found suitable to determine 27 peaks of flavonoids, alkaloid, phenolic acids, and other compounds detected as [M+H]+ ions, at different retention times (Fig. 1). The fragmentation patterns and other details of identified compounds are summarized in (Table 2).
8 4 13 14 21 18 2 23 1 25 24
Recovery(n = 3)
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Table 4 Contents (mg/g) of compounds (n = 3) in different samples of Pterocarpus marsupium heart wood (PMH). Samples Compound No. 8 4 13 14 21 18 2 23 1 25 24 b c d e f g
Reference standards (mg/g) Marsuposide Vijyoside Epicatechin Vanillic acid Pterocarposide Pteroside Pterosupol Quercetin 2,6-dihydroxyphenyl glucopyranoside Naringenin Formononetin
PMb(Bc1) 17.8 4.30 0.34 0.65 0.31 0.029 bdlg 0.65 0.079 0.025 0.77
PM (B2) 45.4 5.30 0.33 1.57 0.39 0.27 bdl 0.39 0.11 bdl 2.08
PM(BHd1) 72.8 7.40 0.34 0.49 1.02 1.12 0.66 0.39 bdl bdl 0.30
PM(BH2) 52.5 5.90 0.58 1.09 0.29 bdl 0.46 0.38 0.22 0.12 1.63
PM(Ale) 56.6 19.5 0.43 3.90 0.61 0.18 bdl 0.43 0.074 0.69 bdl
PM(Npf) 15.6 10.7 0.81 0.88 3.49 3.80 4.10 0.41 0.052 0.017 bdl
PM: Pterocarpus marsupium. B: District Bhinga. BH: District Baharaich. Al: District Allhabad. Np: District Nanpara. bdl: below the detection level.
Fig. 7. (A) Oil Red O images of 3T3-L1 adipocytes either without differentiation (MDI-ve), fully differentiated without extracts (MDI + ve) or differentiated in presence of 50 μg/ml of each extracts at 10X and 40 × . (B) Relative absorbance of captured Oil Red O stain measured at 492 nm (n = 3). Statistical significance was compared with MDI + by using one way anova analysis followed by Dunnet’s multiple comparison test. “*” signifies p < 0.05, “**” signifies p < 0.01, “***” signifies p < 0.001.
3.1.1. Identification of flavonoids Peak 3 was identified and characterized as fisetin-8-C-glucoside (m/ z 449.1078 [M+H]+; C21H20O11). It produced major fragment ions at m/z 431.0943, 329.0648, and 299.0549 in the MS/MS spectra due to consecutive losses of H2O, C4H8O4 and C8H6O3, respectively. Peak 4, 5 and 6 were tentatively identified as vijyoside, 8-C-glucosyl-5-deoxykaempferol and aureusidin-6-rhamnoside respectively. Peaks 4, 5 and 6 showed [M+H]+ ion at m/z 433.1138 (C21H20O10) produced the same fragment ions in the MS/MS scan, representing their isomeric structures. The fragment ions were at m/z 415.1132, 397.0924 and 379.0665 due to consecutive loss of H2O molecules and base peak ion at m/z 313.0602 due to RDA (Retro-Diels–Alder reaction) cleavage in peaks 4 and 5 while in peak 6 showed fragment ion at m/z 329.0700
due to RDA cleavage which further on fragmentation gave base peak ion at m/z 313.0602 due to loss of H2O molecules. Peak 4 was confirmed by comparing with the authentic standard vijyoside. Peaks 5 and 6 were tentatively identified as 8-C-glucosyl-5-deoxykaempferol and aureusidin-6-rhamnoside, respectively. Peak 7 showed [M+H]+ ion at m/z 451.1235 (C21H22O11) and was tentatively identified as macrocarposide. It gave fragments at m/z 433.1094 and 331.0818 due to loss of H2O and RDA cleavage, respectively. Further fragmentation of ion m/z 433.1094 and m/z 331.0818 produced fragment ions at m/z 405.1151 and m/z 313.0689 respectively due to losses of CO and H2O, respectively. Fragment ion at m/z 313.0689 yielded an ion at m/z 285.0736 due to CO loss. Peak 8 exhibited [M+H]+ ion at m/z 435.1281 (C21H20O10) and produced fragment ion at m/z 417.1147 due 33
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to loss of H2O. Further fragmentation of m/z 417.1147 gave ions at m/z 389.1196 and 297.0738 due to loss of CO and C4H8O4, respectively. Peak 8 was identified as marsuposide using authentic standard. Peak 13 was identified and characterized as epicatechin (m/z 291.0856 [M +H]+; C15H14O6) by comparing with authentic standard. It produced major fragment ions at m/z 273.0725 and 139.0383 due to loss of H2O and RDA cleavage, respectively (Pandey and Kumar, 2016). Peak 15 [M +H]+ ion at m/z 255.0653 (C15H10O4) yielded fragment ion at m/z 137.0218 due to RDA cleavage. Fragment ion at m/z 255.0653 generated ion at m/z 227.0679 due to loss of CO. Peak 15 was tentatively identified as 7,4′-Dihydroxyflavone on the basis of fragmentation pattern. Peaks 19, 23 and 24 showing similar fragmentation pathways were identified and characterized as resokaempferol (m/z 271.0604 [M +H]+; C15H10O5) quercetin m/z 303.0856 (C15H10O7) and formononetin m/z 269.0809 (C16H12O4) respectively. Peaks 23 and 24 were further confirmed by matching with their authentic standards. Peaks 16, 25 and 26 gave [M+H]+ ions at m/z 241.0858 (C15H12O3), 273.0752 (C15H12O5) and 257.0809 (C15H12O4), respectively and produced fragment ions at m/z 137.0220, 153.0174 and 147.0439, respectively due to RDA cleavage and were tentatively identified as 7hydroxyflavanone, naringenin and liquirtigenin (Fig. 2). Peak 25 naringenin was also confirmed by matching with authentic standard. Peak 18 identified and characterized as pteroside (m/z 403.1388 [M+H]+; C21H20O8) was confirmed by the authentic standard. Peaks 20 and 21 showing [M+H]+ ion at m/z 417.1181 were identified as retusin-8-Oarabinoside and pterocarposide, respectively and pterocarposide was confirmed with authentic standard. All the identified flavonoids except compound 3, 16, 18, 20, 24 and 25 discussed above were detected in samples PM (B1), PM (BH2), PM (AL), PM (Np) and PM (B2) respectively.
(Fig. 6).All the above compounds were detected in all the samples except compound 1 not detected in PM (BH2), 2 not detected in PM (B1) PM (BH2), 9 not detected in PM (MP), PM (AL) and 27 not detected in sample PM (B2). 3.2. Quantitative analysis of bioactive compounds in PM heartwood using UPLC-ESI-QqQLIT-MS/MS A suitable UPLC-ESI-QqQLIT-MS/MS condition was optimized for quantitation of selected bioactive compounds. Compound dependent parameters were optimized by direct infusion of each analyte to achieve the most specific and stable multiple reaction monitoring (MRM) transitions. MRM extracted ion chromatogram of analytes are shown in Fig. 3. 3.2.1. Linearity, precision and recovery results of the validated method The stock solution was diluted with LC–MS grade methanol for different working concentrations to prepare the calibration curves. The calibration curves were constructed using eleven different concentrations with a weight (1/x2) factor by least-squares linear regression analysis. For each reference compound the linearity of calibration curve was determined on the basis of five experiments (n = 5). The linearity was evaluated by the regression coefficient (r2) of the respective calibration curves which showed r2 within the test ranges (Table 3). The calibration curve showed good linearity with regression coefficient ( r2) of ≥0.9999 over the tested concentration range (10–1000 ng/mL) (Table 3). The limits of detections (LODs) and limits of quantitation’s (LOQs) were measured by signal to noise ratio (S/N) of 3 and 10, respectively. The LODs and LOQs were in the range of 0.36–2.81 ng/mL and 1.10–8.53 ng/mL, respectively. Relative standard deviation (RSD) values for precision were in the range of 0.77–2.32% for intraday assays, 0.77–2.13% for interday assays. RSD for stability was analyzed by replicating injections of the sample solution at 0, 2, 4, 8, 12 and 24 h. The RSD values for stability and recovery were found ≤ 2.82% and ≤ 2.60%, respectively. The recoveries of the analytes were 95.21–102.90% (n = 5).
3.1.2. Identification of isoflavonoids and phenolic acid Peaks 12 (1,3,7-trihydroxyxanthone) or genistein and 17 (5,6,7trihydroxyisoflavone) showed [M+H]+ ion at m/z 271.0601 (C15H10O5) and produced ions at m/z 137.0211 and 153.0182 respectively due to RDA cleavage. They also generated fragment ion at m/z 243.0642 due to loss of CO followed by ion at m/z 225.0646 due to loss of H2O. Peak 12 was further confirmed by comparison with its authentic standard. Peak 14 identified and characterized as vanillic acid (m/z 169.0459 [M+H]+; C8H8O4) was also confirmed by comparison with its authentic standard. All these isoflavonoids and phenolic acids were detected in all the samples.
3.3. Method application The established UPLC-ESI-QqQLIT-MS/MS analytical method was applied to determine eleven bioactive compounds, namely 2,6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside, pterocarposide, epicatechin, vanillic acid, quercetin, formononetin and naringenin in the ethanolic extracts of heart woods of P. marsupium is summarized in (Table 4). The antidiabetic compound marsuposide (Perera, 2016) determined in the range of 15.6–72.8 mg/g was the most abundant constituent in all the analysed samples. The highest content of marsuposide was observed in sample PM (BH1) (72.8 mg/g). Content of vijyoside detected in range 7.40–19.50 mg/g was the second most abundant constituent of PM (BH1). All the quantified compounds were in high abundance in samples PM (BH1), PM (AL) and PM (BH2) respectively among selected samples. The total content of all selected bioactive compounds varied significantly in samples collected from different locations.
3.1.3. Identification of other compounds Peak 1 was identified and characterized as 2,6-dihydroxyphenyl glucopyranoside and showed [M+H]+ ion at m/z 273.0960 (C12H16O7) and produced ions at m/z 255.0762 and 153.0182 due to loss of H2O and RDA cleavage, respectively. It was also confirmed by its authentic standards. Peak 2 identified and tentatively characterized as pterosupol, showed [M+H]+ ion at m/z 245.1173 (C15H16O3) and produced ions at m/z 227.0994, 137.0603 and 108.0875 due to loss of H2O, C7H8O and C8H9O2, respectively. Peak 9 showed [M+H]+ ion at m/z 247.1442 (C14H18N2O2) and produced ion at m/z 189.0795 due to loss of N(CH3)3 and tentatively identified as hypaphorin. Peaks 10 and 11 showed the same [M+H]+ ion at m/z 437.1441 (C21H24O10) and produced ions at m/z 317.1011 due to RDA cleavage. They also generated fragment ions at m/z 419.1326 due to loss of H2O and 299.0913 due to loss of C8H9O2, and tentatively identified as pterosupin (Fig. 5) and coatlin A, respectively. Peak 22 gave [M+H]+ ion at m/z 303.0865 (C16H14O6) and produced the fragment ion at m/z 285.0747 due to loss of H2O. Fragment ion at m/z 285.0747 generated ion m/z 254.0800 due to loss of OCH3. Peak 22 was tentatively identified as carpusin. Peak 27 [M+H]+ ion at m/z 257.1170 (C16H16O3) produced the fragment ions at m/z 226.0988 and 137.0640 due to loss of and C8H8O, respectively. The fragment ion at m/z 137.0640 lost OCH3 to produce the ion at m/z 106.0497 (Fig. 4). It was tentatively identified as pterostilbene
3.4. P. marsupium extracts possess in-vitro lipid lowering activity Excess fat accumulation has well been correlated with obesity (Smith, 2015), accompanied with elevated levels of non-esterified fatty acids, glycerol and pro-inflammatory markers marking diabetic phenotype. In addition, weight gain and increased body mass raises incidence of Type 1 and Type 2 diabetes (Al-Goblan et al., 2014). Reporetd literature suggest that reduction in lipid load has promising therapeutical benefitis, ameliorating obesity and related disordres (Murphy and Yvan-Charvet, 2015). In light of the earlier studies, we assessed the potential of isolated extracts of P. marsupium for their lipid 34
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lowering activity. Assessment of lipid accumulation in 3T3-L1 adipocytes revealed significant attenuation of lipid content in the cells treated with extracts isolated form PM (B2), PM (BH1) as well as PM (BH2). However, extract from PM (B1) was found to be toxic to cells. The results suggest that the extract from PM (BH2) have certain constituents that are capable of reducing obesity and hence could prove to be beneficial in diabetes. (Fig. 7A and B)
Indian herbs and herbal drugs used for the treatment of diabetes. J. Clin. Biochem. Nutr. 40 (3), 163–173. Tiwari, M., Sharma, M., Khare, H.N., 2015. Chemical constituents and medicinal uses of Pterocarpus marsupium Roxb. Flora and Fauna 21 (1), 55–59. Rizvi, S.I., Mishra, N., 2013. Traditional Indian medicines used for the management of diabetes mellitus. J. Diabetes Res. 2013, 11. Bajpai, V., Kumar, S., Singh, A., Bano, N., Pathak, M., Kumar, N., Misra-Bhattacharya, S., Kumar, B., 2017a. Metabolic fingerprinting of dioecious Tinospora cordifolia (Thunb) Miers stem using DART TOF MS and differential pharmacological efficacy of its male and female plants. Ind. Crops Prod. 101, 46–53. Bajpai, V., Kumar, S., Singh, A., Singh, J., Negi, M.P.S., Bag, S.K., Kumar, N., Konwar, R., Kumar, B., 2017b. Chemometric based identification and validation of specific chemical markers for geographical, seasonal and gender variations in Tinospora cordifolia stem using HPLC ESI QTOF MS analysis. Phytochem. Anal. 28 (4), 277–288. Chandra, P., Kannujia, R., Pandey, R., Shukla, S., Bahadur, L., Pal, M., Kumar, B., 2016. Rapid quantitative analysis of multi-components in Andrographis paniculata using UPLC-QqQLIT-MS/MS: application to soil sodicity and organic farming. Ind. Crops Prod. 83, 423–430. Pandey, R., Chandra, P., Srivastva, M., Arya, K.R., Shukla, P.K., Kumar, B., 2014. A rapid analytical method for characterization and simultaneous quantitative determination of phytoconstituents in Piper betle landraces using UPLC-ESI-MS/MS. Anal. Methods 6 (18), 7349–7360. Bajpai, V., Pandey, R., Negi, M.P.S., Bindu, K.H., Kumar, N., Kumar, B., 2012. Characteristic differences in metabolite profile in male and female plants of dioecious Piper betle L. J. Biosci. 37 (1), 1061–1066. Bajpai, V., Singh, A., Chandra, P., Negi, M.P.S., Kumar, N., Kumar, B., 2016. Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT‐MS/MS. Phytochem. Anal. 27 (2), 92–99. Kumar, S., Singh, A., Kumar, B., 2017. Identification and characterization of phenolics and terpenoids from ethanolic extracts of Phyllanthus species by HPLC-ESI-QTOF-MS/ MS. J. Pharm. Anal. 7 (4), 214–222. Singh, A., Bajpai, V., Srivastava, M., Arya, K.R., Kumar, B., 2014. Rapid profiling and structural characterization of bioactive compounds and their distribution in different parts of Berberis petiolaris Wall. Ex G. Don applying hyphenated mass spectrometric techniques. Rapid Commun. Mass Spectrom. 28 (19), 2089–2100. Tolonen, A., Uusitalo, J., 2004. Fast screening method for the analysis of total flavonoid content in plants and foodstuffs by high performance liquid chromatography/electrospray ionization time of flight mass spectrometry with polarity switching. Rapid Commun. Mass Spectrom. 18 (24), 3113–3122. Maurya, R., Singh, R., Deepak, M., Handa, S.S., Yadav, P.P., Mishra, P.K., 2004. Constituents of Pterocarpus marsupium: an ayurvedic crude drug. Phytochemistry 65 (7), 915. Handa, S.S., Singh, R., Maurya, R., Satti, N.K., Suri, K.A., Suri, O.P., 2000. Pterocarposide, an isoaurone C-glucoside from Pterocarpus marsupium. Tetrahedron Lett. 41 (10), 1579–1581. Shao, X., Chen, X., Badmaev, V., Ho, C.T., Sang, S., 2010. Structural identification of mouse urinary metabolites of pterostilbene using liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 24 (12), 1770–1778. Guideline, I.H.T, 2005. Validation of Analytical Procedures: Text and Methodology. Q2 (R1). pp. 1. Gupta, A., Beg, M., Kumar, D., Shankar, K., Varshney, S., Rajan, S., Srivastava, A., Singh, K., Sonkar, S., Mahdi, A.A., Dikshit, M., 2017a. Chronic hyper-leptinemia induces insulin signaling disruption in adipocytes: implications of NOS2. Free Radic. Biol. Med. 112, 93–108. Gupta, A., Singh, V.K., Kumar, D., Yadav, P., Kumar, S., Beg, M., Shankar, K., Varshney, S., Rajan, S., Srivastava, A., Choudhary, R., 2017b. Curcumin-3, 4-Dichloro Phenyl Pyrazole (CDPP) overcomes curcumin’s low bioavailability, inhibits adipogenesis and ameliorates dyslipidemia by activating reverse cholesterol transport. Metab.-Clin. Exp. 73, 109–124. Gupta, A., Kumar, A., Kumar, D., Singh, R., Shankar, K., Varshney, S., Rajan, S., Srivastava, A., Gupta, S., Narender, T., Gaikwad, A.N., 2018. Ecliptal, a promising natural lead isolated from Eclipta alba modulates adipocyte function and ameliorates metabolic syndrome. Toxicol. Appl. Pharmacol. 338, 134–147. Pandey, R., Kumar, B., 2016. HPLC–QTOF–MS/MS-based rapid screening of phenolics and triterpenic acids in leaf extracts of Ocimum species and their interspecies variation. J. Liq. Chromatogr. Relat. Technol. 39 (4), 225–238. Perera, H.K.I., 2016. Antidiabetic effects of Pterocarpus marsupium (Gammalu). Eur. J. Med. Plants 13 (4). Smith, U., 2015. Abdominal obesity: a marker of ectopic fat accumulation. J. Clin. Invest. 125 (5), 1790–1792. Al-Goblan, A.S., Al-Alfi, M.A., Khan, M.Z., 2014. Mechanism linking diabetes mellitus and obesity. Diabetes Metab. Syndr. Obes. 7, 587. Murphy, A.J., Yvan-Charvet, L., 2015. Adipose modulation of ABCG1 uncovers an intimate link between sphingomyelin and triglyceride storage. Diabetes 64 (3), 689–692. Mishra, A., Srivastava, R., Srivastava, S.P., Gautam, S., Tamrakar, A.K., Maurya, R., Srivastava, A.K., 2013. Antidiabetic Activity of Heart Wood of Pterocarpus marsupium Roxb. And Analysis of Phytoconstituents. Jahromi, M.F., Ray, A.B., Chansouria, J.P.N., 1993b. Antihyperlipidemic effect of flavonoids from Pterocarpus marsupium. J. Nat. Prod. 56 (7), 989–994. Devgun, M., Nanda, A., Ansari, S.H., 2009. Pterocarpus marsupium Roxb.–a comprehensive review. Pharmacogn. Rev. 3 (6), 359.
4. Conclusions In conclusion, a rapid and sensitive HPLC-ESI-QTOF-MS/MS in positive ionization mode and UPLC-ESI-QqQLIT-MS/MS method in negative ionization mode were developed and successfully applied for identification, characterization and quantitation of bioactive phytochemicals present in heart woods of P. marsupium. Twenty seven compounds including flavonoids, alkaloid and phenolic acid, were identified in different samples. An UPLC-ESI-QqQLIT-MS/MS analytical method was applied to determine contents of eleven bioactive compounds, namely 2,6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside, pterocarposide, epicatechin, vanillic acid, quercetin, formononetin and naringenin in the ethanolic extracts samples. The total content of eleven selected bioactive compounds was found high in the sample collected from Bhinga district (U.P.). Nevertheless, these methods are simple, rapid and sensitive, provide useful qualitative and quantitative fingerprint, to be used for grading bioactive compounds commercially. Plant samples were screened for their potential to suppresses lipid accumulation in adipocytes in-vitro, in which results were widely varied and could contribute to their application to different therapeutic areas. Acknowledgements Grateful acknowledgement is made to SAIF, CSIR-CDRI, Lucknow, where the mass spectrometry studies were carried out. Pratibha Singh and Vikas Bajpai are thankful to Department of Science and Technology (DST), New Delhi for grant GAP0003. References Therrell, M.D., Stahle, D.W., Mukelabai, M.M., Shugart, H.H., 2007. Age, and radial growth dynamics of Pterocarpus angolensis in southern Africa. For. Ecol. Manage. 244 (1-3), 24–31. Chatterjee, A., Pakrashi, S.C., 1991. The treatise on Indian medicinal plants: vol. 1. New Delhi: Publications and Information Directorate, CSIR 172p.-illus., col. illus. ISBN 8172360118 En Icones. Includes authentic Sanskrit slokas in both Devnagri and Roman scripts. Plant Rec. Geogr. 6. Satyavati, G.V., Gupta, A.K., Tandon, N., 1987. Med. Plants India 27, 574–575. Satyavati, G.V., Neeraj, T., Madhu, S., 1989. Indigenous plant drugs for diabetes mellitus. Indian J. Diabetes Dev. Countries. 1–35. Anis, M., 2005. In vitro plantlet regeneration of Pterocarpus marsupium Roxb., an endangered leguminous tree. Curr. Sci. 88 (6), 86. Kapai, V.Y., Kapoor, P., Rao, I.U., 2010. In Vitro propagation for conservation of rare and threatened plants of India–a Review. Int. J. Biol. Technol. 1 (2), 1–14. Sheehan, E.W., Zemaitis, M.A., Slatkin, D.J., Schiff Jr, P.L., 1983. A constituent of Pterocarpus marsupium,(-)-epicatechin, as a potential antidiabetic agent. J. Nat. Prod. 46 (2), 232–234. Jahromi, M.F., Ray, A.B., Chansouria, J.P.N., 1993a. Antihyperlipidemic effect of flavonoids from Pterocarpus marsupium. J. Nat. Prod. 56 (7), 989–994. Manickam, M., Ramanathan, M., Farboodniay Jahromi, M.A., Chansouria, J.P.N., Ray, A.B., 1997. Antihyperglycemic activity of phenolics from Pterocarpus marsupium. J. Nat. Prod. 60 (6), 609–610. Badkhane, Y., Yadav, A.S., Sharma, A.K., Raghuwanshi, D.K., Uikey, S.K., Mir, F.A., Lone, S.A., Murab, T., 2010. Pterocarpus marsupium Roxb-Biological activities and medicinal properties. Int. J. Adv. Pharm. Sci. 1 (4). Khan, M.Y., Aziz, I., Bihari, B., Kumar, H., Roy, M., Kumar, V., 2014. A reviewPhytomedicines used in treatment of diabetes. Diabetes 101, 126. Modak, M., Dixit, P., Londhe, J., Ghaskadbi, S., Devasagayam, T.P.A., 2007. Recent advances in Indian herbal drug research guest editor: thomas Paul Asir Devasagayam
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Identification and quantification of secondary metabolites of Pterocarpus marsupium by LC–MS techniques and its in-vitro lipid lowering activity
T
Pratibha Singha, Vikas Bajpaia, Abhishek Guptab, Anil N. Gaikwadb, Rakesh Mauryac, ⁎ Brijesh Kumara, a
Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow 226001, India Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India c Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India b
ARTICLE INFO
ABSTRACT
Keywords: HPLC-ESI-QTOF-MS/MS Lipid accumulation Phenolic compounds Pterocarpus marsupium Quantitation UPLC-ESI-QqQLIT-MS/MS 3T3-L1
Pterocarpus marsupium (Vijaysar) is a deciduous tree, considered to have high insulinogenic properties. Since ancient times Ayurveda practitioners believed that heartwood of vijaysar significantly reduced blood sugar levels in diabetes. Marsupsin, pterosupin and pterostilbene are the three important phenolic constituents of the heartwood of this plant reported to significantly lower the blood glucose level. For the dereplication of bioactive phytochemicals present in heartwood of Pterocarpus marsupium a method was developed to identify and elucidate the main fragmentation pathways of phytoconstituents in positive electrospray ionization mode using HPLC-ESIQTOF-MS/MS. Further, an UPLC-ESI-QqQLIT-MS/MS method was developed to determine the contents of bioactive compounds, namely 2,6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside, pterocarposide, epicatechin, vanillic acid, quercetin, formononetin and naringenin in heartwood of this plant. Twenty seven compounds were tentatively identified based on their retention time, accurate mass and MS/MS patterns. Invitro assessment of compounds in 3T3-L1 adipocytes revealed significant attenuation of lipid accumulation in extract from Pterocarpus marsupium, hence it may have certain phytochemicals which are responsible for reducing obesity. The analytical method for quantitation was validated and successfully applied for the simultaneous determination of eleven bioactive compounds. In conclusion, rapid and accurate HPLC-ESI-QTOF-MS/MS and UPLCESI-QqQLIT-MS/MS methods developed in positive ionization mode were successfully applied for identification and quantitation of bioactive phytochemicals present in heartwood of Pterocarpus marsupium.
1. Introduction Pterocarpus is a genus of pan tropical trees in the Fabaceae family and it contains about 35 species. Pterocarpus marsupium (Roxb.), one of the most well-known members of this genus, is a large tree common in central, western and southern parts of India and Sri Lanka (Therrell et al., 2007). P. marsupium (PM), is one of the most valuable multipurpose and important commercial forest trees which yields excellent timber for the international trade market. It is also a valuable medicinal plant, used mainly in Ayurveda, for the treatment of diabetes (Chatterjee and Pakrashi, 1991; Satyavati et al., 1987, 1989). Due to the high commercial significance in timber industry and its use in the preparation of anti-diabetic herbal drugs, the plant has been overexploited (Anis, 2005; Kapai et al., 2010). P. marsupium bark is very effective in preventing cataract formation and reducing hyperglycemia in alloxanized diabetic rats (Therrell et al., 2007). and heartwood
⁎
(PMH) is reported potentially useful as hypoglycemic agents (Sheehan et al., 1983). The heartwood of P. marsupium is more commonly used in the treatment of diabetes by Ayurvedic physicians (Chatterjee and Pakrashi, 1991). Pterostilbene is one of the most active ingredients of PMH extract along with other significant components like epicatechin, marsupin and pterosupin reported for their anti-diabetic potential. The active hypoglycemic principal of the bark of this plant is (-)-epicatechin (Sheehan et al., 1983). Phenolic compounds namely marsupsin, pterosupin, pterostilbene and liquiritigenin have shown anti-hyperlipidemic and anti-hyperglycemic activities (Jahromi et al., 1993a, 1993b; Manickam et al., 1997). Laboratory studies showed that gum resin of Pterocarpus helps to regenerate the beta cells in the pancreas (Badkhane et al., 2010; Khan et al., 2014). P. marsupium is the only pure herb ever found to regenerate beta cells in the pancreas (Khan et al., 2014). Other uses of Pterocarpus as herbal supplement include antioxidant support and skin health (Modak et al., 2007). It is a very rich source of bioactive
Corresponding author. E-mail address: [email protected] (B. Kumar).
https://doi.org/10.1016/j.indcrop.2018.10.047 Received 23 June 2018; Received in revised form 26 September 2018; Accepted 15 October 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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Table 1 Compound dependent parameters (MRM) of analytes. S. No.
Compound. No.
Compound Name
Retention time (min)
Precursor (Q1) mass (Da)
Product (Q3) mass (Da)
Dwell time (ms)
DP (eV)
EP (eV)
CE (eV)
CXP (eV)
1. 2. 3. 4. 5. 6. 7. 8. 9.
8 4 13 14 21 18 2 23 1
2.15 2.37 2.41 2.43 3.09 3.65 4.29 6.06 6.20
433.2 431.1 289.0 167.0 415.0 401.0 243.2 301.0 271.2
313.0 311.2 203.0 108.0 295.0 281.0 135.0 151.0 150.0
200 200 200 200 200 200 200 200 200
−15.0 −21.0 −12.0 −55.0 −21.0 −89.0 −86.0 −107.0 −42.0
−6.0 −8.0 −9.0 −6.0 −5.0 −6.0 −8.0 −9.0 −8.0
−21.0 −29.0 −29.0 −22.0 −32.0 −26.0 −28.0 −31.0 −20.0
−7.0 −35.0 −6.0 −9.0 −16.0 −7.0 −9.0 −12.0 −17.0
10. 11.
25 24
Marsuposide Vijyoside Epicatechin Vanillic acid Pterocarposide Pteroside Pterosupol Quercetin 2,6 dihydroxyphenyl glucopyranoside Naringenin Formononetin
6.22 6.35
271.3 267.0
119.0 251.9
200 200
−58.0 −108
−9.0 −9.3
−38.0 −29.0
−6.0 −23.0
DP-declustering potential; EP-entrance potential; CE-collision energy; CXP-cell exit potential.
Fig. 1. Base peak chromatogram of Pterocarpus marsupium heart woods (PMH) collected from different geographical locations.
phenolic compounds such as pterosupin, pterostilbene, marsupin and epicatechin (Tiwari et al., 2015). P. marsupium also exhibits strong astringent property which is used in the treatment of inflammation and non-insulin dependent diabetes mellitus (Rizvi and Mishra, 2013). The composition and quantity of phytochemicals present in medicinal plants may vary due to different localities, (Bajpai et al., 2017a, b) growing conditions, (chandra et al., 2016), age (Pandey et al., 2014) and gender (Bajpai et al., 2012, 2016). Thus, the phytochemical investigation of raw plants/parts and their finished products is crucial in order to ensure efficacy, safety, authenticity and quality (Kumar et al., 2017). The rapid qualitative and quantitative analysis of known compounds and identification of unknown compounds in trace amounts of extracts and formulations has been effectively and successfully
completed by LC–MS (Singh et al., 2014; Tolonen and Uusitalo, 2004). There is no LC–MS report available for the identification of phytochemicals in P. marsupium except for identification of pterostilbene metabolite in mouse urine (Shao et al., 2010). Therefore, HPLC-ESIQTOF-MS/MS and UPLC-QqQLIT-MS/MS methods are required to study qualitative and quantitative chemical profiles of secondary metabolites of the heartwood of P. marsupium. Hence, a rapid, sensitive, and efficient HPLC-ESI-QTOF-MS/MS method was developed for the qualitative analysis followed by an UPLC-QqQLIT-MS/MS method in MRM (multiple reaction monitoring) mode for the simultaneous determination of selected bioactive phytochemicals. Phytochemical variation was also investigated to see the variations among the selected samples according to different locations. 27
28
4.5 4.6
5.9
5.10
5.11
6.7
8.5
8.9 9.4
9.5
9.7 11.7 10.5
10.7 11.0 12.4
14.7 17.8
11.3 22.2 24.4
12.3
12.8 12.9
13.7 24.0
2 3
4
5
6
7
8
9 10
11
12 17 13
14 15 19
23 24
16 25 26
18
20 21
22 27
C16H14O6 C16H16O3
C21H20O9 C21H20O9
C21H20O8
C15H12O3 C15H12O5 C15H12O4
C15H10O7 C16H12O4
C8H8O4 C15H10O4 C15H10O5
C15H10O5 C15H10O5 C15H14O6
C21H24O10
C14H18N2O2 C21H24O10
C21H22O10
C21H22O11
C21H20O10
C21H20O10
C21H20O10
C15H16O3 C21H20O11
C12H16O7
Molecular formula
303.0863 257.1172
417.1180 417.1180
403.1387
241.0859 273.0757 257.0808
303.0858 269.0808
169.0459 255.0652 271.0601
271.0601 271.0601 291.0853
437.1442
247.1441 437.1442
435.1286
451.1235
433.1135
433.1135
433.1135
245.1172 449.1078
273.0969
Calculated mass [M+H]+
matched with authentic standards and quantified also.
4.2
1
a
tR (min)
S. No.
303.0865 257.1170
417.1181 417.1181
403.1388
241.0858 273.0752 257.0809
303.0856 269.0809
169.0459 255.0653 271.0604
271.0606 271.0601 291.0856
437.1441
247.1442 437.1441
435.1281
451.1235
433.1138
433.1138
433.1138
245.1173 449.1078
273.0960
Measured mass [M+H]+ 255.0762 (1.8), 237.0723 (3), 153.0540 (51.3), 147.0432 (10.4), 135.0439 (100), 123.0442(43.7), 226.0994 (17.2), 137.0603 (100), 108.0875 (98) 431.0943 (29), 413.0849 (34), 329.0648 (100), 299.0549 (27) 415.1030 (25.7), 397.0922 (39.2), 379.0767 (23), 313.0700 (100) 415.1030 (25.7), 397.0922 (39.2), 379.0767 (23), 313.0700 (100) 415.1030 (25.7), 397.0922 (39.2), 379.0767 (23), 329.0700 (25.1), 313.0700 (100) 433.1094 (61), 405.1151 (100), 331.0818 (8.8), 313.0689 (93), 285.0736 (79) 417.1147 (18), 389.1196 (100), 297.0738 (42), 269.0787 (51) 189.0795 (100) 419.1326 (39), 317.1011 (100), 299.0913 (35), 281.0779 (33.8) 419.1326 (39), 317.1011 (100), 299.0913 (35), 281.0779 (33.8) 242.0642 (7), 225.0646 (8), 137.0211(100) 242.0642 (7), 225.0646 (8),169.0131 (8), 153.0182 (12), 273.0725 (8), 165.0527 (23), 139. 0383 (100), 123.0440 (45) 151.0395 (14), 125.0591 (13), 93.0336 (100) 137.0218 (100), 127.0679 (7), 121.0284 (8) 242.0642 (7), 225.0646 (8), 151.0401 (15), 137.0211(100), 121.0279 (82) 285.0718 (30), 257.0777 (100), 194.0215 (15) 253.0477 (89), 237.0513 (62), 226.0602 (69), 197.0588 (100), 137.0229 (23) 223.0743 (7), 137.0220 (100), 195.0791 (13) 153.0174 (100), 147.0437 (55), 119.0490 (7) 239.0701 (8), 211.0749 (9), 147.0439 (40), 239.0701 (8), 211.0749 (9), 119.0491 (7) 386.1366 (22), 368.1260 (58), 313.1071 (100), 295.0965 (16), 283.0965 (9) 399.0931 (12), 371.1180 (10), 353.0977 (100), 279.0763 (8) 399.0931 (21), 371.1180 (14), 353.0977 (100), 279.0763 (11) 285.0747 (49.5), 257.0800 (100), 229.0850 (17.7) 226.0988 (62), 137.0640 (92), 107.0497 (100)
MS/MS Fragments
Table 2 List of identified compounds of Pterocarpus marsupium heart wood (PMH) samples.
−0.64 −0.61
−0.26 −0.26
0.19
0.56 0.39 0.08
0.26 0.33
0.05 −0.57 0.50
−1.01 0 −0.17
0.21
−0.54 0.21
−0.16
−0.08
0.12
0.12
0.12
0.32 −0.06
0.21
Error (Δppm)
1-benzofurans stilbenoid
Flavonoid Flavonoid
Flavonoid
Flavonoid Flavonoid Flavonoid
Flavonoid Flavonoid
phenolic acid Flavonoid Flavonoid
xanthone isoflavone Flavonoid
indole alkaloids 1-benzofurans
Flavonoid
Flavonoid
Flavonoid
Flavonoid
Flavonoid
Flavonoid Flavonoid
Flavonoid
Class of compounds
Carpusin Pterostillbene (Devgun et al., 2009)
Retusin-8-O-arabinoside Pterocarposidea
Pterosidea
7-Hydroxyflavanone Naringenina Liquirtigenin (Jahromi et al., 1993a)
Quercetina Formononetina
Vanillic acida 7,4'-Dihydroxyflavone Resokaempferol
1,3,7-trihydroxyxanthone 5,6,7-trihydroxyisoflavone Epicatechina(Jahromi et al., 1993a)
Coatlin A
Hypaphorin Pterosupin (Jahromi et al., 1993a)
Marsuposidea(Mishra et al., 2013)
Macrocarposide
Aureusidin-6-rhamnoside
8-C-glucosyl-5-deoxykaempferol
Vijyosidea
2,6-dihydroxyphenyl glucopyranosidea (Mishra et al., 2013) Pterosupola(Mishra et al., 2013) Fisetin-8-C-glucoside
Identifications
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Fig. 2. Proposed fragmentation pathway of Liquirtigenin.
Fig. 3. Proposed fragmentation pathway of Pterosupin.
Fig. 4. CID MS/MS spectra of [M+H]+ ions of antidiabetic compounds reported from Pterocarpus marsupium heart wood (PMH).
2. Materials and methods
purchased from the Sigma Aldrich (St. Louis, MO, USA) were used in sample preparation and LC–MS studies. Ultra-pure water was produced by a Direct-Q system (Millipore, Milford, MA, USA). The standard reference (purity≥95%) samples of 2, 6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside and pterocarposide were isolated from the heart wood of P. marsupium and their structures
2.1. Reagents, chemicals and materials Ethanol used for extraction was AR grade and purchased from Merck (Darmstadt, Germany). LC–MS grade acetonitrile and methanol 29
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Fig. 5. Proposed fragmentation pathway of Pterostillbene.
were unambiguously characterized by direct comparison of their 1H and 13C NMR spectral data with reported literature (Maurya et al., 2004; Handa et al., 2000). The standard reference samples of epicatechin, vanillic acid, quercetin, formononetin, genistein and naringenin were purchased from Sigma Aldrich (St. Louis, MO, USA). One sample of heart woods of Pterocarpus marsupium was purchased from district Allahabad (PMAL), one from district Nanpara (PMNp), two samples from district Bhinga (PMB1&B2) and two samples from district Baharaich (PMBH1&BH2) of Uttar Pradesh State, India in March 2017.
autosampler (G1329 A), thermostated column compartment (G1316C) and diode array detector (G1315D). The outlet of the diode array detector was introduced into the electrospray (ESI) interface of the mass spectrometer through a flow splitter (1:1). The HPLC separation was carried out on an Agilent Poroshell 120 EC C18 column (150 mm × 4.6 mm, 2.7 μm) operated at 25 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile with 0.1% formic acid (B) with flow rate of 0.5 mL/min under the gradient program of 10 to 30% (B) for initial 12 min, 30–45% (B) from 12 to 20 min, 45–90% (B) from 20 to 28 min, 90–90% (B) from 28 to 32 min, 90–10% (B) from 32 to 35 min followed by initial 10% (B) in 35–38 min. The sample injection volume was 2 μL. Mass spectrometric analysis was performed on Agilent 6520 QTOF mass spectrometer in positive ESI mode. The resolving power of QTOF analyser was above 10,000 (FWHM) and spectra were acquired within the mass range m/z 50–2000. Nitrogen was used as nebulizing, drying and collision gas. Capillary temperature was set to 350 °C and nebulizer pressure to 40 psi and the drying gas flow rate was 10 L/min. Ion source parameters such as VCap, fragmentor, skimmer and octapole rf peak voltage were set to 3500 V, 150 V, 65 V and 750 V, respectively. Collision energy for various MS/MS experiments ranged from 10 to 40 eV for all the selected masses. Accurate mass measurements were carried out by the auto mass calibration method using external mass calibration solution (ESI-L Low Concentration Tuning Mix; Agilent calibration solution B). The chromatographic and mass spectrometric analysis and prediction of chemical formula including the exact mass calculation were performed by Mass Hunter software version B.04.00 build 4.0.479.0 (Agilent Technology).
2.2. Extraction and sample preparation Hundreded g powder of the dried heart wood of P. marsupium sample was suspended with 20 mL ethanol (100%) and sonicated for 30 min at 25 °C in an ultrasonic water bath (Bandelin SONOREX, Berlin), left for 24 h at room temperature. The extract was collected and filtered through filter paper (Whatman No. 1) and the residue was reextracted three times with fresh solvent following the same procedure. The combined filtrates of each sample was concentrated using a Buchi rotary evaporator (Flawil, Switzerland) under reduced pressure at 20–50 kPa at 40 °C yielding dark yellow-brown semi-solid extract of P. marsupium heartwood (PMH). Yield of samples PM (AL), PM (Np), PM (B1&B2), PM (BH1 and BH2) were 6.336 g, 6.408 g, 6.300 g, 6.225 g 6.378 g and 6.4103 g respectively. All the extracts were prepared in same way from same method. One mg/mL solution of the dried ethanolic extract was prepared in methanol and filtered through a 0.22 μm polyvinylidene difluoride (PVDF) membrane (MILLEX GV filter unit, Merck Millipore, Darmstadt, Germany) prior to LCeMS analysis.
2.4.2. UPLC-MS/MS analysis for quantitative study All sample analysis for quantitative study was accomplished in multiple reaction monitoring mode. AB Sciex Analyst 1.6.2 software (Applied Biosystems) was used for data collection. The UPLC-ESIQqQLIT-MS/MS analysis was performed on a Waters Acquity UPLC™ system (Waters, Milford, MA, USA) interfaced with hybrid linear ion trap triple quadrupole mass spectrometer (API 4000 QTRAP™ MS/MS system from AB Sciex, Concord, ON, Canada) equipped with electrospray (Turbo V™) ion source. The Waters Acquity UPLC™system was equipped with a binary solvent manager, sample manager, column oven and photodiode array detector (PDA). The compound dependent MRM parameter optimization was performed by direct infusion using Harvard apparatus (USA) and flow injection analysis (FIA).
2.3. Preparation of standard and stock solutions Stock solutions (1.0 mg/mL) of eleven reference standards namely 2, 6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside, pterocarposide, epicatechin, vanillic acid, quercetin, formononetin and naringenin were prepared separately in LC–MS grade methanol. Then, methanol stock solution containing the mixture of eleven analytes was prepared and diluted in appropriate concentration to yield a series of concentrations from 0.1 to 1000 ng/mL. All stock solutions were stored in the refrigerator at −20 °C until further use. 2.4. Instrumentation and analytical conditions 2.4.1. HPLC-MS/MS analysis for qualitative study Qualitative analysis was performed with an Agilent 6520 quadrupole time-of-flight (QTOF) mass spectrometer connected with Agilent 1200 HPLC system via Dual electrospray ionization (ESI) interface (Agilent technologies, USA). The Agilent 1200 HPLC system consisted of quaternary pump (G1311 A), online vacuum degasser (G1322 A),
2.4.2.1. Ultra-performance liquid chromatography conditions. Chromatographic separation of compounds was obtained with an ACQUITY UPLC BEH™ C18 column (100 mm × 2.1 mm, 1.7 μm) operated at 25 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B), at a flow rate of 0.3 mL/ min under a gradient program: 30
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Fig. 6. Extracted ion chromatograms (EIC’s) of analytes quantified from Pterocarpus marsupium heart wood (PMH) in MRM mode.
5-5% (B) initial to 1.0 min, 5–20% (B) from 1.0 min to 1.5 min, 20–24 (B) from 1.5 min to 4.5 min, maintained at 24% (B) from 4.5 min to 5.2 min, 24–90% (B) from 5.2 min to 6.0 min, maintained at 90% (B) from 6.0 min to 6.5 min back to initial condition from 6.5 min to 7.5 min and maintained at 5% (B) from 7.5 min to 8.0 min. The sample injection volume used was 2 μL.
spectra were recorded by scanning in the range of m/z 100–1000 at a cycle time of 9 s with a step size of 0.1 Da. Nitrogen was used as the nebulizer, heater, and curtain gas as well as the collision activated dissociation gas. The compound-dependent parameters such as declustering potential (DP), entrance potential (EP), collision energy (CE) and cell exit potential (CXP) were optimized for each compound by direct infusion of 20 ng/mL solutions of the each analyte using a Harvard 144 ‘22’ syringe pump (Harvard Apparatus, South Natick, MA, USA) in negative ionization mode. The optimized parameters are shown in Table 1. Quadrupole 1 and quadrupole 2 were maintained at unit resolution. Quantitative analysis was performed using multiple reactions monitoring (MRM) mode. The optimized source dependent
2.4.2.2. Mass spectrometric conditions. Electrospray ionization in negative mode (-ESI) was used for sample ionization. Precursor ion scan mode was used for screening and MRM acquisition mode for quantitation of eleven bioactive compounds in six PMH samples. All the analytes were detected in -ESI using precursor ion scan and mass 31
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RSD(%)
2.24 1.30 1.01 2.20 1.50 2.60 1.10 2.20 1.15 1.82 0.97
Mean
98.3 102.9 99.60 101.35 95.72 97.40 101.20 100.6 95.21 105.1 99.60
Stability %RSD(n = 5)
2.5. Validation of quantitative analytical method
1.20 0.89 1.25 1.40 2.25 1.22 2.32 0.77 0.98 1.02 0.96 1.80 3.92 3.01 8.36 8.53 1.10 2.48 1.15 5.30 2.10 1.86
1.22 1.71 0.86 1.63 1.25 0.76 0.77 2.13 1.17 0.83 0.94
Intraday(n = 6) LOQ(ng ml−1)
Interday(n = 6)
1.72 0.74 1.80 2.82 2.12 1.24 1.93 1.22 0.97 0.96 1.01
The developed MRM method was validated for linearity, limits of detection (LOD), limits of quantitation (LOQ), precisions, stability and recovery studies according to the International Conference on Harmonization (ICH, Q2R1) guidelines, 2005 (Guideline, I.H.T., 2005). The linearity of calibration curve was performed by the analyte peak area ratio versus the nominal concentration. The linearity calibration curves were made using at least five experiments of each reference compound and evaluated by squared linear correlation coefficient ( r2). The LOD and LOQ were defined as a signal-to-noise ratio (S/N) equal to 3 and 10, respectively. The intra- and inter-day precisions were determined by analyzing known concentrations of the eleven analytes in the nine replicates during a single day and by triplicating the experiments on five successive days. For the recovery test, three spike levels were set as 50%, 100% and 200% of each reference standard. For comparison an unspiked sample was concurrently prepared and analyzed. 2.6. 3T3-L1 cell culture and Oil Red O staining
0.9999 0.9999 0.9999 0.9999 1 0.9999 0.9999 1 1 0.9999 0.9999 10-1000 10-1000 20-1000 25-1000 10-1000 10-1000 10-1000 20-1000 10-1000 10-1000 10-500
Y = 815x + 147 Y = 362x + 142 Y = 388x + 117 Y = 122x + 102 Y = 630x + 538 Y = 4230x + 466 Y = 446x + 111 Y = 938x + 108 Y = 820x + 435 Y = 2520x + 530 Y = 3090x + 577
0.59 1.29 0.99 2.75 2.81 0.36 0.82 0.37 1.75 0.69 0.61
In order to evaluate the effect of extracts isolated form heartwood of P. marsupium of different years, Oil Red O staining was performed. 3T3L1 pre-adipocytes were cultured in DMEM media supplemented with 10% Fetal bovine serum as described earlier (Gupta et al., 2017a, b). 3T3-L1 pre-adipocytes were seeded in 24 well cell culture plates either in presence or absence of 50 μg/ml of each extracts. After two days, fully confluent cells were induced DMEM (Dulbecco's Modified Eagle's medium) media (+10% FBS) (Fetal Bovine Serum) supplemented with 0.5 mM IBMX, 250 nM Dexamethasone and 5 μg/ml insulin (MDI) (Gupta et al., 2018). After 72 h, media replaced with DMEM media (+10% FBS) supplemented with 5 μg/ml insulin. After 48 h, media was changed with DMEM media (+10% FBS) for next 48 h for differentiation into mature adipocytes. Now, cells were stained with 0.36% Oil Red O in 60% isopropanol for 30 min. Excess Oil Red O was washed with PBS and images were captured using Leica inverted microscope at 10X and 40 × . Thereafter, captured Oil Red O was extracted using isopropanol and absorbance was measured at 492 nm for bargraph. 2.7. Statistical analysis
Marsuposide Vijyoside Epicatechin Vanillic acid Pterocarposide Pteroside Pterosupol Quercetin 2,6-dihydroxyphenyl glucopyranoside Naringenin Formononetin
r2 Linear range(ng ml−1)
Regression equation
LOD(ng ml−1)
Precision (%RSD) Linearity Reference standards Compound. No.
Table 3 Validation parameters of the developed method for quantitation of eleven phytochemicals in Pterocarpus marsupium heart wood (PMH) samples.
parameters were as follows: the ion spray voltage (IS) was set to −4200 V; the turbo spray temperature (TEM), 450 °C; nebulizer gas (GS 1) at 50 psi; heater gas (GS 2) at 50 psi; the curtain gas (CUR) at 20 psi and the collision-activated dissociation gas (CAD) was set as medium with the interface heater on. High-purity nitrogen was used for all the processes. AB Sciex Analyst software version 1.6.2 was used to control the UPLC–QqQLIT MS/MS system and for data acquisition and processing.
For in vitro experiments, the experiment was performed in triplicate and statistical significance was compared with MDI + by using Oneway ANOVA (analysis of variance) analysis followed by Dunnet’s multiple comparison test using GraphPad Prism software. “*” signifies p < 0.05, “**” signifies p < 0.01, “***” signifies p < 0.001. 3. Results and discussion 3.1. Analysis of phytochemicals using HPLC-ESI-QTOF-MS/MS The ESI in positive ionization mode was found suitable to determine 27 peaks of flavonoids, alkaloid, phenolic acids, and other compounds detected as [M+H]+ ions, at different retention times (Fig. 1). The fragmentation patterns and other details of identified compounds are summarized in (Table 2).
8 4 13 14 21 18 2 23 1 25 24
Recovery(n = 3)
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Table 4 Contents (mg/g) of compounds (n = 3) in different samples of Pterocarpus marsupium heart wood (PMH). Samples Compound No. 8 4 13 14 21 18 2 23 1 25 24 b c d e f g
Reference standards (mg/g) Marsuposide Vijyoside Epicatechin Vanillic acid Pterocarposide Pteroside Pterosupol Quercetin 2,6-dihydroxyphenyl glucopyranoside Naringenin Formononetin
PMb(Bc1) 17.8 4.30 0.34 0.65 0.31 0.029 bdlg 0.65 0.079 0.025 0.77
PM (B2) 45.4 5.30 0.33 1.57 0.39 0.27 bdl 0.39 0.11 bdl 2.08
PM(BHd1) 72.8 7.40 0.34 0.49 1.02 1.12 0.66 0.39 bdl bdl 0.30
PM(BH2) 52.5 5.90 0.58 1.09 0.29 bdl 0.46 0.38 0.22 0.12 1.63
PM(Ale) 56.6 19.5 0.43 3.90 0.61 0.18 bdl 0.43 0.074 0.69 bdl
PM(Npf) 15.6 10.7 0.81 0.88 3.49 3.80 4.10 0.41 0.052 0.017 bdl
PM: Pterocarpus marsupium. B: District Bhinga. BH: District Baharaich. Al: District Allhabad. Np: District Nanpara. bdl: below the detection level.
Fig. 7. (A) Oil Red O images of 3T3-L1 adipocytes either without differentiation (MDI-ve), fully differentiated without extracts (MDI + ve) or differentiated in presence of 50 μg/ml of each extracts at 10X and 40 × . (B) Relative absorbance of captured Oil Red O stain measured at 492 nm (n = 3). Statistical significance was compared with MDI + by using one way anova analysis followed by Dunnet’s multiple comparison test. “*” signifies p < 0.05, “**” signifies p < 0.01, “***” signifies p < 0.001.
3.1.1. Identification of flavonoids Peak 3 was identified and characterized as fisetin-8-C-glucoside (m/ z 449.1078 [M+H]+; C21H20O11). It produced major fragment ions at m/z 431.0943, 329.0648, and 299.0549 in the MS/MS spectra due to consecutive losses of H2O, C4H8O4 and C8H6O3, respectively. Peak 4, 5 and 6 were tentatively identified as vijyoside, 8-C-glucosyl-5-deoxykaempferol and aureusidin-6-rhamnoside respectively. Peaks 4, 5 and 6 showed [M+H]+ ion at m/z 433.1138 (C21H20O10) produced the same fragment ions in the MS/MS scan, representing their isomeric structures. The fragment ions were at m/z 415.1132, 397.0924 and 379.0665 due to consecutive loss of H2O molecules and base peak ion at m/z 313.0602 due to RDA (Retro-Diels–Alder reaction) cleavage in peaks 4 and 5 while in peak 6 showed fragment ion at m/z 329.0700
due to RDA cleavage which further on fragmentation gave base peak ion at m/z 313.0602 due to loss of H2O molecules. Peak 4 was confirmed by comparing with the authentic standard vijyoside. Peaks 5 and 6 were tentatively identified as 8-C-glucosyl-5-deoxykaempferol and aureusidin-6-rhamnoside, respectively. Peak 7 showed [M+H]+ ion at m/z 451.1235 (C21H22O11) and was tentatively identified as macrocarposide. It gave fragments at m/z 433.1094 and 331.0818 due to loss of H2O and RDA cleavage, respectively. Further fragmentation of ion m/z 433.1094 and m/z 331.0818 produced fragment ions at m/z 405.1151 and m/z 313.0689 respectively due to losses of CO and H2O, respectively. Fragment ion at m/z 313.0689 yielded an ion at m/z 285.0736 due to CO loss. Peak 8 exhibited [M+H]+ ion at m/z 435.1281 (C21H20O10) and produced fragment ion at m/z 417.1147 due 33
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to loss of H2O. Further fragmentation of m/z 417.1147 gave ions at m/z 389.1196 and 297.0738 due to loss of CO and C4H8O4, respectively. Peak 8 was identified as marsuposide using authentic standard. Peak 13 was identified and characterized as epicatechin (m/z 291.0856 [M +H]+; C15H14O6) by comparing with authentic standard. It produced major fragment ions at m/z 273.0725 and 139.0383 due to loss of H2O and RDA cleavage, respectively (Pandey and Kumar, 2016). Peak 15 [M +H]+ ion at m/z 255.0653 (C15H10O4) yielded fragment ion at m/z 137.0218 due to RDA cleavage. Fragment ion at m/z 255.0653 generated ion at m/z 227.0679 due to loss of CO. Peak 15 was tentatively identified as 7,4′-Dihydroxyflavone on the basis of fragmentation pattern. Peaks 19, 23 and 24 showing similar fragmentation pathways were identified and characterized as resokaempferol (m/z 271.0604 [M +H]+; C15H10O5) quercetin m/z 303.0856 (C15H10O7) and formononetin m/z 269.0809 (C16H12O4) respectively. Peaks 23 and 24 were further confirmed by matching with their authentic standards. Peaks 16, 25 and 26 gave [M+H]+ ions at m/z 241.0858 (C15H12O3), 273.0752 (C15H12O5) and 257.0809 (C15H12O4), respectively and produced fragment ions at m/z 137.0220, 153.0174 and 147.0439, respectively due to RDA cleavage and were tentatively identified as 7hydroxyflavanone, naringenin and liquirtigenin (Fig. 2). Peak 25 naringenin was also confirmed by matching with authentic standard. Peak 18 identified and characterized as pteroside (m/z 403.1388 [M+H]+; C21H20O8) was confirmed by the authentic standard. Peaks 20 and 21 showing [M+H]+ ion at m/z 417.1181 were identified as retusin-8-Oarabinoside and pterocarposide, respectively and pterocarposide was confirmed with authentic standard. All the identified flavonoids except compound 3, 16, 18, 20, 24 and 25 discussed above were detected in samples PM (B1), PM (BH2), PM (AL), PM (Np) and PM (B2) respectively.
(Fig. 6).All the above compounds were detected in all the samples except compound 1 not detected in PM (BH2), 2 not detected in PM (B1) PM (BH2), 9 not detected in PM (MP), PM (AL) and 27 not detected in sample PM (B2). 3.2. Quantitative analysis of bioactive compounds in PM heartwood using UPLC-ESI-QqQLIT-MS/MS A suitable UPLC-ESI-QqQLIT-MS/MS condition was optimized for quantitation of selected bioactive compounds. Compound dependent parameters were optimized by direct infusion of each analyte to achieve the most specific and stable multiple reaction monitoring (MRM) transitions. MRM extracted ion chromatogram of analytes are shown in Fig. 3. 3.2.1. Linearity, precision and recovery results of the validated method The stock solution was diluted with LC–MS grade methanol for different working concentrations to prepare the calibration curves. The calibration curves were constructed using eleven different concentrations with a weight (1/x2) factor by least-squares linear regression analysis. For each reference compound the linearity of calibration curve was determined on the basis of five experiments (n = 5). The linearity was evaluated by the regression coefficient (r2) of the respective calibration curves which showed r2 within the test ranges (Table 3). The calibration curve showed good linearity with regression coefficient ( r2) of ≥0.9999 over the tested concentration range (10–1000 ng/mL) (Table 3). The limits of detections (LODs) and limits of quantitation’s (LOQs) were measured by signal to noise ratio (S/N) of 3 and 10, respectively. The LODs and LOQs were in the range of 0.36–2.81 ng/mL and 1.10–8.53 ng/mL, respectively. Relative standard deviation (RSD) values for precision were in the range of 0.77–2.32% for intraday assays, 0.77–2.13% for interday assays. RSD for stability was analyzed by replicating injections of the sample solution at 0, 2, 4, 8, 12 and 24 h. The RSD values for stability and recovery were found ≤ 2.82% and ≤ 2.60%, respectively. The recoveries of the analytes were 95.21–102.90% (n = 5).
3.1.2. Identification of isoflavonoids and phenolic acid Peaks 12 (1,3,7-trihydroxyxanthone) or genistein and 17 (5,6,7trihydroxyisoflavone) showed [M+H]+ ion at m/z 271.0601 (C15H10O5) and produced ions at m/z 137.0211 and 153.0182 respectively due to RDA cleavage. They also generated fragment ion at m/z 243.0642 due to loss of CO followed by ion at m/z 225.0646 due to loss of H2O. Peak 12 was further confirmed by comparison with its authentic standard. Peak 14 identified and characterized as vanillic acid (m/z 169.0459 [M+H]+; C8H8O4) was also confirmed by comparison with its authentic standard. All these isoflavonoids and phenolic acids were detected in all the samples.
3.3. Method application The established UPLC-ESI-QqQLIT-MS/MS analytical method was applied to determine eleven bioactive compounds, namely 2,6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside, pterocarposide, epicatechin, vanillic acid, quercetin, formononetin and naringenin in the ethanolic extracts of heart woods of P. marsupium is summarized in (Table 4). The antidiabetic compound marsuposide (Perera, 2016) determined in the range of 15.6–72.8 mg/g was the most abundant constituent in all the analysed samples. The highest content of marsuposide was observed in sample PM (BH1) (72.8 mg/g). Content of vijyoside detected in range 7.40–19.50 mg/g was the second most abundant constituent of PM (BH1). All the quantified compounds were in high abundance in samples PM (BH1), PM (AL) and PM (BH2) respectively among selected samples. The total content of all selected bioactive compounds varied significantly in samples collected from different locations.
3.1.3. Identification of other compounds Peak 1 was identified and characterized as 2,6-dihydroxyphenyl glucopyranoside and showed [M+H]+ ion at m/z 273.0960 (C12H16O7) and produced ions at m/z 255.0762 and 153.0182 due to loss of H2O and RDA cleavage, respectively. It was also confirmed by its authentic standards. Peak 2 identified and tentatively characterized as pterosupol, showed [M+H]+ ion at m/z 245.1173 (C15H16O3) and produced ions at m/z 227.0994, 137.0603 and 108.0875 due to loss of H2O, C7H8O and C8H9O2, respectively. Peak 9 showed [M+H]+ ion at m/z 247.1442 (C14H18N2O2) and produced ion at m/z 189.0795 due to loss of N(CH3)3 and tentatively identified as hypaphorin. Peaks 10 and 11 showed the same [M+H]+ ion at m/z 437.1441 (C21H24O10) and produced ions at m/z 317.1011 due to RDA cleavage. They also generated fragment ions at m/z 419.1326 due to loss of H2O and 299.0913 due to loss of C8H9O2, and tentatively identified as pterosupin (Fig. 5) and coatlin A, respectively. Peak 22 gave [M+H]+ ion at m/z 303.0865 (C16H14O6) and produced the fragment ion at m/z 285.0747 due to loss of H2O. Fragment ion at m/z 285.0747 generated ion m/z 254.0800 due to loss of OCH3. Peak 22 was tentatively identified as carpusin. Peak 27 [M+H]+ ion at m/z 257.1170 (C16H16O3) produced the fragment ions at m/z 226.0988 and 137.0640 due to loss of and C8H8O, respectively. The fragment ion at m/z 137.0640 lost OCH3 to produce the ion at m/z 106.0497 (Fig. 4). It was tentatively identified as pterostilbene
3.4. P. marsupium extracts possess in-vitro lipid lowering activity Excess fat accumulation has well been correlated with obesity (Smith, 2015), accompanied with elevated levels of non-esterified fatty acids, glycerol and pro-inflammatory markers marking diabetic phenotype. In addition, weight gain and increased body mass raises incidence of Type 1 and Type 2 diabetes (Al-Goblan et al., 2014). Reporetd literature suggest that reduction in lipid load has promising therapeutical benefitis, ameliorating obesity and related disordres (Murphy and Yvan-Charvet, 2015). In light of the earlier studies, we assessed the potential of isolated extracts of P. marsupium for their lipid 34
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lowering activity. Assessment of lipid accumulation in 3T3-L1 adipocytes revealed significant attenuation of lipid content in the cells treated with extracts isolated form PM (B2), PM (BH1) as well as PM (BH2). However, extract from PM (B1) was found to be toxic to cells. The results suggest that the extract from PM (BH2) have certain constituents that are capable of reducing obesity and hence could prove to be beneficial in diabetes. (Fig. 7A and B)
Indian herbs and herbal drugs used for the treatment of diabetes. J. Clin. Biochem. Nutr. 40 (3), 163–173. Tiwari, M., Sharma, M., Khare, H.N., 2015. Chemical constituents and medicinal uses of Pterocarpus marsupium Roxb. Flora and Fauna 21 (1), 55–59. Rizvi, S.I., Mishra, N., 2013. Traditional Indian medicines used for the management of diabetes mellitus. J. Diabetes Res. 2013, 11. Bajpai, V., Kumar, S., Singh, A., Bano, N., Pathak, M., Kumar, N., Misra-Bhattacharya, S., Kumar, B., 2017a. Metabolic fingerprinting of dioecious Tinospora cordifolia (Thunb) Miers stem using DART TOF MS and differential pharmacological efficacy of its male and female plants. Ind. Crops Prod. 101, 46–53. Bajpai, V., Kumar, S., Singh, A., Singh, J., Negi, M.P.S., Bag, S.K., Kumar, N., Konwar, R., Kumar, B., 2017b. Chemometric based identification and validation of specific chemical markers for geographical, seasonal and gender variations in Tinospora cordifolia stem using HPLC ESI QTOF MS analysis. Phytochem. Anal. 28 (4), 277–288. Chandra, P., Kannujia, R., Pandey, R., Shukla, S., Bahadur, L., Pal, M., Kumar, B., 2016. Rapid quantitative analysis of multi-components in Andrographis paniculata using UPLC-QqQLIT-MS/MS: application to soil sodicity and organic farming. Ind. Crops Prod. 83, 423–430. Pandey, R., Chandra, P., Srivastva, M., Arya, K.R., Shukla, P.K., Kumar, B., 2014. A rapid analytical method for characterization and simultaneous quantitative determination of phytoconstituents in Piper betle landraces using UPLC-ESI-MS/MS. Anal. Methods 6 (18), 7349–7360. Bajpai, V., Pandey, R., Negi, M.P.S., Bindu, K.H., Kumar, N., Kumar, B., 2012. Characteristic differences in metabolite profile in male and female plants of dioecious Piper betle L. J. Biosci. 37 (1), 1061–1066. Bajpai, V., Singh, A., Chandra, P., Negi, M.P.S., Kumar, N., Kumar, B., 2016. Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT‐MS/MS. 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Constituents of Pterocarpus marsupium: an ayurvedic crude drug. Phytochemistry 65 (7), 915. Handa, S.S., Singh, R., Maurya, R., Satti, N.K., Suri, K.A., Suri, O.P., 2000. Pterocarposide, an isoaurone C-glucoside from Pterocarpus marsupium. Tetrahedron Lett. 41 (10), 1579–1581. Shao, X., Chen, X., Badmaev, V., Ho, C.T., Sang, S., 2010. Structural identification of mouse urinary metabolites of pterostilbene using liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 24 (12), 1770–1778. Guideline, I.H.T, 2005. Validation of Analytical Procedures: Text and Methodology. Q2 (R1). pp. 1. Gupta, A., Beg, M., Kumar, D., Shankar, K., Varshney, S., Rajan, S., Srivastava, A., Singh, K., Sonkar, S., Mahdi, A.A., Dikshit, M., 2017a. Chronic hyper-leptinemia induces insulin signaling disruption in adipocytes: implications of NOS2. Free Radic. Biol. Med. 112, 93–108. Gupta, A., Singh, V.K., Kumar, D., Yadav, P., Kumar, S., Beg, M., Shankar, K., Varshney, S., Rajan, S., Srivastava, A., Choudhary, R., 2017b. Curcumin-3, 4-Dichloro Phenyl Pyrazole (CDPP) overcomes curcumin’s low bioavailability, inhibits adipogenesis and ameliorates dyslipidemia by activating reverse cholesterol transport. Metab.-Clin. Exp. 73, 109–124. Gupta, A., Kumar, A., Kumar, D., Singh, R., Shankar, K., Varshney, S., Rajan, S., Srivastava, A., Gupta, S., Narender, T., Gaikwad, A.N., 2018. Ecliptal, a promising natural lead isolated from Eclipta alba modulates adipocyte function and ameliorates metabolic syndrome. Toxicol. Appl. Pharmacol. 338, 134–147. Pandey, R., Kumar, B., 2016. HPLC–QTOF–MS/MS-based rapid screening of phenolics and triterpenic acids in leaf extracts of Ocimum species and their interspecies variation. J. Liq. Chromatogr. Relat. Technol. 39 (4), 225–238. Perera, H.K.I., 2016. Antidiabetic effects of Pterocarpus marsupium (Gammalu). Eur. J. Med. Plants 13 (4). Smith, U., 2015. Abdominal obesity: a marker of ectopic fat accumulation. J. Clin. Invest. 125 (5), 1790–1792. Al-Goblan, A.S., Al-Alfi, M.A., Khan, M.Z., 2014. Mechanism linking diabetes mellitus and obesity. Diabetes Metab. Syndr. Obes. 7, 587. Murphy, A.J., Yvan-Charvet, L., 2015. Adipose modulation of ABCG1 uncovers an intimate link between sphingomyelin and triglyceride storage. Diabetes 64 (3), 689–692. Mishra, A., Srivastava, R., Srivastava, S.P., Gautam, S., Tamrakar, A.K., Maurya, R., Srivastava, A.K., 2013. Antidiabetic Activity of Heart Wood of Pterocarpus marsupium Roxb. And Analysis of Phytoconstituents. Jahromi, M.F., Ray, A.B., Chansouria, J.P.N., 1993b. Antihyperlipidemic effect of flavonoids from Pterocarpus marsupium. J. Nat. Prod. 56 (7), 989–994. Devgun, M., Nanda, A., Ansari, S.H., 2009. Pterocarpus marsupium Roxb.–a comprehensive review. Pharmacogn. Rev. 3 (6), 359.
4. Conclusions In conclusion, a rapid and sensitive HPLC-ESI-QTOF-MS/MS in positive ionization mode and UPLC-ESI-QqQLIT-MS/MS method in negative ionization mode were developed and successfully applied for identification, characterization and quantitation of bioactive phytochemicals present in heart woods of P. marsupium. Twenty seven compounds including flavonoids, alkaloid and phenolic acid, were identified in different samples. An UPLC-ESI-QqQLIT-MS/MS analytical method was applied to determine contents of eleven bioactive compounds, namely 2,6-dihydroxyphenyl glucopyranoside, pterosupol, vijyoside, marsuposide, pteroside, pterocarposide, epicatechin, vanillic acid, quercetin, formononetin and naringenin in the ethanolic extracts samples. The total content of eleven selected bioactive compounds was found high in the sample collected from Bhinga district (U.P.). Nevertheless, these methods are simple, rapid and sensitive, provide useful qualitative and quantitative fingerprint, to be used for grading bioactive compounds commercially. Plant samples were screened for their potential to suppresses lipid accumulation in adipocytes in-vitro, in which results were widely varied and could contribute to their application to different therapeutic areas. Acknowledgements Grateful acknowledgement is made to SAIF, CSIR-CDRI, Lucknow, where the mass spectrometry studies were carried out. Pratibha Singh and Vikas Bajpai are thankful to Department of Science and Technology (DST), New Delhi for grant GAP0003. References Therrell, M.D., Stahle, D.W., Mukelabai, M.M., Shugart, H.H., 2007. Age, and radial growth dynamics of Pterocarpus angolensis in southern Africa. For. Ecol. Manage. 244 (1-3), 24–31. Chatterjee, A., Pakrashi, S.C., 1991. The treatise on Indian medicinal plants: vol. 1. New Delhi: Publications and Information Directorate, CSIR 172p.-illus., col. illus. ISBN 8172360118 En Icones. Includes authentic Sanskrit slokas in both Devnagri and Roman scripts. Plant Rec. Geogr. 6. Satyavati, G.V., Gupta, A.K., Tandon, N., 1987. Med. Plants India 27, 574–575. Satyavati, G.V., Neeraj, T., Madhu, S., 1989. Indigenous plant drugs for diabetes mellitus. Indian J. Diabetes Dev. Countries. 1–35. Anis, M., 2005. In vitro plantlet regeneration of Pterocarpus marsupium Roxb., an endangered leguminous tree. Curr. Sci. 88 (6), 86. Kapai, V.Y., Kapoor, P., Rao, I.U., 2010. In Vitro propagation for conservation of rare and threatened plants of India–a Review. Int. J. Biol. Technol. 1 (2), 1–14. Sheehan, E.W., Zemaitis, M.A., Slatkin, D.J., Schiff Jr, P.L., 1983. A constituent of Pterocarpus marsupium,(-)-epicatechin, as a potential antidiabetic agent. J. Nat. Prod. 46 (2), 232–234. Jahromi, M.F., Ray, A.B., Chansouria, J.P.N., 1993a. Antihyperlipidemic effect of flavonoids from Pterocarpus marsupium. J. Nat. Prod. 56 (7), 989–994. Manickam, M., Ramanathan, M., Farboodniay Jahromi, M.A., Chansouria, J.P.N., Ray, A.B., 1997. Antihyperglycemic activity of phenolics from Pterocarpus marsupium. J. Nat. Prod. 60 (6), 609–610. Badkhane, Y., Yadav, A.S., Sharma, A.K., Raghuwanshi, D.K., Uikey, S.K., Mir, F.A., Lone, S.A., Murab, T., 2010. Pterocarpus marsupium Roxb-Biological activities and medicinal properties. Int. J. Adv. Pharm. Sci. 1 (4). Khan, M.Y., Aziz, I., Bihari, B., Kumar, H., Roy, M., Kumar, V., 2014. A reviewPhytomedicines used in treatment of diabetes. Diabetes 101, 126. Modak, M., Dixit, P., Londhe, J., Ghaskadbi, S., Devasagayam, T.P.A., 2007. Recent advances in Indian herbal drug research guest editor: thomas Paul Asir Devasagayam
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