УДК 543



© 2015 Somendu Kumar Roy, Khemraj Bairwa, Jagdeep Grover, Amit Srivastava, Sanjay Madhukar Jachak1

Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER) Sector-67, SASNagar (Mohali), 160062, Punjab, India 1E-mail: sanjayjachak@niper.ac.in Received 31.07.2013; in final form 28.09.2014

Negundoside and agnuside are reported as marker constituents for standardization of Vitex negundo (V. ne-gundo) leaves. This is the first report on determination and quantification of negundoside and agnuside by qNMR in the leaves of V. negundo, collected from various regions of India. The developed qNMR method showed good linearity (r2 > 0.9994), high precision (RSD < 5%) and a good recovery (99.79 and 101.35%) for both the compounds. Therefore, the developed qNMR method is accurate and reliable for the quality control of V. negundo. The qNMR method is useful in quality control of plant extracts without the need of extensive sample preparation and chromatographic separation.

Keywords: Vitex negundo, qNMR, negundoside, agnuside, method validation.

DOI: 10.7868/S0044450215050151

Nuclear magnetic resonance spectroscopy is a quantitative tool because the intensity of a signal is directly proportional to the number of resonating nuclei. This fact enables the accurate and precise quantitative determination of compounds in different matrices [1]. This application is further pertinent to quantitation of natural products in plant extracts, extending the scope of NMR. Various literature reports are available on the application of qNMR for the quantitation of metabolites in pharamaceutical preparations, herbal products, plant extracts, etc. [2—4]. The major advantage of this technique over other metrological techniques is the non-destructive nature of the sample, especially in case of natural products where availability of pure standard is limited. The level of the major chemical ingredient could be determined with accuracy and precision significantly better than 1%, and impurities could be quantified at the 0.1% level or below [5]. The worldwide increase in regulations governing pharmaceutical and environmental sectors that require increased efforts toward validation of analytical and pharmacological standards, necessitates the use of qNMR as one of the important tools for the analysis of these reference materials.

Vitex negundo Linn. (Lamiaceae) is an ingredient of several commercially available herbal formulations, that are used as anti-inflammatory, anti-arthritic, analgesic, and hepatoprotective drugs [6—8]. The sub-effective dose (50 mg/kg) of ethanolic extract of V. ne-gundo leaves when administred orally in rats, showed

potentiation of anti-inflammatory activity of phenylbutazone and ibuprofen [9]. These findings indicated that V. negundo may be used as an adjuvant therapy with anti-inflammatory drugs. Negundoside was reported as an hepatoprotective agent against calcium-mediated toxicity, induced by carbon tetrachloride [10]. Agnuside demonstrated significant suppression of inflammatory mediators and T-cell mediated cytokines [11].

HPTLC and HPLC determination of negundoside, and HPLC determination of vitexin in V. negundo leaves are previously reported [12—14]. Recently we have reported determination of iridoids and flavonoids in V. negundo leaves by HPLC, and validated the developed HPLC method [15]. As negundoside and agnuside are reported as markers and bioactive constituents from V. negundo leaves and also as a part of our continuing research programme on the standardization of medicinal plants [16, 17], we aimed to develop a qNMR method as an alternative of HPLC method for V. negundo.

A simple, accurate, and robust qNMR method for the determination and quantification of negundoside (1) and agnuside (2) in V. negundo leaves sample from three different regions of India is described here-in.The developed qNMR method was validated by determining selectivity, linearity, precision, accuracy, and robustness.


Plant materials. A sample of V. negundo leaves was collected from NIPER campus, SAS Nagar, Punjab (sample A), and two other samples of V. negundo leaves were purchased from Abirami Botanicals Corporation, Tuticorin, Chennai (sample B), and Srijee Ayurved, Mumbai (sample C). The samples were authenticated by Dr. A.S. Sandhu, botanist, Department of Natural Products (DNP), NIPER, and voucher specimens (NIP-H-176, NIP-NPM-CD-021, and NIP-NPM-CD-022 for samples A, B, and C, respectively) were deposited in the herbarium of DNP, NIPER, SAS. Nagar, India. Diaion HP-20 resin was purchased from Supelco (PA, USA). Ultra pure water (18 mQ) was obtained from the Millipore system (Billerica, USA).

Instrumentation. Bruker Avance III 400 NMR

spectrometer (Bruker Biospin AG, Faellanden, Switzerland) operating at a frequency of 400.13 MHz for protons, equipped with a 5 mm multinuclear inverse probe head was used for acquisition of NMR spectra. The spectra were acquired in a non-spinning mode at a calibrated probe temperature of 293 K. Total 16 scans of 32 K data points for free induction decay were acquired with a spectral width of 8012 Hz (16 ppm), preacquisition delay of 6 ^s, acquisition time of 4.0 s, recycle delay of 5.0 s and a flip angle of 30°. The spectra were Fourier transformed after zero filling to 64.1 K, giving a digital resolution in the frequency domain of 0.125 Hz/pt. Manual phase and baseline correction were performed prior to integration. Preliminary data processing was accomplished using Bruker software, TOPSPIN 3.2.

Isolation of marker constituents (1 and 2). Powdered leaves (500 g) of V. negundo were extracted for 24 h with 2.5 L methanol using Soxhlet apparatus. The methanol extract was filtered and dried under reduced pressure to give 82 g of extract. The part of methanol extract (10 g) was dissolved in 10 mL of methanol, and subjected to column chromatography on Diaion HP-20 resin. The sample was eluted gradient wise using water—methanol (0—100% methanol) as mobile phase. A fraction eluted with 20% methanol (4 g) contained mainly a mixture of 1 and 2. A part of this fraction (0.5 g) was separated on Waters HPLC system (Waters 600, USA) eluted with methanol-water (30 : 70), at a flow rate of 5 mL/min on RP-18 column (XTerra®, 19 x 250 mm, 10 mm), with the detection at 254 nm using Waters 2996 PDA detector (MA, USA), to give 1 (67 mg) and 2 (54 mg).

Sample preparation for qNMR analysis. Weighed amount (25 g) of coarsely powdered leaves of three samples (A, B, and C) of V. negundo was refluxed with methanol (100 mL) separately for 2 h; after reflux, the extract solution was cooled to room temperature and filtered through Whatmann filter paper and dried under reduced pressure. Weighed amounts (26.4, 33.2, and 22.0 mg) of extract of samples A, B, and C, re-

spectively, were mixed with 2 mg of 1,3,5-trimethoxybenzene (TMB) and dissolved in 0.6 mL of methanol-d4 and 1H NMR spectra were acquired (parameters of NMR are described under Instrumentation). The sample preparation method was repeated two more times for all the three samples to check the reproducibility of the qNMR analysis. These methanol extracts were used for quantitative analysis of compounds 1 and 2 by NMR.

Validation of qNMR analytical method. The developed method for quantification of marker constituents (1 and 2) was validated in accordance with the International Conference on Harmonization (ICH) guidelines for the selectivity, linearity, precision, recovery, robustness and stability studies [18-20].

Selectivity. The 1H NMR spectrum of blank sample (0.6 mL of methanol-d4) and the sample without reference standard, TMB were recorded.

Linearity, limit of detection (LOD) and limit of quantification (LOQ).The linearity was performed for each compound separately and the concentration range was chosen on the basis of the expected values in the study. The calibration curves were generated from four concentrations of the compound each in duplicate, and from calibration curves, regression coefficient, slope, and intercept were calculated. The calibration curves thus obtained were used to quantify the respective compounds in methanol extracts of the three samples. LOD and LOQ of 1 and 2 were calculated based on equation of the standard deviation of the response and

the slope as per ICH guidelines: LOD = ,



LOQ = —, where s and S are the standard deviation S

of the response and slope of the calibration curve, respectively.

Precision. For intra-day precision, the individual compound (1 and 2) was determined at three different concentrations in triplicate within one day. While for inter-day precision, the above mentioned compounds were determined in triplicate for three consecutive days. The precision values were expressed as the relative standard deviation (RSD, %).

Accuracy. Accuracy was determined by performing recovery study, which was performed by adding a known amount of compounds (1 and 2) at low, medium, and high concentration into the methanol extract (23 mg/mL concentration) of sample C. The three solutions were analyzed in triplicate, and the results were expressed as the percentage recovery of the added compounds.

Robustness. The robustness of the method was studied by making deliberate changes in two parameters: number of scans (16, 32, and 64) and operating frequency of the instrument (400 and 500 MHz). The variations were analyzed and expressed as RSD of the three determinations.

Table 1. Linearity, LOQ and LOD study of compounds 1 and 2 Table 2. Precision (RSD, %) study of compounds 1 and 2

Parameter 1 2

Concentration range, mg/mL 3.3-10 3.3-10

Intercept -0.003 -0.002

Slope 0.103 0.108

Correlation coefficient 0.9999 0.9999

LOQ, mg/mL 0.6 0.8

LOD, mg/mL 0.2 0.3

Compound Concentration, mg/mL Intra-day (n = 3) Inter-day (n = 6)

1 0.67 1.30 0.

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