A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations Bulletin of Faculty of Pharmacy, Cairo University xxx (2016) xxx–xxx[.]
Bulletin of Faculty of Pharmacy, Cairo University xxx (2016) xxx–xxx Contents lists available at ScienceDirect Bulletin of Faculty of Pharmacy, Cairo University journal homepage: www.sciencedirect.com Original Article A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations Kedar Kalyani Abhimanyu a,b,⇑, Chaudhari Sanjay Ravindra c, Rao Srinivasa Avanapu d a Pharmacognosy Department, P E Society’s Modern College of Pharmacy, Nigdi, Pune, Maharashtra 411044, India Jawaharlal Nehru Technological University (JNTU), Hyderabad, Andra Pradesh 500072, India c Amrutvahini College of Pharmacy Amrutnagar, Sangamner SK, Tal-Sangamner, Dist Ahmednagar 422608, India d Bhaskar Pharmacy College, Yeknapally, Moinabad (Mandal), R.R (Dt), Hyderabad 500075, India b a r t i c l e i n f o Article history: Received 12 August 2016 Received in revised form October 2016 Accepted 14 November 2016 Available online xxxx Keywords: Putranjiva roxburghii Wall Euphorbiaceae Friedelin Polyherbal formulation HPTLC Method validation a b s t r a c t In present study HPTLC method was developed and validated for the determination of friedelin in Putranjiva roxburghii Wall (family: Euphorbiaceae) leaf, bark extract and in polyherbal formulations Analysis of samples were performed on TLC aluminium precoated plate (60 F254) by using mobile phase toluene: chloroform (9:1 v/v) Plate was derivatized with vanillin sulphuric acid and scanned at 580 nm Developed method found to give compact spot for friedelin at Rf value 0.43 ± 0.01 The method was validated using International Council for Harmonization (ICH) guidelines including linearity, precision, accuracy, and robustness Friedelin was found to be present in leaf extract of Putranjiva roxburghii Wall (0.003% w/w), in bark (0.04% w/w), formulation (0.002% w/w) and formulation (0.035% w/w) A good linearity relationship was found to be (100–500 ng spot 1) with correlation coefficient (r2) value of 0.9892 for friedelin Limit of detection and limit of quantitation was found to be 32.15, 97.44 ng/band respectively for friedelin The developed method was found to be accurate and precise with 0.78%, 0.9% (%RSD) for interday and intraday precision Accuracy of the method was performed by recovery studies at three different concentration levels and the average percentage recovery was found to be 98.55% for friedelin The proposed method for the quantitation of friedelin was found to be simple, specific, accurate and robust in Putranjiva roxburghii Wall and polyherbal formulations Ó 2016 Publishing services provided by Elsevier B.V on behalf of Faculty of Pharmacy, Cairo University This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/) Introduction Euphorbiaceae family having 220 genera and 4000 plant species found in various tropical regions of India [1,2] Following genera of Euphorbiaceae were reported as medicinal plants: Acalypha, Aleurites, Bridelia, Jatropha, Phyllantus, Putranjiva, Ricinus [2–4] Species of Euphorbiaceae family was commonly seen in India is Putranjiva roxburghii Wall which is known as child’s amulet tree or child-life tree [5] Putranjiva is a mostly dioeciously, evergreen tree, growing up to 18 m in height The Putranjiva roxbourghii Wall mentioned frequently recorded folk remedy claims in that the plant leaf, bark, seed, nuts are Peer review under responsibility of Faculty of Pharmacy, Cairo University ⇑ Corresponding author at: Department of Pharmacognosy, Progressive Education Society’s Modern College of Pharmacy, Sector-21, Yamunanagar, Nigdi, Pune 411044, Maharastra, India E-mail addresses: kk_pharma20@rediffmail.com (K.K Abhimanyu), dr_srchaudhari@yahoo.com (C.S Ravindra), dravanapu@yahoo.com (R.S Avanapu) medicinally useful Nuts are taken orally by women (sterile) in villages near Renuka forest division in Himachal to effect conception and attributed with the birth of child [5] Bark and the seeds of this plants are useful in antidotal treatment of snake-bite Its leaves and fruits, stones of this plant have been traditionally used for the treatment of fever, muscle twisting, aphrodisiac, arthralgia and rheumatism [6–8] It was used as antinociceptive, antipyretic, anti-inflammatory, antioxidant [9] Puntranjiva roxburghii bark contained important phytoconstituents such as triterpenoids (putranjivanonol, putranjic acid, putranjivadione, roxburgholone), pentacyclic triterpene (friedelin, friedelanol) [10–12] A friedelane triterpenoid keto acid (roxburghonic acid), biflavonoid (putraflavone) were isolated from the leaves part of Puntranjiva roxburghii [13] Previous analytical work includes that amentoflavone, b-amyrin and stigmasterol determined from Putranjiva roxburghii Wall by HPTLC method [14,15] So an attempt has been made to carry out detail chromatographic analysis of leaves and bark of Putranjiva roxbourghii Wall http://dx.doi.org/10.1016/j.bfopcu.2016.11.002 1110-0931/Ó 2016 Publishing services provided by Elsevier B.V on behalf of Faculty of Pharmacy, Cairo University This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: K.K Abhimanyu et al., A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations, Bulletin Facult Pharmacy Cairo Univ (2016), http://dx.doi.org/10.1016/j.bfopcu.2016.11.002 K.K Abhimanyu et al / Bulletin of Faculty of Pharmacy, Cairo University xxx (2016) xxx–xxx Friedelin was pentacyclic triterpene isolated from several plant species such as Terminalia avicennioides, Cissus quadrangularis, Acer mandshuricum, Cassia siamea Lam, Celastrus vulcanicola, Maytenus aquifolium, Salacia campestris, Maytenus macrocarpa, Alangium salvifolium (Linn), Holoptelea integrifolia (Roxb) [16–22] Structure of friedelin has shown in (Fig 1) Friedelin compound shows effective estrogenic activity in female rat and this compound is potential candidate for hormonal replacement therapy in postmenopausal woman Now a day’s various ayurvedic formulations are available in market to treat female menstrual problems, it helps to improve hormonal therapy, use as uterine tonic etc From this Femiforte and Femiplex tablets (Charak Pharma Pvt Ltd Dist Solan) are useful as uterine tonic in female; these tablets contain extract of Putranjiva roxbourghii Wall So present study helps to explore quantization and identification of friedelin in Putranjiva roxbourghii Wall in polyherbal formulation by high performance thin layer chromatography and this study also help in analysis of friedelin in other several plant species Materials and methods 2.1 Plant material tion of developing chamber, derivatization reagents carried out as per guideline mention in United States Pharmacopeia (USP Chapter, 203) According to this chromatographic analysis was done on pre-coated silica gel 60 F254 plates (10 10 cm with 200 lm thickness HPTLC) Samples of extracts, formulations and standards were applied by using microsyringe (Linomat syringe, Hamilton-Bonaduz schweiz, Camag, Switzerland) in band length mm wide and mm apart by Camag Linomat sample applicator (Camag, Muttenz, Switzerland) The application rate of sample on plate was 150 nl The plate was developed in previously saturated 10 10 cm twin-trough glass chamber (Camag, Muttenz, Switzerland) at room temperature Initially different mobile phases were used for chromatogram development from this best resolution was observed in the composition of Toluene: Chloroform (9:1 v/v) for friedelin Dry thin layer chromatographic plate derivatized with vanillin sulphuric acid reagent, heat plate at 105 °C and observed separation of bands it helps in analysis of friedelin in leaf and bark Extracts and formulations [23] Analysis carried out at 580 nm in absorbance remission mode by TLC scanner III (Camag, Muttenz, Switzerland) and win CATS version 1.4.0 software (Camag, Muttenz, Switzerland) were used in this study Microsoft excel was also used to treat data statistically 2.4 Preparation of standard solution The leaves and trunk bark material of grown tree of Putranjiva roxburghii Wall was collected from Khadaki region of Maharashtra The taxon was authenticated from Botanical Survey of India, Pune dated 18/08/2014 with Voucher number BSI/WRC/Cert./2014 and collection No KKA 01 The herbarium specimen is deposited in the Modern college of pharmacy, Nigdi, Pune A stock solution of friedelin (100 lg/ml) was prepared by dissolving 10 mg of accurately weighed friedelin in 100 ml chloroform For calibration 1–5 ll standard solution was applied to HPTLC plate in the range 100–500 ng per band 2.2 Chemicals and reagents 2.5 Preparation of sample solution Friedelin was purchased from Sigma-Adrich (USA) (product code-101669048) all other solvents, reagents and precoated silica gel 60 F254 high performance thin layer chromatography plates (HPTLC, 20 20 cm) were purchased from Merck (Germany) Now a days in market we can get ayurvedic formulation Femiforte tablet (formulation 1) containing extracts of Putranjiva roxburghii (40 mg each tablet), Femiplex tablet (13.05 mg each tablet) (formulation 2) was procured from local market, Pune (Charak Pharma Pvt Ltd Baddhi, Dist Solan, Himachal Pradesh, India) Based on solubility of the marker compounds, chloroform extract leaf and bark were prepared by weighing 50 g of dried powdered of Putranjiva roxburghii and extraction was carried out by soxhlet extraction assembly for h Solution was filtered, concentrated and use for HPTLC analysis From this weigh 10 mg leaf and bark extract transferred to a 10 ml volumetric flask Chloroform was added to volumetric flask to make final concentration (1000 lg/ml) Weigh five femiforte and ten femiplex tablets, crushed it, removed tablet coating and powdered were kept for maceration for overnight in 10 ml chloroform, filtered it and use for HPTLC analysis 2.3 HPTLC instrumentation and experimental conditions Method development parameters includes sample and test solution preparation, HPTLC instrumentation condition, prepara- H3C CH3 CH3 H3C H H H CH3 O CH3 CH3 Fig Chemical structure of Friedelin CH3 2.6 Method validation The analytical method was validated for linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of quantization (LOQ) according to the International Council for Harmonization (ICH, 2005) guidelines The linearity was carried out by applying different concentration of standard friedelin Quantization’s of marker in samples were carried out by calibration curve Limit of detection (LOD) and limit of quantitation (LOQ) were calculated based on standard deviation (SD) and slope (S) of the calibration curve at levels approaching to the LOD according to formula {LOD = 3.3(SD/S) and LOQ = 10 (SD/S)} Precision studies include, repeatability and system precision Accuracy by recovery studies were carried out by spiking known concentration of standard to pre-analyzed samples The robustness was carried out by making small variation in optimized method parameters such as variation in composition of mobile phase, chamber saturation time etc The specificity of the method was determined by comparing Rf values and ultraviolet– visible (UV) spectra of peaks of components in sample, formulation and standard chromatogram Please cite this article in press as: K.K Abhimanyu et al., A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations, Bulletin Facult Pharmacy Cairo Univ (2016), http://dx.doi.org/10.1016/j.bfopcu.2016.11.002 K.K Abhimanyu et al / Bulletin of Faculty of Pharmacy, Cairo University xxx (2016) xxx–xxx Results and discussion 3.1 Solvent system optimization For optimization of solvent system various compositions of mobile phases were use When mobile phase consisting toluene: ethyl acetate: formic acid (9:1:0.1 v/v/v) component in samples not get resolved and Rf of standard was very close to solvent front, other composition of mobile as toluene: ethyl acetate (9:1 v/v) was not shows better resolution in peaks in samples In order to improve resolution in between peaks mobile phase in composition of toluene: chloroform (9:1 v/v), gives compact peak of standard and standard in samples and formulations Observation shows the same Rf value (Fig 3) for friedelin in standard and samples 3.2 Method validation 3.2.1 Linearity For determining the linearity range of standard friedelin, a series of spots of different volumes (1, 2, 3, 4, ll) were applied so as to get 100–500 ng quantity of standard per band, respectively Linearity was evaluated in triplicate The plate was scanned at 580 nm and curve was prepared with respect to area vs amount per spot (Fig 2) A good linearity relationship was found to be with correlation coefficient (r2) value of 0.9892 for friedelin (Table and Fig 2) 3.2.2 Quantification of friedelin in the leaves, bark and in polyherbal formulation containing Putranjiva roxburghii Wall ll of the plant leaf, bark extract and 50 ll of formulation and were applied to HPTLC plate in triplicate and the amount of friedelin in samples were determined by using calibration curve of standard Previously HPTLC determination of friedelin was reported in Alangium salvifolium (Linn), Holoptelea integrifolia (Roxb) [24] This method shows the presence of 0.003% w/w for friedelin in leaf extract, 0.04% w/w in bark extract, 0.002% w/w in formulation and 0.035% w/w in formulation 3.2.3 Limit of detection and quantitation In order to determine limit of detection (LOD) and limit of quantitation (LOQ) were calculated according to formula {LOD = 3.3(SD/ S) and LOQ = 10 (SD/S)} and found to be 32.15, 97.44 ng spot for friedelin (Table 1) 3.2.4 Precision Precision studies were carried out to show the reproducibility of the proposed developed method Intraday precision study was carried out by applying six times 300 ng per band of same concentration It can be analyzing at three different times in a day for intraday precision and the same procedure was followed for three different days to determine interday precision The results were reported as SD (%RSD) (Table 2) The %RSD was found to be 0.78%, 0.9% for interday and intraday precision The low %RSD indicated the method is precise for the analysis (Table 2) 3.2.5 Specificity The specificity of the method was determined by analysing standard drug and sample The presence of friedelin in leaf, bark and formulations were confirmed by comparing Rf and ultraviolet–visible spectra of sample with standard Purity of sample spot corresponding to friedelin in sample and both formulations were analyzed by superimposing the spectrum of standard and sample peaks (Fig.4) 3.2.6 Recovery studies (Accuracy) Accuracy of method was studied by performing recovery studies at levels of friedelin The pre-analyzed samples were spiked with 80%, 100% and 120% of the standard friedelin and analyzed by the proposed HPTLC method The experiment was conducted six times the percentage recovery at three different levels of friedelin was found to be 98.24, 98.59, 98.82% respectively (Table 3) 3.2.7 Robustness Robustness was studied in triplicate at 300 ng band by making small variation in optimised method parameters such as variation in composition of mobile phase, chamber saturation time The results were examined in terms of relative standard deviation (% RSD) and standard error of peak area (Table 4) Mobile phase prepared by solvent system such as Toluene: chloroform in composition (9:1 v/v), (9.2:0.8 v/v), (8.8:1.2 v/v) etc Duration of saturation time change during chromatograph development (18, 20 and 22 min) respectively The plate was activated at 110 °C for 20 and analyzed at 580 nm By introducing small changes into TLC method % RSD was obtained less than 2% proved the robustness of proposed method Conclusion In present study HPTLC method was developed and validated for the determination of friedelin in Putranjiva roxburghii Wall leaf, bark and in polyherbal formulation, which shows 0.003% w/w for friedelin in leaf extract, 0.04% w/w in bark extract, 0.002% w/w in formulation and 0.035% w/w in formulation The proposed Fig Calibration curve of Standard Friedelin at 580 nm Please cite this article in press as: K.K Abhimanyu et al., A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations, Bulletin Facult Pharmacy Cairo Univ (2016), http://dx.doi.org/10.1016/j.bfopcu.2016.11.002 K.K Abhimanyu et al / Bulletin of Faculty of Pharmacy, Cairo University xxx (2016) xxx–xxx (a) (b) (d) (c) (e) Fig HPTLC Chromatogram of standard Friedelin (a), leaf extract (b), Bark extract (c), formulation (Femiforte Tablet) (d), formulation (Femiplex tablet) (e) at 580 nm Table Method validation parameters for the quantitation of friedelin by HPTLC Parameters Results Range of linearity (ng/band) Regression of equation Slope Correlation coefficient (r2) LOD (ng/band) LOQ (ng/band) 100–500 Y = 1642.47 + 17.11x 17.11 0.9892 ± 0.003 32.15 ng 97.44 ng method was found to be simple, accurate, specific and robust for the analysis of friedelin in crude drug sample and polyherbal formulations So these studies shade on chromatographic analysis of Putranjiva roxburghii Wall and this plant containing polyherbal formulations Based on these results bark of this plant contain higher friedelin as compare to leaf, so concentrated fractions or extract of bark of Putranjiva roxburghii Wall is a rich source of friedelin and may be more useful for formulations to treat female infertility Please cite this article in press as: K.K Abhimanyu et al., A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations, Bulletin Facult Pharmacy Cairo Univ (2016), http://dx.doi.org/10.1016/j.bfopcu.2016.11.002 K.K Abhimanyu et al / Bulletin of Faculty of Pharmacy, Cairo University xxx (2016) xxx–xxx Table Interday and intraday precision of HPTLC Amount (ng/band) Interday precision a Friedelin 300 a Intraday precision Mean area SD %RSD Meana area SD %RSD 8123.43 63.85 0.78 7754.5 70.54 0.9 Mean of six determinations Fig Overlay UV absorption spectra of Friedelin in peaks of standard and extracts (a), Friedelin in peaks of standard and formulations (b) at 580 nm Please cite this article in press as: K.K Abhimanyu et al., A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations, Bulletin Facult Pharmacy Cairo Univ (2016), http://dx.doi.org/10.1016/j.bfopcu.2016.11.002 K.K Abhimanyu et al / Bulletin of Faculty of Pharmacy, Cairo University xxx (2016) xxx–xxx Table Results of accuracy study a Level of recovery (%) Theoretical content (lg/band) Experimental content (lg/band) % RSD % meana recovery 80 100 120 0.57 0.71 0.85 0.56 ± 0.005 0.70 ± 0.005 0.84 ± 0.005 0.89 0.7 0.58 98.24 98.59 98.82 Mean of three determinations Table Robustness study for the HPTLC method Factors a Chromatographic changes Level Peak areaa SD %RSD Mobile phase composition 9.2:0.8 9:1 8.8:1.2 (±2) +2 8771.48 7721.16 8361.76 145.54 132.19 125.58 1.65 1.70 1.50 Saturation time 18 20 22 (±10%) 10% +10% 7754.5 8621.48 8295.1 111.75 96.05 128.58 1.44 1.11 1.55 Mean of three determinations Conflicts of interest There are no conflicts of interest [13] [14] Acknowledgments Authors are thankful to Botanical Survey of India (BSI), Pune, Board of College & University Development (BCUD) Savitribai Phule Pune University and Dr D.Y Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune for providing instrumental facility [15] [16] [17] References [1] Anonymous, The Wealth of India: Raw Materials, New Delhi Publications & 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Friedelin in peaks of standard and formulations (b) at 580 nm Please cite this article in press as: K.K Abhimanyu et al., A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii. .. 246–255 Please cite this article in press as: K.K Abhimanyu et al., A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations,