A validated HPTLC method for the quantification of friedelin in putranjiva roxburghii wall extracts and in polyherbal formulations

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[.]

Original Article

A validated HPTLC method for the quantification of friedelin in Putranjiva

roxburghii Wall extracts and in polyherbal formulations

a Pharmacognosy Department, P E Society’s Modern College of Pharmacy, Nigdi, Pune, Maharashtra 411044, India

b

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

a r t i c l e i n f o

Article history:

Received 12 August 2016

Received in revised form 3 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 Rfvalue 0.43 ± 0.01 The method was val-idated using International Council for Harmonization (ICH) guidelines including linearity, precision, accu-racy, 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 1 (0.002% w/w) and formulation 2 (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, speci-fic, 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-nc-nd/4.0/)

1 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,

Aleu-rites, 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

medicinally useful Nuts are taken orally by women (sterile) in vil-lages 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 phytocon-stituents such as triterpenoids (putranjivanonol, putranjic acid, putranjivadione, roxburgholone), pentacyclic triterpene (friedelin, friedelanol) [10–12] A friedelane triterpenoid keto acid (rox-burghonic 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 chromato-graphic 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/ ).

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).

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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

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

salv-ifolium (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

chromatog-raphy and this study also help in analysis of friedelin in other

sev-eral plant species

2 Materials and methods

2.1 Plant material

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

2.2 Chemicals and reagents

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

Femi-forte tablet (formulation 1) containing extracts of Putranjiva

rox-burghii (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)

2.3 HPTLC instrumentation and experimental conditions

Method development parameters includes sample and test

solution preparation, HPTLC instrumentation condition,

prepara-tion of developing chamber, derivatizaprepara-tion 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

200lm thickness HPTLC) Samples of extracts, formulations and standards were applied by using microsyringe (Linomat syringe, Hamilton-Bonaduz schweiz, Camag, Switzerland) in band length

8 mm wide and 8 mm apart by Camag Linomat 5 sample applicator (Camag, Muttenz, Switzerland) The application rate of sample on plate was 150 nl 1 The plate was developed in previously satu-rated 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 resolu-tion was observed in the composiresolu-tion of Toluene: Chloroform (9:1 v/v) for friedelin Dry thin layer chromatographic plate deriva-tized 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

A stock solution of friedelin (100lg/ml) was prepared by dis-solving 10 mg of accurately weighed friedelin in 100 ml chloro-form For calibration 1–5ll standard solution was applied to HPTLC plate in the range 100–500 ng per band

2.5 Preparation of sample solution Based on solubility of the marker compounds, chloroform extract leaf and bark were prepared by weighing 50 g of dried pow-dered of Putranjiva roxburghii and extraction was carried out by soxhlet extraction assembly for 6 h Solution was filtered, concen-trated 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 (1000lg/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

2.6 Method validation The analytical method was validated for linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of quantiza-tion (LOQ) according to the Internaquantiza-tional Council for Harmoniza-tion (ICH, 2005) guidelines

The linearity was carried out by applying different concentra-tion of standard friedelin Quantizaconcentra-tion’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 sam-ples 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 Rfvalues and ultraviolet– visible (UV) spectra of peaks of components in sample, formulation and standard chromatogram

CH3

H3C

CH3

CH3

H3C

H

CH3

CH3

CH3

H

H O

Fig 1 Chemical structure of Friedelin.

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

3 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 Rfof 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 Rfvalue (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

ser-ies of spots of different volumes (1, 2, 3, 4, 5ll) were applied so as

to get 100–500 ng quantity of standard per band, respectively

Lin-earity 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

correla-tion coefficient (r2) value of 0.9892 for friedelin (Table 1andFig 2)

3.2.2 Quantification of friedelin in the leaves, bark and in polyherbal

formulation containing Putranjiva roxburghii Wall

3ll of the plant leaf, bark extract and 50ll of formulation 1 and

2 were applied to HPTLC plate in triplicate and the amount of

frie-delin 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 1 and 0.035% w/w in formulation 2

3.2.3 Limit of detection and quantitation

In order to determine limit of detection (LOD) and limit of

quan-titation (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 1for

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 concen-tration 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 indi-cated 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 Rfand ultravio-let–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 stud-ies at 3 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 friede-lin 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 1by mak-ing small variation in optimised method parameters such as varia-tion in composivaria-tion of mobile phase, chamber saturavaria-tion time The results were examined in terms of relative standard deviation (% RSD) and standard error of peak area (Table 4) Mobile phase pre-pared by solvent system such as Toluene: chloroform in composi-tion (9:1 v/v), (9.2:0.8 v/v), (8.8:1.2 v/v) etc Duracomposi-tion of saturacomposi-tion time change during chromatograph development (18, 20 and

22 min) respectively The plate was activated at 110°C for

20 min and analyzed at 580 nm By introducing small changes into TLC method % RSD was obtained less than 2% proved the robust-ness of proposed method

4 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 1 and 0.035% w/w in formulation 2 The proposed

Fig 2 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

method was found to be simple, accurate, specific and robust for the analysis of friedelin in crude drug sample and polyherbal for-mulations 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

(a)

Fig 3 HPTLC Chromatogram of standard Friedelin (a), leaf extract (b), Bark extract (c), formulation 1 (Femiforte Tablet) (d), formulation 2 (Femiplex tablet) (e) at 580 nm.

Table 1

Method validation parameters for the quantitation of friedelin by HPTLC.

Range of linearity (ng/band) 100–500

Regression of equation Y = 1642.47 + 17.11x

Correlation coefficient (r 2

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

Table 2

Interday and intraday precision of HPTLC.

Amount (ng/band) Interday precision Intraday precision

Mean a

a Mean of six determinations.

Fig 4 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

Conflicts of interest

There are no conflicts of interest

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

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Table 3

Results of accuracy study.

Level of recovery (%) Theoretical content (lg/band) Experimental content (lg/band) % RSD % mean a

recovery

a

Mean of three determinations.

Table 4

Robustness study for the HPTLC method.

Factors Chromatographic changes

Mobile phase composition (±2)

a

Mean of three determinations.

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