Simultaneous Quantification of Lupeol, Stigmasterol and β- Sitosterol in Extracts of Adhatoda vasica Nees Leaves and its Marketed Formulations by a Validated RP-HPLC Method

Standardization is a significant tool for ensuring the quality of herbal drugs. The drug identity, purity, content, chemical and other biological properties determine the quality of the products. Quantitative estimation of chemical markers within the crude drugs is usually troublesome. Separation of markers using optimal separation techniques offer high resolution of the compounds and least interferences.1 Herbal medicine has been utilized by 85% of the world’s population since prehistoric eras for the treatment of a variety of diseases.2 Therapeutic efficacy and safety depend upon the quality of herbal medicine. Lack of quality control profiles created obstacles in the approval of ayurvedic medicines for healthcare needs. Establishment of quality control parameters gets affected due to the complexion nature in the chemical compounds and its variability of herbal medicines. Advancement in the modernized analytical methods is increased to subdue these complications by developing an effective separation of marker compounds.3 The approaches in chromatographic separation techniques made quantification of chemical markers in crude drugs with very limited clean-up.4 Mainly, reverse phaseliquid chromatography (RP-HPLC) used for the estimation of various chemical compounds existing in single drug or mixture of drugs and its herbal formulations.5


INTRODUCTION
Standardization is a significant tool for ensuring the quality of herbal drugs. The drug identity, purity, content, chemical and other biological properties determine the quality of the products. Quantitative estimation of chemical markers within the crude drugs is usually troublesome. Separation of markers using optimal separation techniques offer high resolution of the compounds and least interferences. 1 Herbal medicine has been utilized by 85% of the world's population since prehistoric eras for the treatment of a variety of diseases. 2 Therapeutic efficacy and safety depend upon the quality of herbal medicine. Lack of quality control profiles created obstacles in the approval of ayurvedic medicines for healthcare needs. Establishment of quality control parameters gets affected due to the complexion nature in the chemical compounds and its variability of herbal medicines. Advancement in the modernized analytical methods is increased to subdue these complications by developing an effective separation of marker compounds. 3 The approaches in chromatographic separation techniques made quantification of chemical markers in crude drugs with very limited clean-up. 4 Mainly, reverse phaseliquid chromatography (RP-HPLC) used for the estimation of various chemical compounds existing in single drug or mixture of drugs and its herbal formulations. 5 Adhatoda vasica Nees (Acanthaceae), also known as Vasaka in Ayurveda, is a perennial shrub prevalent in the hot regions of Southeast Asia. 6 The plant is extensively used in Indian system of medicine (ISM) for the medication of respiratory system related ailments such as cough, asthma, allergeninduced bronchial obstruction and it has been used as an herbal remedy for tuberculosis. 7 The medicinal properties of A. vasica characteristic to the presence of active phytoconstituents such as alkaloids, glycosides, flavonoids, coumarins, quinines triterpenes, polysaccharides, polyphenols, vitamin C, proteins and essential oils etc. 8 A. vasica leaves contain tricyclic quinazoline alkaloids (pyrroquinazoline alkaloids) viz. a major alkaloid, vasicine, vasicinone and deoxyvasicine. 7 Roots of A. vasica possess vasicol and vasicinolone, kaempferol and quercetin were present in the plant flowers. 9 Apart from these, leaves and roots found to the presence of other metabolites including, terpenoids, steroids, saponins, proteins, amino acids, fats, gum resins and sugars. Various chromatographic methods were reported for the estimation of alkaloids such as vasicine and vasicinone by HPTLC and HPLC in crude drug and its formulations. UHPLC/QqQLIT-MS/MS reported for the simultaneous determination alkaloids and flavonoids in A. vasica leaves. 10 Several methods were available for the quantification of lupeol and stigmasterol, lupeol and β-sitosterol by both HPTLC and HPLC. 11,12 and sterols by HPLC 13,14 Literature survey revealed that there is no method available for the simultaneous estimation of lupeol, stigmasterol and β-sitosterol.
In the current investigation, we have established a simple, rapid and optimized HPLC method for the simultaneous estimation of lupeol, stigmasterol and β-sitosterol ( Figure 1) in A. vasica extracts. A. vasica containing Ayurvedic formulations were also evaluated for lupeol, stigmasterol and β-sitosterol. The developed method was evaluated for different parameters and validated according to the International Conference on Harmonization (ICH) guidelines. 15 Validation has been carried out based on its linearity, accuracy, precision, limit of detection and limit of quantification. This is the first report on simultaneous quantification of three markers in A. vasica leaf extracts. Kancheepuram, Tamil Nadu, India. The collected materials were cleaned, shade dried and pulverized in a blender, sieved through 60 mesh sieve and well-preserved in amber coloured airtight container at room temperature.

Standards and reagents
Lupeol (97%), stigmasterol (99%) and β-sitosterol (98%) standards were procured from Natural Remedies Pvt. Ltd, Bangalore, India. Formulation I, II and III were purchased from Ayurvedic pharmacy, Metabolic ward, Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM Institute of Science and Technology, Kattankulathur. Formic acid (analytical grade), methanol (HPLC grade) were procured from Ranchem Private Limited. Mill-Q-water from Millipore was used. Chemicals of analytical grade were obtained from Merck Specialities Private Limited, Mumbai.

Extraction of A. vasica
Each 150g of powdered A. vasica were extracted in 750ml of hexane, chloroform, ethyl acetate and methanol for 72h at room temperature for non-successive extraction. The extracts were filtered and re-extracted thrice by following the same procedure. The filtrates were combined and concentrated under reduced pressure using a Buchi rotary evaporator.

Preparation of Ayurvedic formulations
Each 5 tablets of Formulation I and II were grinded in a mortar and pestle and 5ml of Formulation III were evaporated to dryness. 100mg of each sample were dissolved in 1mL of methanol and sonicate for 15min. The sample was then filtered and made up to 1mL of methanol.

Instrumentation and chromatographic conditions
Shimadzu LC-20AD HPLC system equipped with CT0-20A controller and the column oven. A Rheodyne 7725 injection valve with 20 µL loop volume, an SPD-M20A photo-diode array detector and a Lab solution version 7.1 software was used for data acquisition and interpretation. Chromatographic separation was achieved on RP C 18 Phenomenex column (250mm x 4.6mm; i.d., 5µm pore size). The solvent mixture of 0.1% formic acid in water: methanol (28:82%v/v) at a flow rate 0.8mL/ min, the column was maintained at room temperature and the PDA detector was fixed at 208 nm.

Statistical analysis
The data were statistically analysed using the one-way analysis of variance (ANOVA). The results were presented as mean of three replicates ± standard error of mean (SEM) n=3

RESULTS AND DISCUSSION
Optimization of HPLC chromatographic conditions HPLC chromatographic conditions were optimized after running with a C 18 reverse phase column using different mobile phases. All the three marker compounds showed a satisfactory response of detection wavelength at 208nm. Ambient temperature was maintained in the column till the completion of the analysis. A mobile phase of 28%v/v of 0.1% formic acid in water (A) and 82%v/v of methanol (B) used with 0.8mL/min flow rate in an isocratic elution. An HPLC fingerprint was developed for A. vasica extract with an optimized chromatographic condition.

Preparation of standards and samples
Standard solutions of lupeol, stigmasterol and β-sitosterol were prepared and diluted in the concentration range of 12.5 to 200µg/mL in methanol for construction of calibration curve.
Methanolic stock solutions (10mg/mL) of hexane, chloroform, ethyl acetate and methanolic extracts of A. vasica were prepared and filtered through a 0.22μm PVDF membrane and stored at -20°C.

Development of calibration curve
Calibration was achieved with six dilutions of standards, at concentrations ranging from 12.5 to 200µg/mL and triplicate measurement was performed. The linearity of each standard attained by plotting peak areas versus standard concentrations. Identification of peak was attained by comparing both retention time (R t ) and UV absorbance (λ max ).

Method validation
According to the ICH guidelines, 15 validation was performed for the developed method for linearity, accuracy, precision, LOD and LOQ. Linearity was performed in the concentration range of 12.5-200µg/ mL of standards. The accuracy was calculated by means of percentage recovery, which was determined by the standard addition method. Standard solutions of 80%, 100% and 120% were spiked into the samples prior to the extraction and analysed under the optimized method to estimate the spiked recoveries. Precision was determined using repeatability and intermediate precision. Repeatability or intraday precision was determined by injecting the standards six times on the same day. Intermediate precision was performed with the same sample in the same way on different day. Limit of Detection (LOD) and Limit of Quantification (LOQ) of standards were estimated separately with a standard calibration curve.

Quantification of marker compounds in marketed formulation
The marker compounds were identified and quantified in the ayurvedic formulations depicted in Figure 3 and Table 2. Formulation III revealed the presence of high content of lupeol (23.72ng/g) whereas stigmasterol (2.57ng/g) and β-sitosterol (0.98ng/g) content were more in Formulation I.

Method validation for HPLC fingerprinting analysis
The developed RP-HPLC methodology was validated and evaluated by determining the range and linearity, accuracy, precision, LOD and LOQ. The technique was also assessed for qualitative analysis by considering the precision and selectivity of eluted chemical marker. Repeatability was achieved with a high concentration of standards and extracts. Linearity, precision, accuracy, LOD and LOQ were estimated for the quantification of markers. The determination of LOD and LOQ were 0.66 and 2.01µg/mL for lupeol, 5.64 and 17.10µg/mL for stigmasterol and 12.08 and 36.62µg/mL for β-sitosterol, correspondingly. The linearity of three markers was obtained with concentrations versus peak areas in the concentration range of 12.5-200µg/mL and R 2 were higher than 0.99 (Table 3). 98.19-102.31% of recovery of the compounds showed an acceptable accuracy. Relative standard deviation (RSD) of all compounds was less than 2%, a representative for high repeatability (Table 4). Precision and intermediate precision have a very low coefficient of variation reveals the method is precise ( Table 5). The method validation parameters were shown in Figure 4.     Data are represented as mean ± SD (n=3)   Data are represented as mean ± SD (n=6)