INTRODUCTION:

Pterostilbene (trans-3, 5-dimethoxy-4-hydroxystilbene) is a naturally derived compound found primarily in Pterocarpus marsupium heartwood and blueberries^1,2. It is an active constituent in Pterocarpus marsupium and the primary active constituents of blueberries^3-6. It was first isolated from Pterocarpus santalinus⁷. It commonly grows in India and Sri Lanka. It is also known as Vijaysaar in Hindi, Sarfaka in Sanskrit and Indian kino in English Heartwood of P. marsupium widely used for diabetes mellitus^8-16.

Other pharmacological activities of various parts of P. marsupium were also reported like antioxidant^17-20, hypolipidemic, antifungal activities^21-24.

Pterostilbene was identified as the major phenolic compound in darakchasava, a traditional Ayurvedic medicinal drink from India used to treat cardiovascular and other ailments²⁵. Pterostilbene has several therapeutic properties in a vast range of human diseases that includes neurological, cardiovascular and hematologic disorders. It was shown to be potent anticancer agent in several malignancies²⁶.

Several spectroscopic and chromatographic methods were developed for the determination of Pterostilbene. Amongst available liquid chromatographic methods one with fluorescence detection²⁷ and other two are based on HPLC analysis^28-42. One spectroscopic method is also reported.

In present research a new rapid and sensitive HPLC method was developed and validated according to ICH guidelines. Proposed work also includes forced degradation studies of Pterostilbene.

Fig. 1: Chemical Structure of Pterostilbene

MATERIAL AND METHODS:

Chemicals and Reagent:

Pterostilbene was obtained from TCI chemicals (India) Pvt. Ltd. HPLC grade Acetonitrile was purchased from Rankem. HPLC grade water was used for proposed study.

Instruments:

High Performance Liquid Chromatography (Agilent Technologies) was used for chromatographic analysis. The system was equipped with a G1329B auto sampler system. G1315F variable wavelength detector (Agilent Technologies) was used for the analysis. HPLC grade water was obtained from “Extra pure” water purification system (Lab link). Mobile phase was degassed by using Ultrasonicator (PCi Analyticals). Vibra HT (Essae) analytical balance was used for weighing of chemicals,

Preparation of Mobile Phase:

Mobile phase composed of Water: ACN (35:65 v/v) used for HPLC analysis. It was filtered through 0.22μm filter and degassed by ultrasonication for 10min.

Chromatographic conditions:

All analyses were done under isocratic condition at ambient temperature. Before analysis sample was filtered through 0.2μm filter. The mobile phase was run at a flow rate of 1mL/minute for ten minutes. The column eluent was monitored at 306nm with UV detection and injection volume was 20μL.

Preparation of standard stock solution:

Stock solutions-I (1mg/mL) of pterostilbene were separately prepared in HPLC grade ACN and filtered through 0.45µm nylon membrane syringe filter.

Preparation of standard calibration curve:

Calibration curve was prepared to achieve the six different calibration standards representing 0.5, 1, 2, 3, 5 ng/mL strength of Pterostilbene. It was injected into HPLC column and the peak area was measured. The standard calibration curves of peak area Vs concentration (ng) were plotted.

Method Validation:

The method was validated according the Q2(R1) guidelines of International Conference on Harmonization (ICH) with respect to system suitability, linearity, range, LOD, LOQ, accuracy, precision. The forced degradation was validated according to ICH guidelines.

System Suitability:

System suitability test is generally used to ensure that the complete system including reagents, instrument, columns analysts etc. is suitable for intended analysis. Parameters usually determines are: resolution, theoretical plate count, tailing factor and reproducibility. The system suitability test was carried out using freshly prepared solutions of 1ng/mL of Pterostilbene solution was repeatedly analyzed by using proposed HPLC conditions. Acceptable upper limit of %RSD for peak area and retention time was set at 2 whereas acceptable lower limit of number of theoretical plates was set at 2000. System was considered to be suitable only when obtained values were within the set limits.

Linearity and Range:

Linearity of the proposed method was calculated by using six different calibration standards representing 0.5, 1, 2, 3, 5ng/mL strength of Pterostilbene. Concentration vs. peak areas were plotted, subjected to linear regression analysis and linearity in terms of R-squared values and respective range were reported.

Accuracy (% Recovery):

Accuracy of pre-optimized HPLC method was assessed using recovery studies by standard addition method. To the solutions with predefined amount of Pterostilbene (1, 2.5 and 4.5ng/mL), its 80, 100 and 120% amount was added externally and the % recovery was calculated.

Precision:

The precision of the analytical method was evaluated by performing Intra-day and Inter-day studies. Intra-day precision study was carried out by analyzing five replicates of three different concentrations (1, 2.5 and 4.5ng/mL of Pterostilbene) at morning, afternoon and evening time of the same day. Similarly, inter-day precision study was carried out by analyzing the samples on three consecutive days. Intra- and inter-day precision results were expressed in terms of %RSD.

Robustness:

Robustness of the proposed HPLC method was evaluated by making slight changes in the given chromatographic conditions. Modified chromatographic conditions for the assessment of robustness were ±1^oC deviation in column temperature, ±0.05ml/min deviation in flow rate of mobile phase and ±1unit deviation in volume of ACN and water. For the robustness study, a solution (2ng/mL) was repeatedly (n=5) analyzed for retention time and peak area of Pterostilbene using above-mentioned modified chromatographic conditions. Results of the robustness study were expressed in terms of %RSD. Proposed method was considered to be robust only when the %RSD values for both retention time and peak areas were below 2.

Limit of detection (LOD) and Limit of quantification (LOQ):

The LOD and LOQ were calculated by analyzing the CAL STD-1. Chromatogram of CAL STD-1 was processed using HPLC software settings “Annotations” and obtained values were reported as LOD and LOQ for Pterostilbene.

Force degradation studies:

Forced degradation studies provide information about possible degradation product and degradation pathways of molecule. These are undertaken to degrade active drug. These studies are used to evaluate an analytical methods ability to measure an active ingredient and its degradation product. Sample or drug substances are exposed to acid, base, oxidizing agent, water and UV light.

Acid and alkali hydrolysis:

125µl of Pterostilbene solution (1mg/ml) was mixed with 5ml of 0.1N HCL and 0.1 N NaOH. The resulting solution was subjected to reflux in a boiling water bath (80⁰C) for 2 hrs and sample was cooled to room temperature. The treated solution was neutralized with acid or base. From the resulting solution 1μl was injected into the column under above mentioned chromatographic conditions.

Oxidation:

125µl of Pterostilbene solution (1mg/ml) was mixed with 5ml of 10% hydrogen peroxide solution. The resulting solution was subjected to reflux in a boiling water bath (80⁰C) for 2 hrs and sample was cooled to room temperature. The treated solution was neutralized with HPLC grade water from the resulting solution 1μl was injected into the column under above mentioned chromatographic conditions.

Thermal conditions:

125µl of Pterostilbene solution (1mg/ml) was mixed with 5ml of HPLC grade water. The resulting solution was subjected to reflux in a boiling water bath (80⁰C) for 2hrs and sample was cooled to room temperature. The treated solution was neutralized with HPLC grade water from the resulting solution 1μl was injected into the column under above mentioned chromatographic conditions.

Photolytic conditions:

1mg of Pterostilbene was exposed to UV light (254nm) for 2hrs. The material was dissolved in 1ml water to prepare solution of 1mg/ml concentration. From the resulting neutral solution, 1μl was injected into the column under above mentioned chromatographic conditions.

Application of proposed HPLC method:

Proposed HPLC method was used for the estimation of Pterostilbene in various extracts of Pterocarpus marsupium. Three different types of extracts of Pterocarpus marsupium viz. aqueous, aqueous-ethanolic and 95% ethanolic were prepared. In order to prepare aqueous extract, 20gm of Pterocarpus powder was placed for hot maceration technique with 200ml of distilled water for 8 hrs. Obtained aqueous extract was filtered and lyophilized using lab Lyophilizer. Aqueous-ethanolic extract of Pterocarpus was prepared using cold maceration. Twenty gm of Pterocarpus powder was imbibed in 200ml mixture of distilled water and 95% ethanol (50:50 v/v) for 12 hrs. After completion of extraction period, extract was filtered and evaporated at 60^oC to dryness using rotary vacuum evaporator. Ninety five % ethanolic extract of Pterocarpus powder was prepared using Soxhlet apparatus. Twenty gm of Pterocarpus powder was placed in thimble and extracted repeatedly for 8hrs using 95% ethanol as extraction solvent. The extract was then evaporated to dryness using rotary vacuum evaporator. All the extracts were stored in vacuum desiccator until analyzed.

Five mg of aqueous, aqueous-ethanolic and 95% ethanolic extracts was weighed separately using analytical weighing balance. Each extract was dissolved in 5ml of HPLC mobile phase and placed for ultra-sonication. Samples were further diluted with HPLC mobile phase so as to achieve solutions with strength of 100µg/ml (based on dry weight of extract) and filtered through 0.22um syringe filter. Filtered samples were analyzed for the Pterostilbene content using proposed HPLC method.

RESULTS AND DISCUSSION:

Optimization of RP-HPLC Method:

Resolution was considered to be the most important criteria for the method and was imperative to achieve good resolution among the both compounds. Based on pKa and solubility of both the compounds, various compositions of mobile phase were tried and best resolution was obtained with mobile phase consisting of water and Acetonitrile in the ratio of 35:65 v/v. Better resolutions of the peaks with clear base line was found. Detection was carried out at 306nm. Optimized chromatographic conditions are given in Table 1. Under these conditions retention time for Pterostilbene were 3.19min shown in figure 2.

Table 1: The optimized chromatographic conditions

Separation variable	Optimized conditions
Chromatography	Agilent 1260 Series
Column	C-18, 150 x 4.6 mm, 3 um, ACE
Mobile phase	Water: ACN (35:65 v/v)
Flow rate	1 mL/min
Total Run Time	6 min
Pressure	165-170 bar
Temperature	40 degree Celsius
Detection wavelength	306 nm
Retention time of Pterostilbene	3.19 min

Fig 2. HPLC Chromatogram of Pterostilbene

System suitability:

On the basis of obtained results, it was found that system is suitable for the analysis. The details of system suitability results are summarized in Table 2.

Table 2: System suitability parameters for Pterostilbene

Sr. No.	Parameter	Acceptance criteria	Results
Sr. No.	Parameter	Acceptance criteria	Pterostilbene	% RSD
1	Retention Time	%RSD ≤ 2%	3.195	0.057
2	Peak Area	%RSD ≤ 2%	69368	1.40
3	Theoretical plates	≥ 2000	9413	461

Method validation:

Linearity and Range:

Different concentrations and peak area values for Pterostilbene are depicted in Table 3 (Fig 3). From the linearity study, it was revealed that, there is a linear relationship between response and amount of drug within the range 0.5-5ng/ml.

Fig 3. Calibration curve of Pterostilbene

Table 3: Linearity of Pterostilbene

Sr. No	Conc. (ng/mL)	Peak Area
1	0.5	70139
2	1	141165
3	2	293613
4	3	441060
5	4	586792
6	5	74334
7	Slope	6.697
8	y-intercept	0.0426
9	R²	0.9998

Accuracy (percentage Recovery):

From the results of accuracy studies, it was concluded that the proposed method was accurate.

Precision:

The results expressed in terms of % RSD for the intra and inter-day precision study depicted in Table 5 and 6 respectively. It was concluded that the proposed analytical technique showed good repeatability.

Table 4: Recovery studies of Pterostilbene

Sr. No.	Sample	Spiked level	Theoretical Concentration (ng/mL)	Practical Concentration (ng/mL)	% Recovery	Mean % Recovery	% RSD
1	Pterostilbene	80%	1	0.9849	98.49	98.65 ±0.49	1.18 ±0.19
		100%	2.5	2.47	98.97
		120%	4.5	4.54	100.93

Table 5: Intra-day precision data for Pterostilbene

Sr. No.	Pterostilbene
Sr. No.	Amount present (ng/ml)	Amount recovered (ng/ml)	% Assay	% RSD
1	1	0.9974	99.74	0.0410
2	2.5	2.4999	99.99	0.0133
3	4.5	4.5779	101.73	0.0068

Table 6: Inter-day precision data for Pterostilbene

Sr. No.	Pterostilbene
Sr. No.	Amount present (ng/ml)	Amount recovered (ng/ml)	% Assay	% RSD
1	1	0.9988	99.87	0.0335
2	2.5	2.4981	99.23	0.0111
3	4.5	4.5861	101.91	0.0055

Robustness:

Robustness of the proposed method was established by slightly changing the column temperature, mobile phase flow rate and mobile phase composition. The % RSD values were found to be less than 2 (Table 7). Thus, proposed method was found to be robust.

Table 7: Robustness study for Pterostilbene

Sr.	Parameter	Setting	Pterostilbene
Sr.	Parameter	Setting	RT	% RSD	Peak Area	% RSD
1	Column Temperature (°C)	38	3.18	0.18	1.95	1.84
		40	3.19	0.18	2.02	2.05
		42	3.20	0.31	1.98	2.53
2	Mobile phase flow rate (ml/min)	9	3.20	0.18	1.91	2.57
		1	3.19	0.47	2.00	1.75
		1.1	3.18	0.31	2.13	1.69
3	Mobile phase composition (% v/v)	34:66	3.18	0.31	2.01	1.74
		35:65	3.19	0.18	1.99	0.76
		36:64	3.20	0.31	1.97	1.54

Force degradation studies:

Pterostilbene was exposed to water, acid (0.1N Hcl), base (0.1N NaOH), oxidizing agent (10% H₂O₂), and UV light. Results are summarized in Table 8 and Chromatograms are shown in Figure 4.

Table 8: Summery of stressed degradation studies

Drug	% of degradation
Pterostilbene	Acid	Base	Oxidation	Water	UV
	100	98.16	98.24	98.32	21.60

Fig 4. Chromatograms of stressed condition: a) Chromatogram of Acid degradation; b) Chromatogram of Alkali degradation; c) Chromatogram of Oxidation degradation; d) Chromatogram of Hydrolytic degradation; e) Chromatogram of Photolytic degradation

Application of proposed HPLC method:

Proposed HPLC method was successfully used for the estimation of Pterostilbene content of various extracts of Pterocarpus marsupium. Analytical standard of Pterostilbene and the extracts of Pterocarpus marsupium when analyzed by using proposed HPLC method showed the presence of Pterostilbene at the average retention time of 3.2min. The chromatograms showing presence of Pterostilbene in aqueous, aqueous-ethanolic and 95% ethanolic extracts are shown below in the Figure 5. Pterostilbene content of the aqueous extract (extractive yield: 5.25%) of Pterocarpus marsupium powder by using proposed HPLC method was found to be 1.732µg. Similarly, Pterostilbene content of aqueous-ethanolic extract (extractive yield: 10%) was found to be 30.540 µg. Ninety five % ethanolic extract of Pterocarpus marsupium (extractive yield: 3.33%) was found to contain 34.663µg Pterostilbene.

(a)

(b)

(c)

Fig 5 Chromatograms showing Pterostilbene content of Aqueous extract (a), Aqueous-ethanolic extract (b) and 95% ethanolic extract (c) of Pterocarpus marsipium

CONCLUSION:

In this study, an accurate and sensitive HPLC method was developed for Pterostilbene. The method was validated according the Q2 (R1) guidelines of International Conference on Harmonization (ICH). The main feature of the present method is short run time around 6 min. The forced degradation was validated according to International Conference on Harmonization (ICH). The proposed method was robust enough to reproduce accurate and precise results under different chromatographic conditions. The proposed method was successfully applied to various extracts of Pterocarpus marsupium for estimation of Pterostilbene.

CONFLICTS OF INTEREST:

The authors declare that they have no conflict of interest.

ACKNOWLEDGEMENT:

The extra-mural grant support of DST-DPRP, Govt. of India (Ref: VI-D&P/626/2018-19/TDT) sanctioned to P.I. Dr. Sachin S. Bhusari for the proposed research work is highly acknowledged.

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Received on 11.09.2020 Modified on 12.06.2021

Research J. Pharm. and Tech. 2022; 15(7):2969-2975.

DOI: 10.52711/0974-360X.2022.00495