PHYTOCHEMICAL MARKER BASED EVALUATION OF RAJAPRAVARTINI VATI USING HPTLC

PHYTOCHEMICAL MARKER BASED EVALUATION OF RAJAPRAVARTINI VATI USING HPTLC

Sachin Palekar *, Nandini Girish and Shashank Ramakrishnan

Department of Bioanalytical Sciences, Ramnarain Ruia Autonomous College, Matunga, Mumbai, Maharashtra, India.

ABSTRACT: Rajapravartini Vati is an Ayurvedic formulation prescribed for gynaecological and menstrual disorders such as amenorrhea and is comprised of raw materials, namely Shuddha Kasisa (purified green vitriol), Hing (purified Ferula asafoetida), Tankana (purified borax) and Kumari (Aloe vera) mixed in equal proportions. The present work focuses on HPTLC-based evaluation and phytochemical profiling of commercially available Rajapravartini Vati samples. The work proposes a phytobioactive compound detected in the commercial samples as a marker compound for evaluation of Rajapravartini Vati. It also establishes it as a compound different from conventional expected marker compounds such as aloin from Aloe vera and ferulic acid from Ferula asafoetida, which were not detected in the commercial samples but only in the in-house laboratory raw material mixture. Aloin and ferulic acid were subsequently quantified in the prepared sample at concentrations of 0.99 mg/g and 0.92 mg/g respectively. The findings of the study thus provide valuable insights into the phytochemical fingerprint of Rajapravartini Vati and will help propel future work on the marker compounds studied herein.

Keywords: Rajapravartini Vati, Marker compounds, HPTLC, Phytochemical evaluation, Quality, In-process changes

INTRODUCTION: In the last few years, an increasing shift towards the use of traditional medicinal systems is being observed. However, the lack of sufficient scientific validation with respect to the physicochemical and marker compound profile of such polyherbal formulations has deterred significant expansion with respect to their adoption. Thus, Ayurvedic remedies require scientific supplementation to ensure their authenticity, quality, safety and potency.

Rajapravartini Vati is one such Ayurvedic formulation which has long been prescribed for gynaecological and menstrual disorders such as amenorrhea, categorised as Artavakshaya in Ayurvedic literature. In Sanskrit terminology, ‘Rajas ‘refers to the menstrual cycle while ‘Pravartini’ describes its stimulation ^{1, 2}. The principal raw materials and their associated therapeutic roles in Rajapravartini Vati are illustrated below in Table 1 ^{3, 4}.

While the individual components themselves do not have any documented standalone roles to play in treatment of menstrual disorders, synergistic harmony between their composite phyto-constituents is hypothesised to play a role in bringing about the therapeutic effects associated with Rajapravartini Vati.

This is attempted to be scientifically understood through quantification of specific phytobioactives to provide better insights about the formulation.

TABLE 1: RAW MATERIALS USED IN RAJAPRAVARTINI VATI AND THEIR ASSOCIATED ROLES IN THE FORMULATION

This study thus comparatively evaluates commercially available formulations of Rajapravartini Vati by HPTLC fingerprinting and quantitation to provide valuable insights in to the phytobioactives in Rajapravartini Vati as well as the possible in-process changes in its phytochemical profiles. This is crucial due to it being a polyherbal formulation, where conventional marker compounds from the raw materials such as ferulic acid from Ferula asafoetida and aloin from Aloe vera are expected to be identified. Aloin is presently being explored for its anti-inflammatory, laxative, neuroprotective and anti-cancer properties ^{5, 6}. Ferulic acid is an established antioxidant compound with its pharmacological potential to serve as an anti-diabetic, anti-carcinogenic and radioprotectant properties under investigation ⁷. These compounds by themselves may however not be reflective of the synergism between them in the final polyherbal formulation. Exploration for such marker compounds thus attempts to provide a comprehensive understanding of the potential of Rajapravartini Vati in therapeutics.

MATERIALS AND METHODS:

Sample Collection and Reagents: Five different commercially available brands of Rajapravartini Vati tablets were procured from local Ayurvedic stores in Mumbai and were labelled as RPV-1, RPV-2, RPV-3, RPV-4 and RPV-5 respectively.

Raw materials required for formulating the raw material mixture of Rajapravartini Vati such as Ferula asafoetida and Aloe barbadensis powder, and Aloe vera juice were procured from Ayurvedic stores. All chemicals and reagents which were used were of analytical grade. Standard aloin (Sigma Aldrich 1415-73-2), standard ferulic acid (Sigma Aldrich 537-98-4) and standard gallic acid monohydrate (Himedia 5995-86-8) were used for all analyses wherever applicable.

HPTLC Analysis: The detailed HPTLC method parameters are illustrated herein in Table 2.

TABLE 2: COMMON METHOD PARAMETERS FOR HPTLC ANALYSIS

Preliminary Screening: HPTLC screening was carried out for preliminary identification of marker compounds across the commercial Vati samples. Methanolic extracts of the samples (RPV-1 to RPV-5) at a concentration of 10000 ppm were subjected to HPTLC separation in presence of standard gallic acid, standard aloin and standard ferulic acid solutions, each prepared in methanol at a concentration of 1000 ppm. The mobile phase composition for separation of aloin was optimised after referring to multiple literature sources based on similar chromatographic separations of aloin and aloin-related compounds ⁸. Two duplicate plates (20 cm × 10 cm) were run and derivatised with different reagents as described in Table 3.

TABLE 3: PRELIMINARY SCREENING OF COMMERCIAL RAJAPRAVARTINI VATI SAMPLES FOR MARKER COMPOUNDS

Fingerprinting of Raw Materials and Finished Formulations: To gain preliminary insights into the differences between the phytochemical profiles of commercial samples and freshly formulated raw material mixtures, a raw material mixture sample of Rajapravartini Vati in the laboratory was prepared. Each of the four raw material powders – Shuddha Tankana (purified borax), Shuddha Kasisa (purified green vitriol or ferrous sulphate), Shuddha Hing (Ferula asafoetida powder) and Kumari (Aloe barbadensis powder) of Ayurvedic grade were thoroughly pulverised and ground in a mortar and pestle to obtain a homogenous mixture in equal proportions. This was labelled as RPV-L. Based on further literature review, 60% ethanol was chosen as the extracting solvent for subsequent analyses ¹⁰. 60% ethanolic extracts of all commercial samples, the freshly formulated mixture and individual raw materials at 5% w/v concentration were prepared and subjected to HPTLC fingerprinting as illustrated in Table 4.

In addition, to gain further understanding of the impact of commercial manufacturing on phytochemical profiles, 60% ethanolic extracts (5% w/v) of commercial Aloe gel and Aloe juice were also subjected to HPTLC fingerprinting.

TABLE 4: FINGERPRINTING OF RAW MATERIALS AND FINISHED FORMULATIONS OF RAJAPRAVARTINI VATI

Quantitation of Aloin and Ferulic Acid from Raw Material Mixture: Qualitative detection of aloin and ferulic acid markers in the freshly formulated raw material mixture was followed by quantitative estimation of aloin and ferulic acid in the mixture using calibration curve method by HPTLC, as the first step towards preliminary quality control. This is illustrated in Table 5.

TABLE 5: QUANTITATION OF ALOIN AND FERULIC ACID FROM THE FRESHLY FORMULATED RAW MATERIAL MIXTURE OF RAJAPRAVARTINI VATI

RESULTS AND DISCUSSION: Preliminary screening of the commercially available samples of Rajapravartini Vati for presence of prospective marker compounds did not show presence of ferulic acid or aloin in any of the methanolic extracts of the samples, when compared with the separation profiles of standard ferulic acid and standard aloin and confirmed with derivatisation techniques, as is illustrated in Fig. 1 and 2.

The chosen mobile phase composition for aloin separation was subsequently determined and optimised to be suitable for separation of ferulic acid, allowing for simultaneous analysis of both aloin and ferulic acid in subsequent analyses. The peak shapes for the two standard components for the optimised mobile phase are illustrated in Fig. 3. These findings are illustrative of the fact that phytobioactive profiles of the commercially available formulations are likely to be influenced by synergistic in-process changes which might result in non-detection of expected marker compounds characteristic of their raw materials.

To establish whether marker compounds such as aloin and ferulic acid are detectable in raw material mixtures of Rajapravartini Vati which have not been subjected to commercial manufacturing, a fingerprinting analysis was carried out with 60% ethanolic extracts of the commercial samples.

In addition, a freshly prepared raw material mixture of Rajapravartini Vati using Ayurvedic grade raw materials was also subjected to extraction with 60% ethanol and used for fingerprinting, along with individual raw materials, commercial Aloe gel and Aloe juice samples. When subjected to chromatographic separation with the previously optimised mobile phase and standards, aloin was not detected in any of the commercial samples, including Aloe gel and Aloe juice, further highlighting the possibility of in-process phytochemical profile changes during commercial manufacturing. Aloin was however detected in Aloe barbadensis powder as well as the freshly formulated raw material mixture of Rajapravartini Vati. Ferulic acid was similarly detected in Ferula asafoetida as well as the freshly formulated mixture. This is illustrated through the images shown in Fig. 4 and 5.

FIG. 1: HPTLC PLATES AND TRACK PATTERN VISUALISED FOR PRELIMINARY SCREENING OF FERULIC ACID VISUALISED AT (A) 254 NM, (B) 366 NM AND (C) VISIBLE LIGHT POST-DERIVATISATION WITH 1% FECL₃ REAGENT. THE MOBILE PHASE COMPOSITION USED WAS TOLUENE : ETHYL ACETATE : FORMIC ACID (2:7:1) (V/V). PHENOLIC COMPONENTS SUCH AS FERULIC ACID FORM A DEEP BLUE COMPLEX WITH THE DERIVATISATION REAGENT (HIGHLIGHTED IN RED BOXES). (D) COMPARATIVE CHROMATOGRAM VIEW AND RF TABLE OF THE TRACKS SCREENED AT 254 NM.

FIG. 2: HPTLC PLATES AND TRACK PATTERN VISUALISED FOR PRELIMINARY SCREENING OF ALOIN VISUALISED AT (A) 254 NM, (B) 366 NM AND (C) 366 NM POST-DERIVATISATION WITH 10% METHANOLIC BORIC ACID. THE MOBILE PHASE COMPOSITION USED WAS ETHYL ACETATE : METHANOL : WATER (10:1.45:1) (V/V). ALOIN POSSESSES A CHARACTERISTIC RED COLOURED FLUORESCENCE WHICH TURNS ORANGE AFTER DERIVATISATION (HIGHLIGHTED IN RED BOXES). (D) COMPARATIVE CHROMATOGRAM VIEW AND RF TABLE OF THE TRACKS SCREENED AT 254 NM

FIG. 3: HPTLC CHROMATOGRAMS FOR (A) STANDARD ALOIN AND (B) STANDARD FERULIC ACID DEVELOPED USING THE MOBILE PHASE ETHYL ACETATE : METHANOL : WATER (10:1.45:1) (V/V) (AT 254 NM). GOOD SEPARATION AND PEAK QUALITY WERE OBSERVED FOR SIMULTANEOUS ANALYSIS OF BOTH ALOIN AND FERULIC ACID USING THE SAME MOBILE PHASE WHICH WAS OPTIMISED FOR FURTHER ANALYSES

FIG. 4: HPTLC FINGERPRINTS OF RAW MATERIALS AND FINISHED FORMULATIONS VISUALISED AT (A) AND (B) 254 NM, AND (C) AND (D) 366 NM. 60% ETHANOLIC EXTRACTS OF ALL SAMPLES WERE USED

FIG. 5: (A) AND (B) COMPARATIVE CHROMATOGRAM VIEW OF RAW MATERIALS AND FINISHED FORMULATIONS VISUALISED AT 254 NM (PLATE IMAGES ILLUSTRATED IN FIGURE 4). THE PRESENCE OF ALOIN (HIGHLIGHTED BY RED ASTERISK) AND FERULIC ACID (HIGHLIGHTED BY BLUE ASTERISK) IS NOTED IN THE FORMULATED VATI RAW MATERIAL MIXTURE. ADDITIONALLY, A COMMON ALOE MARKER COMPOUND (HIGHLIGHTED BY GREEN ASTERISK) IS NOTED IN THE COMMERCIAL FORMULATIONS AND ALOE GEL

A common unidentified Aloe marker compound was observed to be present in all of the commercial Vati samples as well as Aloe gel, confirmed through spectral scan overlay as shown in Fig. 6. This component requires characterisation through further sophisticated techniques of analysis such as mass spectrometry. It is likely that this compound is reflective of the in-process changes or effect of certain other supplementary manufacturing procedures in these samples. In addition, effect of certain Ayurvedic procedures such as Bhavana and Shodhana, which are likely to be carried out as part of commercial production of Rajapravartini Vati, where certain other plant extracts are employed for enriching the raw material powders through sudation or trituration, may also play a role in impacting their phytochemical marker profiles ¹¹.

FIG. 6: UV SPECTRAL OVERLAY OF (A) STANDARD ALOIN (ORANGE) WITH ALOIN DETECTED IN THE FORMULATED VATI MIXTURE, (B) STANDARD FERULIC ACID (PURPLE) WITH FERULIC ACID DETECTED IN THE FORMULATED VATI MIXTURE AND (C) ALOE MARKER COMPOUND WHICH IS COMMON TO ALL MARKETED FORMULATIONS AND COMMERCIAL ALOE GEL

Quantitative analysis of aloin and ferulic acid was subsequently carried out from the freshly formulated raw material mixture as a preliminary quality control measure as well as to validate the developed method for simultaneous analysis of aloin and ferulic acid from such samples. Calibration curve method was utilised for quantitative analysis. A five-point volume series of standard aloin (1000 ppm) (0.2, 0.4, 0.6, 0.8, 1.0 µL) and standard ferulic acid (1000 ppm) (0.4, 0.8, 1.2, 1.6, 2.0 µL) (1000 ppm) were respectively used to quantitate the two markers from the freshly formulated Vati extract by HPTLC. The plate images are shown in Fig. 7. Appreciable linearity was observed for the two calibration curves with R² = 0.9895 (aloin) and R² = 0.9867 (ferulic acid) respectively. Using the σ/S approach, the LLOD and LLOQ of aloin using the developed method were established at 0.12 µg and 0.37 µg respectively. Similarly, the LLOD and LLOQ of ferulic acid were established at 0.28 µg and 0.85µg respectively (where µg represents the amount of analyte deposited on the plate). The sample extract was applied in triplicates in increasing volumes and the final concentration of the two components was computed as the average from the three sample tracks. These are illustrated in Fig. 8. Aloin and ferulic acid were subsequently quantitated at 0.99 mg/g and 0.92 mg/g in the raw material mixture respectively.

FIG. 7: (A) HPTLC PLATE VISUALISED AT 254 NM AND TRACK PATTERN FOR QUANTITATION OF ALOIN ANDFERULIC ACID FROM THE FORMULATED VATI RAW MATERIAL MIXTURE AND (B) COMPARATIVE CHROMATOGRAM VIEW AND RF TABLE OF THE TRACKS SCREENED AT 254 NM

FIG. 8: CALIBRATION CURVES OF (A) ALOIN AND (B) FERULIC ACID FOR QUANTITATION OF THE TWO COMPONENTS FROM THE LABORATORY FORMULATED VATI RAW MATERIAL MIXTURE

CONCLUSION: HPTLC fingerprinting of the raw materials and formulations of Rajapravartini Vati provides a convenient and precise means for establishing their phytochemical marker profiles. In addition, it is also beneficial for gaining valuable preliminary insights into how in-process changes during commercial manufacturing and Ayurvedic enrichments such as Bhavana and Shodhana samskaras may serve as key factors which impact their phytochemical profiles. This is evident through the differences in the phytochemical profiles between the freshly formulated raw material mixture and the commercial formulations. Quantitative analysis of marker compounds such as aloin and ferulic acid from the raw material mixture serves as the first step towards quality control and raw material authentication in the production of Rajapravartini Vati. The findings provide a significant boost to knowledge insights on traditional Indian systems of medicine, besides opening up future avenues for exploration of these phytochemical profiles through more sensitive techniques of analysis, as well as for providing evidences of the mechanisms of action of phytobioactives in the Rajapravartini Vati.

ACKNOWLEDGEMENTS: The authors express their sincere gratitude to Principal, Ramnarain Ruia Autonomous College and Dean Research, Ramnarain Ruia Autonomous College for granting the seed money for execution of this research work.

CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interest.

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    How to cite this article:

    Palekar S, Girish N and Ramakrishnan S: Phytochemical marker based evaluation of Rajapravartini Vati using HPTLC. Int J Pharm Sci & Res 2026; 17(5): 1621-29. doi: 10.13040/IJPSR.0975-8232.17(5).1621-29.

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