Quantitative Estimation of Piperine in Drakshadi Ghrita by HPTLC Method

Jagriti Chandrakar¹, Neeraj Agrawal¹, LowkeshChandravanshi¹, Satyawati Rathia¹, Arun KS Parihar², Chandan Kumar Sahu², Nagendra Singh Chauhan^2*

¹Department of Kaumarabhritya, Shri Narayan Prasad Awasthi Government Ayurved College Raipur (C.G.), India.

²Drugs Testing Laboratory AvamAnusandhan Kendra, Raipur, Chhattisgarh 402010, India.

Abstract:

The present study focuses on the HPTLC estimation of piperine in Drakshadi Ghrita. The method involves the extraction of piperine from the Drakshadi Ghrita formulation, followed by its separation and quantification using an HPTLC system. The analysis is performed on a silica gel 60 F254 plate, using a suitable mobile phase for the optimal separation of piperine. The HPTLC analysis was performed using a developing solvent system consisting of toluene, ethyl acetate, and formic acid in a ratio of 7:3:0.3 (v/v/v). The HPTLC method accurately quantified piperine in Drakshadi Ghrita with Rf values of 0.43. The technique showed results presented as the coefficient of variance of 24.03. When the plate is visualized under UV light, specific wavelengths detect piperine due to its unique fluorescence properties. The HPTLC method developed offers a reliable approach for the quality control and standardization of Drakshadi Ghrita, facilitating consistent potency and ensuring its therapeutic efficacy.

Keywords: HPTLC, Piperine, Drakshadi Ghrita, Mobile phase, Solvent system.

1. Introduction

Undernutrition is a global health issue in developing countries (Olaf Müller etal., 2005) Reduced dietary intake, failure of absorption and excessive nutrient loss are the primary causes of undernutrition (OP Ghai, 2023). Drakshadi Ghrita is a classic Ayurvedic formulation containing Draksha (grapes), ghrit, pippali and yastimadhu. It is a highly nutritious and excellent source of good fats that nourish, enhance digestion and increase immunity (Manisha Jagtap (2019). According to Vangsen Samhita Drakshadi Ghrita Balmamsavriddhikar refers to increasing body mass and strength (Tripathi Pandit Harihar Prasad 2016). Drakshadi Ghrita is a calorie-dense formulation high in good fats and necessary fatty acids that help nourish the body's tissues (Dhatus). These characteristics make it a good choice for increasing overall energy levels, stimulating muscle mass development and enhancing growth. The involvement of ghrita and pippali makes it a good choice for boosting digestive fire (Jatharagni) which is said to improve digestive health (Sharma P.V.   2003). Combining the action of ghee and herbs increases the immune system, improving resistance to infections and diseases. Drakshadi Ghrita is commonly used to balance the Vata and Pitta doshas and is beneficial for poor digestion, debility, weariness and respiratory problems. It is thought to restore the body's tissues, increase strength and maintain the digestion system. It is known for its health benefits, particularly regeneration and overall strength.

Pippali (Piper longum Linn.) is also known as "Long pepper". It has spread throughout the tropical and subtropical parts of the earth. Pippali is an important medicinal plant from the Piperaceae family that is utilized in Indian traditional medicine. Piperine is one of the primary active ingredients in pippali. It is an alkaloid (1-[5-(1, 3-benzodioxol-5-yl)-1-oxo-2,4-petadienyl].According to some reports, Pippali (Piper longum) contains up to 4-5% piperine(Chopra B. et al, 2016). Pippali (Piper longum) improves both the digestive and respiratory systems (Gurjar Hemwati 2024).

Pippali is a prevalent ayurvedic supplementary integrant that improves the bioavailability and absorption of other active substances (Chaudhri SK2023). Pippali exhibits a variety of actions including carminative, appetizing, cardioprotective, hepatoprotective, immunomodulatory, larvicidal and treatment of asthma, cough and respiratory disorders, as well as weakness, dementia, insomnia, fever, diabetes, rheumatic diseases and spleendisorders (SharmaP.V.2003). Acharya Charak recommended pippalirasayan for the treatment of respiratory illnesses and pippalivardhamanrasayan for digestive issues. It has anti-aging properties promotes longevity, increases well-being and intelligenceand improves health (Tripathi Dr. Brahmanand 2013).

Modern study has established that piperine improves digestion, reduces inflammation and relieves pain and asthma (Singh, A. et al. 2009). The goal of this study was to estimate piperine in Drakshadi Ghrita by HPTLC method and create a quality control tool for its efficacy.

Fig. 1. Chemical structure of piperine

2.  Materials and methods

2.1 Plant Material and Formulations

The Fruit of Piper longum, Vitis vinifera, and Glycyrrhiza glabra was collected from Karnataka in April 2024. The sample was authenticated by Shri B.M.K. Ayurveda Mahavidyalaya A constituent unit of KLE Academy of Higher Education and Research Central Research Facility Belgaum at Karnataka in April 2024. Drakshadi Ghrita (Table 1) was prepared at Government Ayurveda College Raipur under the guidance of Ras Shastra Avam Bhaisajya Kalpana department in June 2024.

Table 1 Representing DrakshadiGhrita ingredients

2.2 Physicochemical Analysis of ingredients

The ingredients of Drakshadi Ghrita and Drakshadi Ghrita formulation were tested in Drugs testing laboratory Avam Anusansdhan Kendra Raipur Chhattisgarh as per protocols given in Ayurvedic Pharmacopeia of India (Table 2-4).

Table 2 Physicochemical Analysis of ingredients of Drakshadi Ghrita

(NMT- Not more than, NLT- Not less than)

Table 3 Physicochemical Analysis of Go- Ghrita

(NMT- Not more than, NLT- Not less than)

Table 4 Physicochemical Parameters of Drakshadi Ghrita

2.3. Chemical and reagents

The authentic marker of Piperine was obtained from Yucca Enterprises Mumbai, Maharashtra and the precoated Silica-G aluminium plate was obtained from Merck, Darmstadt, Germany. Methanol was procured from Loba Chemical Pvt. Ltd. TLC plates coated with silica gel 60F254 were used for HPTLC. All other chemicals, reagentsand solvents were used of Analytical Reagent grade.

2.4 Preparation of the test sample

500 mg of the sample was dissolved in 20 ml of Petroleum ether and left for 24 hours. The next day, the solution was filtered and the extract was dissolved in 10 ml of methanol. Thereafter extract was sonicated for 10 minutes before HPTLC analysis. The extract was filtered through a 0.2 um syringe-filter to obtained a clear solution.

2.5 Standard piperine solution.

10 mg of Piperine was accurately weighed and dissolved in 20 ml of methanol to prepare a (0.5 mg/ml) stock solution. The solution was sonicated for 10 minutes in an ultrasonic bath to achieve a homogeneous solution. The solution was then transferred to a tightly sealed volumetric flask and refrigerated.

Instrumentation and chromatographic parameters

Piperine was detected and quantified in Drakshadi Ghrita by utilizing the CAMAG HPTLC system. HPTLC separation was performed on a TLC plate coated with silica gel 60 F254 (200 × 100 mm) with a thickness of 0.25 mm using the Linomat 5 automatic sample spotter; all solutions were placed with a bandwidth of 7 mm and a distance of 11.4 mm from the bottom border. The application rate remained constant at150 nl/s. The plate was raised to 70 mm in a CAMAG twin-trough glass chamber. The chamber was pre-saturated with mobile phase vapor for 20 minutes at 25 °C before being developed with a solvent mixture of toluene, ethyl acetate and formic acid (7:3:0.3, v/v/v). The plate was visualized using Visualiser 2 (CAMAG, Switzerland). TLC scanner 4 (CAMAG, Switzerland) was utilized for densitometric scanning at 254 nm. The slit measured 5×0.2 mm with a scanning speed of 20 mm/s. The densitometric investigation was analysed using the Vision CATS system.

Calibration curve

As previously described, standard solution aliquots of piperine were applied (0.25µg, 0.5µg, 1.0µg, 1.5µg, 2.0µgand 3.0µg) over the silica gel 60 F254 plate. The plate was created and examined in order to produce a calibration equation for the quantification of piperine in samples (Figure 2).

2.8Method validation

The procedure for the validation of the analytical techniques followed the ICH recommendations (Jha., et al. 2024) the process was found to be accurate, precise and reproducible (Figure 3 to 6).The method's repeatability was evaluated by repeatedly scanning the same piperine with the findings provided as the co-efficient of variation (24.03%). Piperine specimens (0.25-3.0 ug) were analysed many times to investigate the method's variability. The results were presented as a percentage of the confidence interval (CV). To assess the accuracy of the approach, the recovery percentage and average recovery percentage were calculated.

3. Results

The HPTLC analysis was performed out using a developing solvent system that includes toluene, ethyl acetateand formic acid in the ratio 7:3:0.3 (v/v/v). The HPTLC method accurately measured piperine in Drakshadi Ghrita with Rf values of 0.43 and results presented as the coefficient of variance (24.03%).In Drakshadi Ghrita concentration of piperine phytoconstituents in track 3, track 4, track 5 and track 6 is respectively 46.23 (µg/ml),40.03 (µg/ml), 28.80 (µg/ml) and 25.63 (µg/ml) (Table 5). The procedure achieved a linear range of 0.25-3.0 µg/band with high accuracy and minimal fluctuation (Figure 2-6).

Table 5 Concentration of piperine in Drakshadi Ghrita

Fig.2. Calibration curve of Piperine

Fig.3. HPTLC Chromatogram of Piperine and DrakshadiGhrita

Fig.4. TLC Fingerprint at 254 nm                                 Fig.5. Calibration bar

Fig. 6.Spectrum analysis of Piperine

4. Discussion

The process of HPTLC analysis involves several critical steps, beginning with sample preparation. In the case of Drakshadi Ghrita, the formulation is extracted using an appropriate solvent that can effectively dissolve piperine while minimizing interference from other substances present. Once the extract is prepared, it is applied onto a pre-coated silica gel HPTLC plate. The application is conducted using a fine capillary tube or an applicator, ensuring uniform spots for effective resolution during the chromatographic run. After application, the plate is developed in a suitable mobile phase often a combination of solvents such as toluene, ethyl acetate and formic acid in a ratio of 7:3:0.3 (v/v/v) to achieve optimal separation of piperine from other constituents. Following development, the plate is visualized under UV light and specific wavelengths are used to detect piperine due to its unique fluorescence characteristics. The intensity of the spots corresponding to piperine can then be quantified using a densitometer. Calibration curves are established by analyzing standard solutions of known piperine concentrations, enabling accurate quantification in the test samples by comparing their peak areas to those of the standards. HPTLC analysis of piperine in Drakshadi Ghrita was also confirmed by spectrum analysis of piperine in 332-333 wavelengths. The results obtained from the HPTLC analysis of piperine in Drakshadi Ghrita provide crucial information regarding its quality and consistency. Analysis of piperine in Drakshadi Ghrita using High-Performance Thin-Layer Chromatography (HPTLC) not only offers insights into the quality and potency of this formulation but also ensures the advantages of thin-layer chromatography with high-performance capabilities, allowing for precise separation, identification, and quantification of compounds in complex mixtures. The analysis indicates that piperine is present in Drakshadi Ghrita. Due to the presence of piperine the efficacy and bioavailability of Drakshadi Ghrita may increase. It is a traditional Ayurvedic formulation, known for its therapeutic properties, particularly in enhancing digestive health and increasing body weight.

5. Conclusion

The HPTLC analysis of piperine in Drakshadi Ghrita demonstrates the integration of modern analytical techniques with traditional herbal therapy. This approach not only confirms DrakshadiGhrita's legitimacy and efficacy but also establishes a platform for future study and development in herbal pharmacology. As the demand for natural resources grows, the incorporation of modern analytical technologies like HPTLC will be significant in maintaining the standards of quality and safety of ayurvedic products. The use of HPTLC is an important technique in present herbal analysis, ensuring the consistency and efficacy of piperine in Drakshadi Ghrita. The results not only justify the traditional use of Drakshadi Ghrita, but they also suggest that adding piperine can improve the preparation's bioavailability and therapeutic potential. HPTLC analysis of the active components of Drakshadi Ghrita's additional compounds, such as Yastimadhu and Draksha, may be performed in the future.

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