STANDARDIZATION OF AYURVEDIC FORMULATION “ARJUNARISHTA” IN TERMS OF PHYSICOCHEMICAL, SPECTROSCOPY AND CHROMATOGRAPHIC TECHNIQUES

STANDARDIZATION OF AYURVEDIC FORMULATION “ARJUNARISHTA” IN TERMS OF PHYSICOCHEMICAL, SPECTROSCOPY AND CHROMATOGRAPHIC TECHNIQUES

K. S. Thakur ^{* 1}, Priyanka Patil ² and Madhuree Gawhankar ²

Shree Dhootapapeshwar Limited ¹, Shree Dhootapapeshwar Ayurvedic Research Foundation ², Veer Sawarkar Chowk, Panvel, Raigad - 410206, Maharashtra, India.

ABSTRACT: Objective: Arjunarishta is an important Ayurvedic formulation with chief ingredient Arjuna (Terminalia arjuna), which is excellent “Hrudya” Dravya. It nourishes & strengthens heart muscles & promotes cardiac functioning by regulating blood pressure and cholesterol level. This study aims to established quality parameters for standardization of Ayurvedic formulation “Arjunarishta” on the basis of various techniques viz. Physico-chemical screening, Chromatography & IR Spectroscopy. Method: Conventional analysis such as Organoleptic tests, pH, Alcohol content, Brix, and Specific gravity was done. Chromatographic analysis was performed to estimate Arjungenin, Ellagic acid, and Gallic acid using High-Performance Thin Layer Chromatography (HPTLC). IR fingerprint was done using Fourier transform infrared (FT-IR) spectrometer. Result: The above analysis showed the pH of tested formulation ranges between 3.0-5.0, Brix 25-30%, and Alcohol content 6-10% v/v. Organoleptic tests confirmed characteristic odor of self-generated alcohol with sweet, astringent & slightly bitter taste. HPTLC analysis confirmed the presence of Arjungenin, Ellagic acid, and Gallic acid. FT-IR reveals unique transmittance spectra in the range of 4000 - 600 cm^-1. Conclusion: This study can be used for qualitative evaluation of Arjunarishta in terms of modern parameters, which may help in the authenticity of the drug and to compile suitable information for the better utility and safe use of this formulation in therapeutics.

Standardization, Arjunarishta, High-Performance Thin Layer Chromatography (HPTLC), Fourier transform infrared spectroscopy (FT-IR), Arjungenin

INTRODUCTION: Ayurveda is the oldest traditional Indian medicinal system that contributes to a healthy life of human being ¹. The word 'Ayurveda' is comprised of two Sanskrit words, ‘Ayu’ means Life and ‘Veda’ means knowledge, which is originated in India but now practiced all over the world ^2-3. The World Health Organization (WHO) reported that almost 80% of the world populations rely on medicines mainly of herbal sources in their healthcare ^4-7.

The primary focus of Ayurvedic medicine is to prevent illness and promote good health. Various forms of Ayurvedic medicines such as Churna, Avaleha, Bhasmas, Asavas, Arishthas, Tailas are being used since ancient time to present era. Arishtas are an important group of Ayurvedic formulations used as medicines to treat various disorders in Ayurveda for over 3000 years ^8-9.

Arjunarishta also known as Parthadyarishta, is formulated with key ingredient ‘Arjuna twak’ (Arjuna Bark) (Terminalia arjuna) which is excellent ‘Hrudya’ Dravya i.e. Cardio-protective; it nourishes and strengthens heart muscles and promotes cardiac functioning by regulating blood pressure and cholesterol, is one of the ancient Ayurveda formulations prescribed in cardio-vascular disorder in Ayurveda ^10-19.

FIG. 1: ARJUN TWAK (ARJUNA BARK) (TERMINALIA ARJUNA)

Arjun Twak (Terminalia arjuna) has anti-hypotensive effects, boosts anti-oxidant activities, and prevents fibrosis ^20-23. It contains hydrolyzable tannins as gallic acid, ellagic acid, triterpenoid glycosides, Sapogenins as arjunic acid, arjunolic acid, arjungenin; flavonoids, mineral salt and sugar ^24-27. The previous studies reveal the effect of arjunolic acid, arjungenin, and arjunetin on the process of respiratory oxidative burst and cardiac fibrosis ^28-29.

The formulation Arjunarishta also contains Mrudvika (Vitis vinifera), Dhataki Pushpa (Wood-fordia fruticosa), Madhuka Pushpa (Madhuka indica) which are rich sources of antioxidants ^30-31.

In order to achieve consistent, reproducible results in terms of efficacy and safety of drugs, the quality of phyto ingredients should be standardized and duly validated. In the case of polyherbal formulations, phytochemical screening, fingerprint profiling, and quantification of multiple markers need to be developed and implemented for ensuring quality and batch to batch consistency.

In quality control methods for medicinal plant materials, WHO has clearly specified for quanti-fication of marker compounds and development of fingerprint profile for all botanical preparations ^32-33. Every drug should have complete information related to its organoleptic properties, physico-chemical characteristics & phytoconstituents.

Here an attempt was made to standardize ayurvedic formulation Arjunarishta in terms of physico-chemical, spectroscopy, and chromatographic techniques.

MATERIALS AND METHODS: Three batches of Arjunarishta were procured from approved stockist of Shree Dhootapapeshwar Ltd. batch codes designated were as Sample-1, Sample-2, and Sample-3. These batches were analyzed for physico-chemical parameters, IR spectroscopic, and chromatographic evaluations to standardize the formulation.

All chemicals and reagents (Toluene, Chloroform, Ethyl acetate, Acetic acid, Formic acid, Methanol, etc.) used in the analysis were of analytical reagent grade of Merck. Reference standards (RS) of Arjungenin of purity 98.90% (CAS No, 58880-25-4), Gallic acid of purity 98.50% (CAS No, 149-91-7), and Ellagic acid of purity 99.80% (CAS No, 476-66-4) were used for the chromatographic study.

Organoleptic Properties: Various parameters such as color, taste, and odor of the samples of Arjunarishta were observed and recorded.

Physico-chemical Evaluation: In Physico-chemical screening, the samples of Arjunarishta were analyzed for various physicochemical parameters such as pH, alcohol content, specific gravity, and Brix and as per the Ayurvedic Pharmacopoeia of India (API) ³⁴ and as per in-house specifications.

Chromatographic Analysis: HPTLC Instrument Camag with sample applicator Linomat 5, Densitometer TLC Scanner 4, and Wincat Software was used for chromatographic analysis of a methanolic extract of Arjunarishta. A twin trough chamber was used for the development of HPTLC plates. A photo documentation cabinet fitted with a high-resolution camera was used for capturing images at different wavelengths. Densitometer TLC Scanner 4 equipped with Deuterium (D2) & Tungsten (W) lamps were used to obtain spectra for the quantitative determination of Arjungenin, Gallic acid & Ellagic acid. Precoated silica gel G60-F₂₅₄ Aluminium sheets (E. Merck, Germany) 20 × 10 mm, thickness layer 0.2 mm was used as the stationary phase. The solvent systems Toluene: Ethyl acetate: Acetic acid (5: 5: 0.5) v/v for Arjungenin and Chloroform: Ethyl acetate: Formic acid (5: 4: 1.6) v/v for Gallic acid & Ellagic acid was selected for estimation, which gave good resolution. The wavelengths of 600 nm, 290 nm & 278 nm were used for quantification of Arjungenin, gallic acid & ellagic acid, respectively, in the samples.

IR Spectroscopic Evaluation: The samples of Arjunarishta were analyzed in the range of 4000 cm^-1 to 600 cm^-1 using ALPHA FTIR, Bruker with ATR sampling mode and OPUS software

RESULTS AND DISCUSSION: The formulation Arjunarishta characterized as a dark brown colored liquid having characteristic fermented odor with sweet taste followed by slight astringent & bitter Table 1.

The Physicochemical evaluations showed the pH of the formulation ranges between 3.5 to 5.0, Alcohol content between 7 to 10 % v/v, Brix 25 to 35 %, and Specific gravity 1.0 to 1.2. Table1 The pH of the solution provides an indication of the acidity or alkalinity of a solution. The digital pH meter was used for the pH measurement after calibration with buffer solutions. pH was noted for all the samples, and the results have been shown in Table 1. The formulation Arjunarishta contains self-generated alcohol.

The alcohol content was determined as per the method described in The Ayurvedic Pharma-copoeia of India (API) for all the samples ³⁴. Brix was determined using a refractometer, and results were recorded. Brix is the sugar content of an aqueous solution. It represents the strength of the solution as a percentage by mass ³⁵. Specific gravity is the ratio of the specific weight of the material to the specific weight of the distilled water. Specific gravity was determined as per the method described in The Ayurvedic Pharmacopoeia of India (API) and recorded.³⁴ Table 1.

TABLE 1: RESULTS OF ORGANOLEPTIC AND PHYSICO-CHEMICAL EVALUATIONS

The chief ingredient Arjuna twak (Bark) (Terminalia arjuna) contributed to the presence of Arjungenin, Gallic acid, and Ellagic acid in the formulation Arjunarishta. The HPTLC analysis confirms the presence of Arjungenin, Gallic acid, and Ellagic acid at Rf 0.15 ± 0.02, 0.24 ± 0.02, and 0.37 ± 0.02, respectively Fig. 2, 3 & 4. The content of Arjungenin, Gallic acid & Ellagic acid in Sample-1, Sample-2, and Sample-3 of Arjunarishta was estimated, and the results have been shown in Table 2.

FIG. 2: PEAK DISPLAY OF ARJUNGENIN STANDARD AND ARJUNARISHTA FORMULATION (SAMPLE-1 SAMPLE-2 & SAMPLE-3)

FIG. 3: PEAK DISPLAY OF GALLIC ACID & ELLAGIC ACID STANDARD AND ARJUNARISHTA FORMULATION (SAMPLE-1, SAMPLE-2 & SAMPLE-3)

FIG. 4: THE HPTLC ANALYSIS CONFIRMS THE PRESENCE OF ARJUNGENIN, GALLIC ACID & ELLAGIC ACID IN ARJUNARISHTA FORMULATION (SAMPLE-1, SAMPLE-2 & SAMPLE-3)

TABLE 2: PHYTOCHEMICAL CONTENT IN ARJUNA-RISHTA

This technique of HPTLC has been known for the simultaneous estimation of markers compounds in polyherbal formulations. With the application of a very small quantity of samples, various samples can be run on the TLC plate and quantifications can be performed up to very low concentrations ³⁶.

The FT-IR spectrum of Arjunarishta reveals the presence of O-H stretching at 3268 cm^-1, C=C stretching at 1637 cm^-1 & 1418 cm^-1, C-O stret-ching at 1262 cm^-1 & 1242 cm^-1 and C-H bending at 925 cm^-1, 871 cm^-1, 818 cm^-1 & 773 cm^-1. This is shown in Table 3 and Fig. 5. It confirms the presence of alcohol, carboxylic acid, primary alcohol alkenes, and aromatic groups in Arjunarishta formulation ³⁷.

TABLE 3: FT-IR FINGERPRINT OF ARJUNARISHTA

FIG. 5: FT-IR SPECTRUM OF ARJUNARISHTA

CONCLUSION: In this study, the formulation Arjunarishta was analyzed according to the standard pharmacopoeia procedures for its physico-chemical screening, spectroscopic and chromato-graphic evaluations. This study will help to develop a quality control profile for future reference and can be used for qualitative evaluation of Arjunarishta in terms of modern parameters, which may help in the authenticity of the drug and to compile suitable information for the better utility and safe use of this formulation in therapeutics.

With the increasing use of Ayurvedic medicines, there is certainly a need for more advanced techniques that will help drug manufacturer’s to follow specifications & justify therapeutic efficacy, safety, and stability of products.

Besides this, quality assessment of Ayurvedic medicines has to be done in an integrated manner, including both traditional and modern analytical techniques. This will help in validating the ancient wisdom of Ayurveda, which has been in practice for many centuries.

ACKNOWLEDGEMENT: We are thankful to Shree Dhootapapeshwar Ltd. for support throughout the work.

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. Chauhan A, Semwal DK, Mishra SP and Semwal RB: Ayurvedic research and methodology: Present status and future strategies. AYU 2015; 36: 364-9.
2. Agrawal M, Byadgi PS and Dwibedy BK: Importance of ayu pareeksha for the management of diseases. J Clin Diagn Res 2017; 11(1): OE01-OE04.
3. Sihag J and Rani J: A studu of concept of kaala in ayurveda w.s.r. to kaala as a karan dravya. International Journal of Scientific Research 2019, 8(6): 2277-79.
4. World Health Organization, Traditional Medicine Strategy Report, Document WHO/EDM/TRH/2002.
5. Bafor: Effect of laxiflora on female reproductive system. J Sci Pract Pharm 2015; 2(1): 55.
6. Goswami S, Jain R and Masih H: Antifungal, antioxidant and DNA protection potential of Grewia asiatica leaves acetone extract. Journal of Pharmacognosy and Phytochemistry 2018; SP1: 212-17.
7. Jacqui W: Herbal products are often contaminated, study finds. BMJ 2013; 347: f6138.
8. Singh H, Mishra SK and Pande M: Standardization of Arjunarishta formulation by TLC method. Int J of Pharm Sciences Review and Research 2010; 2(1): 007.
9. Darvin SS: Preparation and Standardization of SiddhalepaAyur Elixir 14 Blood Purifier (Nimbaarishta), A Traditional Ayurvedic Formulation”. Acta Scientific Pharmaceutical Sciences 3.12 (2019): 47-51.
10. Amalraj A and Gopi S: Medicinal properties of Terminalia arjuna (Roxb.) Wight & Arn.: A review. J Tradit Complement Med 2017; 7(1): 65-78.
11. Priya N, Mathur KC, Sharma A, Agrawal RP, Agarwal V and Acharya J: Effect of Terminalia arjuna on total platelet count and lipid profile in patients of coronary artery disease. Adv Hum Biol 2019; 9: 98-101.
12. Soni N and Singh VK: Efficacy and Advancement of Terminalia arjuna in Indian Herbal Drug Research: A Review. Trends in Applied Sciences Research 2019; 14: 233-42.
13. Gupta A, Nishteswar K, Shukla VJ and Ashok BK: Evaluation of analgesic activity of Terminalia arjuna (Roxb.) Wight and Arn bark: A tribal claim. AYU 2014; 35: 458-61.
14. Dwivedi S and Jauhari R: Beneficial effects of Terminalia arjuna in coronary artery disease. Department of Medicine, University College of Medical Sciences, Delhi. Indian Heart J 1997; 49(5): 507-10.
15. Bharani A, Ganguli A, Mathur LK, Jamna Y and Raman PG: Efficiency of Terminalia arjuna in chronic stable angina: a double-blind placebo-controlled, crossed over Terminalia arjuna study comparing with Isosorbide mononitrate. Indian Heart J 2002; 54(2): 170-5
16. Bharani A, Ganguli A and Bhargava K: Salutary effect of Terminalia arjuna in patient with severe refractory heart failure. Int J Cardiol 1995; 49(3): 191-99.
17. Singh N, Kapur KK, Singh SP, Shanker K, Sinha JN and Kohli RP: Mechanism of Cardiovascular action of Terminalia arjuna. Planta Medica 1982; 45: 102-04.
18. Kapoor D: Journal of Ethnopharmacology 2014; 155: 1029-45.
19. Ragini H, Amita P and Jain AK: An approach to standardize Arjunarishta: a well-known ayurvedic formulation using UV and Colorimetric method. J Med Pharm Allied Sci 2012; 01: 77-84.
20. Bansal T, Chatterjee E, Singh J, Ray A, Kundu B and Thankamani V: Arjunolic acid, a peroxisome proliferator-activated receptor alpha agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-beta signaling. J Biol Chem 2017; 292: 16440-62.
21. Kumar S, Enjamoori R, Jaiswal A, Ray R, Seth S and Maulik SK: Catecholamine-induced myocardial fibrosis and oxidative stress is attenuated by Terminalia arjuna (Roxb.). Journal of Pharmacy and Pharmacology 2009; 61: 1529-36.
22. Kumar S, Alam MJ, Prabhakar P, Ahmad S, Maulik SK and Sharma M: Proteomic analysis of the protective effects of aqueous bark extract of Terminalia arjuna (Roxb.) on isoproterenol-induced cardiac hypertrophy in rats. J Ethnopharmacol 2017; 198, 98-108.
23. Mohanty IR, Borde M, Kumar S and Maheswari U: Dipeptidyl peptidase IV Inhibitory activity of Terminalia arjuna attributes to its cardioprotective effects in experimental diabetes: In-silico, in-vitro and in-vivo analyses, Phytomedicine 2018. doi: https://doi.org/10.1016 /j.phymed.2018.09.195
24. Rahman Z, Kohli K, Lambha HS, Rathour A and Pahwa R: An overview of Terminalia arjuna: Chemistry and Pharmacological Profile. Indian Drugs 2004; 41(11): 641-48.
25. Saha A, Pawar VM and Jayaraman S: Characterisation of polyphenols in Terminalia arjuna bark extract. Indian J Pharm Sci 2012, 74(4): 339-47.
26. Rana MS, Walia R, Dixit A and Raina K: To compare and evaluate the anti-inflammatory efficacy of Terminalia arjuna (aqueous extract of bark) with diclofenac sodium on rats. Int. J. Basic Clin. Pharmacol 2016; 5: 692-95.
27. Pawar RS and Bhutani KK: Effect of oleanane triterpenoids from a cardio protective drug Terminalia arjuna on the process of respiratory oxyburst. Phytomedicine 2005; 12: 391-93.
28. Maulik SK, Wilson V, Seth S, Bhargava B, Dua P and Ramakrishnan S: Clinical efficacy of water extract of stem bark of Terminalia arjuna (Roxb. ex DC.) Wight & Arn. in patients of chronic heart failure: a double-blind, randomized controlled trial. Phytomed 2016; 23: 1211-19.
29. Meghwani H, Prabhakar P, Mohammed SA, Seth S, Hote MP and Banerjee SK: Beneficial effects of aqueous extract of stem bark of arjuna (Roxb.), an ayurvedic drug in experimental pulmonary hypertension. J Ethnopharmacol 2017; 197: 184-94.
30. Alberto D, Begona B and Carmen G: Antioxidant properties of commercial grape juices and vinegars. Food Chemistry 2005; 93(2): 325-30.
31. Fugh BA: Herbs and Dietary supplements in prevention and treatment of cardiovascular disease prevention. Cardiol. Winter 2000; 3: 24-32.
32. Quality Control Methods for Medicinal Plant Materials. World Health organisation, Genava 1988.
33. Quality Control Methods for Medicinal Plant Materials. World Health organisation, Genava 1992.
34. The Ayurvedic Pharmacopoeia of India, Part-II, Volume-II, First Edition (Formulations), Ministry of Health and Family Welfare Government of India, Department of Ayurveda, Yoga – Naturopathy, Unani, Siddha & Homeopathy (AYUSH), 2010.
35. Wikipedia - Brix. https://en.wikipedia.org/wiki/Brix 2019.
36. Jianga Y, David B, Tu P and Barbin Y: Recent analytical approaches in quality control of traditional Chinese medicines-A review, Anal Chim Acta 2010; 657: 9-18.
37. Pavia DL, Lampman GM, Kriz GS and Vyvyan JR: Introduction to Spectroscopy, Fourth Edition, Department of Chemistry Western Washington University Bellingham, Washington 2009; 29.
    

    ---

    How to cite this article:

    Thakur KS, Patil P and Gawhankar M: Standardization of ayurvedic formulation “Arjunarishta” in terms of physicochemical, spectroscopy and chromatographic techniques. Int J Pharm Sci & Res 2020; 11(12): 6237-42. doi: 10.13040/IJPSR.0975-8232.11(12). 6237-42.

    ---

    All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---