Comparative Phyto-Pharmacognostic study of Field collected and Pharmacy...

[Full title: Comparative Phyto-Pharmacognostic study of Field collected and Pharmacy sample of Arjuna (Terminalia arjuna) Bark]

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ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 7 Comparative Phyto-Pharmacognostic study of Field collected and Pharmacy sample of Arjuna ( Terminalia arjuna ) Bark Dongre Sushma 1 , Pande Shishir 2 1 Ph.D. Scholar & Assistant Professor, Dept. of Rasashashtra and Bhaishjya Kalpana, Govt. Ayurved College, Osmanabad, Maharashtra, India. 2 Ph.D. Guide & HOD, Dept. of Rasashashtra and Bhaishjya Kalpana, ASS Ayurved College, Nashik, Maharashtra, India. I NTRODUCTION Arjuna ( Terminalia arjuna ) is well-known drug in Ayurveda which is widely used for many therapeutic purposes. It consists of the stem bark of Terminalia Address for correspondence: Dr. Dongre Sushma Ph.D. Scholar & Assistant Professor, Dept. of Rasashashtra and Bhaishjya Kalpana, Govt. Ayurved College, Osmanabad, Maharashtra, India. E-mail: sushmaosbd 2007@gmail.com Submission Date: 10/01/2022 Accepted Date: 17/02/2022 Access this article online Quick Response Code Website: www.jaims.in DOI: 10.21760/jaims.7.2.2 arjuna W. & A. (Fam. Combretaceae); a large deciduous tree, commonly found throughout the greater parts of the country [1] It is widely used for therapeutic purposes in Ayurveda in various forms such as Kshirpaka, Siddha Gruta, Arishta etc. It is indicated in Medoroga (Obesity), Vrana (Wounds), Hridroga (heart diseases), Kshatashaya (Debility), Prameha (Diabetes), Vyanga (Chloasma) etc. In Ayurveda, bark is collected under good collection practice, when the potency of drug is optimum at that particular season [2] Although bark is widely available in market, it is hardly selfcollected for pharmaceutical purposes. The market for herbal drugs has grown at an impressive rate due to a global resurgence in traditional and alternative healthcare systems, and therefore medicinal plants have great economic importance. However, loss of biodiversity, over-exploitation and unscientific use of A B S T R A C T Arjuna ( Terminalia arjuna ) is the most widely used drug in Ayurveda due to its potential therapeutic competence. Quality assurance is important issue nowadays due to poor collection and storage practices. So, it needs quality control both for small scale and large-scale preparations of Arjuna formulations. Hence present study is planned to compare both field collected and pharmacy sample of Arjuna bark by analytical testing. Macroscopic, microscopic, physicochemical and high performance thin layer chromatography (HPTLC) by using Arjunic acid as a marker is carried out. Macroscopic and microscopic characteristics observed as per Ayurveda Pharmacopeia (API) except color of field collected bark. In Physicochemical parameters, Ash value was higher in pharmacy sample but within limit whereas percentage of water soluble and alcohol-soluble extractives was high in field collected Arjuna as compare to pharmacy sample. Percentage of extractive values was not as per limits of API. Tannin percentage was three times more in field collected Arjuna bark. In HPTLC study, percentage of Arjunic acid was much higher in pharmacy sample (0.072%) than field collected sample (0.054%). Also, the spots observed were more in this sample. 0.60 was the R f Value for Arjunic acid and one common unknown spot was noticed in both samples. Present study set preliminary data for percentage of Tannins and quantification of Arjunic acid in Arjuna Bark by HPTLC study which is not found in monograph of Arjuna hence can be used as reference for further study. Key words: Arjuna, Terminalia arjuna, Pharmacognostic study, Ayurveda, Physicochemical parameters.

[Find the meaning and references behind the names: Forest, Certified, Raw, Standard, Active, Dish, Odor, Matter, Chain, Brown, Vernier, Bio, Size, Major, Lack, Foreign, Vasu, Green, Self, Stock, Blue, Black, Bath, House, Need]

Dongre Sushma et al. Comparative Phyto-Pharmacognostic study of Arjuna (Terminalia arjuna) Bark ISSN: 2456-3110 ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 8 medicinal plants, industrialization, bio piracy, together with lack of regulation and infrastructure are the major impediments to the growth of herbal medicine. In most countries, herbal products are launched into the market without proper scientific evaluation, and without any mandatory safety and toxicological studies. There is no effective machinery to regulate manufacturing practices and quality standards [3] However, Standards of raw drugs in Pharmaceutical companies are maintained to some extend with inhouse quality control laboratory. Also, storage condition may impact the quality specially before being supplied to pharmacy. The postharvesting process of medicinal plants has the great importance in the production chain, because of its direct influence on quality and quantity of active principles in the product sold [4] Due to large scale demand there need to assure quality of Pharmacy sample. Also, selfcollected samples need quality check by different analytical testing. So, in present study, comparative study has been planned between Pharmacy and Self collected sample of Arjuna bark to ensure quality by pharmacognostic evaluation. M ETHODS AND M ATERIALS Collection and Authentication of drug Arjuna is collected from Forest region of Bhimashanker, near Pune which is authenticated from Botanical survey of India. Whereas pharmacy sample of Arjuna is taken from GMP certified Ayurvedic pharmacy that was authenticated from in-house Quality control department. Phyto-Pharmacognostic study Macroscopic, Microscopic study, Physicochemical study and High performance thin layer chromatography (HPTLC) was carried out at Vasu Research Laboratory, Vadodara, Gujrat Macroscopic and Microscopic study Macroscopic Study [5] Macroscopic study was carried out as per standard operating procedures in mentioned in pharmacognosy. Various Morphological characters and organoleptic characterization such as size, shape, color, odor, taste, external markings, fracture, were observed. The diameter of bark was measured with the help of vernier caliper. Microscopy study [6] Microscopic identification was carried out according to the Ayurveda pharmacopeia of India (Appendix - 2.1.1) Physicochemical analysis Physicochemical analysis namely pH value [7] , Foreign matter [8] , Ash Value [9] , Acid insoluble ash [10] , Water soluble extractives [11] ,and Alcohol soluble extractives [12] , were carried out according to the Ayurvedic Pharmacopeia of India (API). Tannin [13] - Tannin is detected by treating with ferric ammonium sulphate solution (blue-black or green black color shows the presence of Tannin) or with potassium-bichromate solution (brown color indicates the presence of Tannin). HPTLC analysis of Arjuna (Identification and Quantification) [14] Preparation of Test solution 1 g of Arjuna sample was accurately weighed in a conical flask. It was refluxed for 30 minutes with 15 mL of Methanol consecutively for 3 times. Then filtered with the help of Whatman filter paper No. 1 and concentrated the combined extracts in an evaporating dish on water bath. Thereafter, made up the volume up to 10 mL with Methanol. Test solution thus obtained was used for HPTLC fingerprinting. Preparation of Standard solution Accurately weighed 11.7 mg of standard Arjunic acid was taken into 2 mL volumetric flask and dissolved in Methanol to make up the volume up to 2 mL with Methanol. From this stock solution, 0.1 mL of solution was pipetted into 2 mL volumetric flask and made up the volume up to 2 mL with Methanol. The Standard solution thus obtained was used for HPTLC fingerprinting.

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Dongre Sushma et al. Comparative Phyto-Pharmacognostic study of Arjuna (Terminalia arjuna) Bark ISSN: 2456-3110 ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 9 O BSERVATIONS Table 1: HPTLC Chromatographic Conditions Chromatographic Conditions Application Mode CAMAG Linomat 5 - Applicator Filtering System Whatman filter paper No. 1 Stationary Phase MERCK - TLC / HPTLC Silica gel 60 F 254 on Aluminum sheets Application (Y axis) Start Position 10 mm Development End Position 80 mm from plate base Band Length 8 mm Distance Between Tracks 15 mm Sample Application Volume 12.0 μL Standard Application Volume 12.0 μL Development Mode CAMAG TLC Twin Trough Chamber Chamber Saturation Time 30 minutes Mobile Phase (MP) Chloroform : Methanol (9 : 1 v/v ) Visualization @ 254 nm Quantification Wavelength 205 1) Images of Field Collected T. Arjuna (Image I-IV, VII) I. T. Arjuna tree II. Wet bark of T. Arjuna III. Dry bark of T. Arjuna IV. Powder of T. Arjuna

[Find the meaning and references behind the names: Smooth, Light]

Dongre Sushma et al. Comparative Phyto-Pharmacognostic study of Arjuna (Terminalia arjuna) Bark ISSN: 2456-3110 ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 10 2) Images of Pharmacy Collected sample of T. Arjuna (V-VI,VIII) V. Bark of T. Arjuna VI. Powder of T. Arjuna VII. Bark of T. Arjuna (Field collected) VIII. Bark of T. Arjuna (Pharmacy sample) Table 2: Macroscopic study of T. Arjuna Bark Sample Parameters Macroscopy of Arjuna Field Collected Sample (FCS) Macroscopy of Arjuna Pharmacy Sample (PCS) Stem Bark Stem bark pieces flat or slightly curved Stem bark pieces flat and slightly curved Outer surface Smooth and greenish brown in colour; Smooth and light brown in colour Inner surface Brown and longitudinally striated; Brown and longitudinally striated;

[Find the meaning and references behind the names: Rosette, Crystal]

Dongre Sushma et al. Comparative Phyto-Pharmacognostic study of Arjuna (Terminalia arjuna) Bark ISSN: 2456-3110 ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 11 Table 3: Microscopy study of T. Arjuna Bark SN Pharmacognostic Features of Powder Field collected Sample (FCS) Pharmacy Sample (PCS) 1. Compound starch crystals 2. Rosette Crystals of Calcium Oxalate 3. Cluster Crystals of Calcium Oxalate 4. Parenchyma Containing Cluster Crystal of calcium oxalate 5. Parenchyma Containing Tannin

[Find the meaning and references behind the names: Rough, Cork, Rasa, Chemical, Nil, Rupa]

Dongre Sushma et al. Comparative Phyto-Pharmacognostic study of Arjuna (Terminalia arjuna) Bark ISSN: 2456-3110 ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 12 6. Cork Fragment 7. Lignified Phloem Fibers Table 4: Organoleptic Characteristics of Arjuna SN Parameters Arjuna field Collected (FCS) Arjuna Pharmacy collected (PCS) 1. Fracture ( Shabda ) Medium Medium 2. Touch Rough Rough 3. Rupa (color) Smooth and greenish brown Smooth and light brown 4. Rasa (Taste) Astringent Astringent 5. Gandha (Smell) Characteristic Characteristic Table 5: Physico Chemical Analysis of Arjuna SN Parameters Arjuna field Collected Arjuna Pharmacy collected 1. Foreign matter Nil Nil 2. Total Ash 9.72% 10.48 % 3. Acid insoluble Ash Nil Nil 4. Water Soluble extractives 18.40% 16.09% 5. Alcohol Soluble Extractives 10.71% 8.17% 6. Total Tannin 12.10% 4.04%

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Dongre Sushma et al. Comparative Phyto-Pharmacognostic study of Arjuna (Terminalia arjuna) Bark ISSN: 2456-3110 ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 13 Image 3: HPTLC 3 D overlay chromatogram@205 nm of Arjuna Samples. 2 D chromatogram of Arjuna Powder (Field Collection) @ 205 nm 2 D chromatogram of Arjunic Acid @ 205 nm 2 D chromatogram of Arjuna Powder (Pharmacy Sample) @ 205 nm Table 6: HPTLC study of Arjuna Samples Sample Arjunic Acid Arjuna Powder (Field Collection) Arjuna Powder (Pharmacy Sample) Weight per 10 mL 2.9 mg 1013 mg 1130 mg Area 2636.6 510.7 759.6 % Arjunic Acid -- 0.054 % 0.072 % DISCUSSION Termanalia arjuna is widely available all over India. While collecting any drug its authenticity needs to be taken into account as many species of single plants are available in the field. Studied sample of Arjuna was noticeably collected from field by good collection practices which showed distinctive characteristics of T. Arjuna in organoleptic study, whereas pharmacy collected sample (PCS) was already authenticated by quality control department. While comparing both sample of Arjuna Bark, it was observed that field collected sample (FCS) was quite greenish white in colour whereas pharmacy sample was pinkish brown in colour. There also occurred marked difference in powders of both samples in color i.e., field and pharmacy sample which was cream white (Image 1: I, II, III, IV) and faint brown (Image 2: V, VI) respectively. Pinkish brown is the original character of Arjuna stem and Reddish – brown is the color of Arjuna powder. So, this dissimilarity in color may be due to difference in maturity of plant or due to long storage. (Table 2) Microscopic study of both samples shown compound starch crystals, Rosette Crystals of Calcium Oxalate, Cluster Crystals of Calcium Oxalate, and Parenchyma Containing Cluster Crystal of calcium oxalate and tannins. It also observed cork fragment and Lignified Phloem Fibers. (Table 3) Physicochemical analysis have shown more percentage of ash value (10.48%) in PCS but not more than 25% as specified in API. Water soluble and alcohol soluble extractive % is seen higher in FCS than PCS but less than 20% as per API. Tannin is the most important constituent of Arjuna but its

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Dongre Sushma et al. Comparative Phyto-Pharmacognostic study of Arjuna (Terminalia arjuna) Bark ISSN: 2456-3110 ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 14 standards are not mentioned in API. (Table: 5) Percentage of tannin in FCS was seen three times more than PCS. However, in HPTLC study % of Arjunic acid is seen more in PCS than that of FCS. Also, the number of spots which indicates unknown phyto-constituents are more in PCS (03) than FCS (02) other than Arjunic acid spot (0.60). Presence of common spot (0.85) in both samples which suggest potent unknown phytoconstituent of Arjuna . In this study single samples were compared on the basis of quality parameters which are not sufficient to specify the standards, however identification and quantification of Arjunic acid by HPTLC study is the important parameter which is not mentioned in the API. CONCLUSION Field collected sample is qualitatively best in physicochemical parameters than pharmacy sample. Whereas in HPTLC study, pharmacy sample has higher percentage of Arjunic Acid and more spots. This study used single sample for quality comparison which is not sufficient for substantial conclusion. Hence their needs more number of samples for significant inferences. However, Percentage of Tannins and quantification of Arjunic acid by HPTLC is not specified in the monograph of Arjuna which can be used as a preliminary data for further study. REFERENCES 1 Anonymous. The Ayurvedic Pharmacopoeia of India. Government of India, Ministry of Health and Family Welfare Department of Ayush. Part- I, Volume – II, 17- 18. 2 Siddhinandan Mishra. Abhinava Bhaishjya Kalpana Vigyana. Choukhambha Surbharti Prakashana, Varanasi. Reprint 2003, p 101. 3 Kunle, Oluyemisi Folashade, Egharevba, Henry Omoregie, Ahmadu, Peter Ochogu. Standardization of herbal medicines - A review. International Journal of Biodiversity and Conservation. Vol 4(3), March 2012, 101-112. 4 Kulshetra Mayanka Krishna, Kalbhal Kamleshwar Singh. Effect of different Drying Methods on the quality of Stem bark of Terminalia Arjuna Roxb. IJRAP. 3(4), Jul – Aug 2012, p 515-518. 5 Khandelwal KR. Practical Pharmacognosy. Nirali Prakashana; Pune. 2008;p 162-4. 6 Anonymous. The Ayurvedic Pharmacopoeia of India. Government of India, Ministry of Health and Family Welfare Department of Ayush. Part- I, Volume - VI, First Edition, 2008, APPENDIX -22,1,1- Microscopic Identification, 233,274. 7 Anonymous. The Ayurvedic Pharmacopoeia of India. Government of India, Ministry of Health and Family Welfare Department of Ayush. Part- I, Volume – VI, First Edition, 2008, APPENDIX -22,1,1- Microscopic Identification, 233. 8 Anonymous. The Ayurvedic Pharmacopoeia of India (API). Government of India, Ministry of Ayush, Published by Pharmacopoeia Commission for Indian Medicine &Homoeopathy, Ghaziabad. Part-I, Vol-IX (Extracts); (Extracts); Appendices - 2, First Edition, 2016; 114-15. 9 Anonymous. The Ayurvedic Pharmacopoeia of India (API). Government of India, Ministry of Ayush, Published by Pharmacopoeia Commission for Indian Medicine & Homoeopathy, Ghaziabad. Part-I, Vol-IX (Extracts); Appendices-2, First Edition, 2016; 113. 10 Anonymous. The Ayurvedic Pharmacopoeia of India (API). Government of India, Ministry of Ayush, Published by Pharmacopoeia Commission for Indian Medicine & Homoeopathy, Ghaziabad. Part-I, Vol-IX (Extracts); Appendices-2, First Edition, 2016; 113-14. 11 Anonymous. The Ayurvedic Pharmacopoeia of India (API). Government of India, Ministry of Ayush, Published by Pharmacopoeia Commission for Indian Medicine & Homoeopathy, Ghaziabad. Part-I, Vol-IX (Extracts); Appendices-2, First Edition, 2016; 114. 12 Anonymous. The Ayurvedic Pharmacopoeia of India (API), Government of India, Ministry of Ayush, Published by Pharmacopoeia Commission for Indian Medicine & Homoeopathy, Ghaziabad. Part-I, Vol-IX (Extracts); Appendices-2, First Edition, 2016; 114. 13 Anonymous. The Ayurvedic Pharmacopoeia of India. Government of India, Ministry of Ayush, Published by Pharmacopoeia Commission for Indian Medicine &Homoeopathy, Ghaziabad. Part – I, Volume – VII, (Minerals & Metals) Appendix – 2, 2,1, Tests and Determinations, First Edition, 2008, p 238. 14 Pulok K Mukherjee. Quality Control and Evaluation of Herbal Drugs. Elsivier Publication, USA. 2019 High-

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Dongre Sushma et al. Comparative Phyto-Pharmacognostic study of Arjuna (Terminalia arjuna) Bark ISSN: 2456-3110 ORIGINAL ARTICLE March 2022 Journal of Ayurveda and Integrated Medical Sciences | March 2022 | Vol. 7 | Issue 2 15 Performance Thin-Layer Chromatography (HPTLC) for Analysis of Herbal Drugs Chapter - 9,: 381- 385. ******************************* How to cite this article: Dongre Sushma, Pande Shishir. Comparative Phyto-Pharmacognostic study of Field collected and Pharmacy sample of Arjuna (Terminalia arjuna) Bark. J Ayurveda Integr Med Sci 2022;2:7-15. http://dx.doi.org/10.21760/jaims.7.2.2 Source of Support: Nil, Conflict of Interest: None declared. Copyright © 2022 The Author(s); Published by Maharshi Charaka Ayurveda Organization, Vijayapur (Regd). This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by-nc-sa/4.0), which permits unrestricted use, distribution, and perform the work and make derivative works based on it only for non-commercial purposes, provided the original work is properly cited

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