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ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 3  |  Page : 175-180

Identification of adulterants of Terminalia arjuna bark from market samples through a pharmacognostical study


1 Department of Dravyaguna, Sri Jayendra Saraswati Ayurvedic College and Hospital, Chennai, Tamil Nadu, India
2 Department of Rasashastra and Bhaishajya Kalpana, Sri Jayendra Saraswati Ayurvedic College and Hospital, Chennai, Tamil Nadu, India

Date of Submission13-Mar-2021
Date of Decision09-Jul-2021
Date of Acceptance24-Jun-2021
Date of Web Publication28-Sep-2021

Correspondence Address:
Dr. Polepalli Mallikarjuna Rao
Department of Dravyaguna, Sri Jayendra Saraswati Ayurvedic College and Hospital, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISM.JISM_24_21

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  Abstract 

Background: Pharmacognostical parameters, namely macroscopic and microscopic techniques, are very important in the identification of drugs. The macroscopic and microscopic feature of medicinal plants is believable, accurate and involves less cost. So, the study of the morphology and the organoleptic nature of drugs is undertaken using highly sophisticated modern techniques. In Ayurveda, Arjuna bark is an important drug for the treatment of heart disorders and other diseases. There is a great demand for Arjuna bark in the market, and it is very prone to getting adulterated. To find out the correct identity of Arjuna bark from adulterants with the help of pharmacognostical techniques, the present study was undertaken. Materials and Methods: The Arjuna bark was obtained from the source tree and from four raw drug markets of the capital cities of south India. These five Arjuna bark samples were considered for analyzing their organoleptic characteristics; microscopic examination, polarization, fluorescence microscopic examination, powder microscopy, and thin layer chromatography (TLC) of the drug were used for authentication. Results: The Arjuna bark collected from the source tree and the market samples showed the same presentations, thus the market samples were acceptable. It is also found that the bark of the tree Terminalia paniculata is used as an adulterant in a negligible quantity. Conclusion: The present study reveals that, the chances of adulteration for T. arjuna are very less.

Keywords: Arjuna bark, pharmacognostic studies, identification


How to cite this article:
Mallikarjuna Rao P, Virupaksha Gupta K L. Identification of adulterants of Terminalia arjuna bark from market samples through a pharmacognostical study. J Indian Sys Medicine 2021;9:175-80

How to cite this URL:
Mallikarjuna Rao P, Virupaksha Gupta K L. Identification of adulterants of Terminalia arjuna bark from market samples through a pharmacognostical study. J Indian Sys Medicine [serial online] 2021 [cited 2021 Dec 2];9:175-80. Available from: https://www.joinsysmed.com/text.asp?2021/9/3/175/326836




  Introduction Top


The tree Arjuna (Terminalia arjuna [Roxb.] Wight and Arn, of Combretaceae family) bark has been traditionally used to treat heart disease since long ago and it is also known as the guardian of the heart.[1]Kshirapaka or Arjuna milk decoction is useful in heart diseases, as mentioned by Vrinda vaidyaka.[2]Arjuna tree is known since the Vedic period. Arjuna is used in the treatment of diseases such as Medoroga (obesity), Mutraghata (urinary disorder), Mukharoga (mouth disease), Pittaja, Vataja, and Kaphaja Prameha (poly uric disorders) as per various Samhitas. Arjuna is used in different dosage forms, such as Lepa, Churna, Ghrita, Tail, Kwath, Asava, Aristha, Dhupa, Phanta, Modaka etc.[3]

Arjuna bark consists of various chemical compounds, including Myristyl oleate, Gallocatechol, epicatechol, epigallocatechol, 6, 10, ellagic acid 7, 8, 9, arjunin 7, 9, 10, epicatechin, Arjunic acid 6, 8, 9, Arjunin, Calcium oxaide, 34% of Calcium carbonate, Sodium, oleanolic acid, Tannins, Triterpinoids, Magnesium oxide and Beta sitosterol, Arjunglucoside, arjunolone, baicaleins, leucodelphinidin, leucocyanidin, arjunosides I, II, III, and IV, and reducing sugars. Because of the earlier stated chemical active components, the stem bark is astringent to bland in taste; removes excess bodily heat; increases sperm count and quality; acts as a demulcent; is known as being good for the heart; is hemostatic, vermicidal, a urinary alkaliner, a bronchodilator, lithotriptic, and strength promoting in nature. It is also useful in the treatment of fractures, ulcers, white discharge, diabetes, anemia, fatigue, ear disorders, inflammation, hemorrhagic conditions such as bleeding piles and fistula in ano, cirrhosis of liver, hypertension etc. The properties of Arjuna bark are proved both clinically and experimentally. Their major therapeutic uses are as antihyperlipidemics and antihypertensives in heart diseases.[4]

As per classics, Arjuna and Kakubha are two variants of Arjuna; as white and black varieties, they are T. arjuna and T. tomentosa or T. alata, respectively. The barks of both T. arjuna and T. alata are sold as Arjuna in the market.

There are more than 10 species of Terminalia, namely T. bialata, T. belerica, T. alata, T. manni, T. myriocarpa, T. chebula, T. catappa, T. purifolia, T. travancorensis, T. pallid, T. citrina, T. paniculata etc barks are available.[5] As per a few scholars, the bark of these trees is very similar in appearance that there is very great likelihood of their being mistaken for one another and all of them are being sold indiscriminately as Arjuna, as drug sellers cannot distinguish between these varieties in the market.[6] However, till date, there have been no studies on the market samples to identify the adulteration. Hence, this study was required.


  Materials and Methods Top


Collection

Sample Arjuna bark was collected from Arjuna tree; market Arjuna bark samples were collected from Chennai, Hyderabad, Trivandrum, and Bangalore cities to confine this study to south Indian states. All the five samples were identified in the department Dravyaguna, Sri Jayendra Saraswathi Ayurveda College and Hospital, Chennai, Tamil Nadu, and they were subjected to screening for the present study. Bark sample was collected from a source tree in Sri Jayendra Saraswathi Ayurveda College herb garden. The quantity of each source purchased was 5 kg, from which bark samples were randomly subjected for study.

  • Sample 1: Bark collected from Arjuna tree


  • Sample 2: Collected from Chennai raw drug market


  • Sample 3: Collected from Hyderabad raw drug market


  • Sample 4: Collected from Trivandrum raw drug market


  • Sample 5: Collected from Bangalore raw drug market


Morphology of Arjuna

The tree is common in tropical parts of India, at the river side, Nallamalai forest of Andhra, Mangalore and on Chamunda hills of Karnataka. Arjuna is a moderate-sized deciduous tree that grows up to 20 m in height. The stem of Arjuna can be identified with its peel-off nature and with its creamy white stem. Its leaves are simple, sub-opposite oblong, crenulated, and coriaceous being a Combreteaceae family tree. The leaf margins are serrated, every leaf possesses one or two red glands at the base of the leaf, and the leaf is leathery in touch. Flowers are small, white and they are arranged in spike influorescence. Fruits are round-ovate, 2–5 cm long, fibrous woody, and glabrous with five to seven hard wings. It has a smooth gray bark [Figure 1]. It has a fleshy hard trunk and fast-spreading branches. Arjuna flowers between March and June, and it bears fruits between September and November.
Figure 1: Terminalia Arjuna bark and fruit

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Macroscopy

The dried stem bark that was obtained in pieces is flat, square in shape, slightly curved, and 0.2–15 cm thick; its outer surface is smooth and creamy white to gray in color; its inner surface is fibrous, pink in color; its transverse cut surface is pink to brown in color just as a muscle fiber; on fracture, it is too hard to break, astringent in taste; and its scents are mild and characteristic in all the five samples.

Microscopy

Transverse sections (TS) of Arjuna bark were taken by using a microtome. The permanent mount of bark was prepared by using safranin fast green stain by the double staining technique. Images are obtained with the help of a camera attached to a microscope for the study of cells in the section cutting. Microtome TS of the bark transverse section shows cork, followed by thin-walled parenchymatous tissue embedded with crystals of calcium oxalate. Cork is composed of five to eight layers of thick-walled, lignified rectangular cells with tannins; it is followed by two layers of thin-walled rectangular and narrow cells of phellogen. The cortex is made up of six to eight layers of thin-walled, radially flat cells and secondary phloem with sclerenchymatous fibers, secreting ducts, and tanniferous cells. Mature bark has a phloem consisting of phloem parenchyma, sieve tubes, companion cells, and phloem fibers with rosette crystals of calcium oxalate and tannin.

Phloem parenchyma cells are polygonal in shape and with rosette crystals of calcium oxalate, starch grains, and tannin. Phloem fibers are broad stained in horizontal stripes with thick-walled elongated cells which are present tangentially, alternate to that parenchyma of medullary rays are found[2] Medullary rays are uniseriate, radially elongated cells that are full of tannin. Uniseriate medullary rays dilate radially to the cortical region and form a broad medullary ray. They are tangentially elongated polygonal cells containing large-sized rosette crystals of calcium oxalates, tannin, and starch grains. Very large-sized rosette crystals of calcium oxalate and starch grains in some of the cortical cells are seen in the five samples [Figure 2]. The morphological characteristics were reconfirmed by using various Floras of Gamble.[7],[8],[9]
Figure 2: Microscopy of Terminalia arjuna bark. (A) TS of bark, ×40; (B) cork region enlarged, ×400; (C) and (D) phloem region enlarged, ×400; (E) histochemical staining for starch, ×40; (F) histochemical staining for lignin, ×40; (G) histochemical staining for tannin, ×40. ck = cork, ct = cortex, lc = lignified cells, mr = medullary ray, ph = phloem, phf = phloem fibers, prcr = prismatic crystals of calcium oxalate, rer = rosette crystals of calcium oxalate, sg = starch grains, stc = stone cells, te = tannin cells

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Among five samples, Hyderabad sample had less than 5% of bark appeared different. So, special concentration was paid to this sample and it was evaluated. Microscopically, the bark sample was compared; it was observed that the bark of Terminalia paniculata. TS of the bark, medullary rays, and cork tissue looked unique and different from other samples. Its presentation is shown in [Figure 3].
Figure 3: TS of bark Terminalia paniculata. ck = cork, ct = cortex, mr = medullary rays, Ph = phloem

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Polarization and Fluorescence Microscopy

In polarization microscopy, numerous rosette and cluster crystals of calcium oxalates of different sizes were scattered throughout the bark, especially below the cork region and medullary rays. In fluorescence microscopy, groups of phloem fibers and stone cells are seen in the five samples [Figure 4].
Figure 4: Fluorescence microscopy of T. arjuna bark. (A) TS of lignified cork cells and stone cells, ×200; (B) group of phloem fibers in the phloem region, ×200. ck = cork, phf = phloem fibers, stc = stone cells

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  Powdered Crude Drug Microscopy Top


Method

The bark powder of Arjuna was boiled separately with a small volume of chloral hydrate solution. Cleaved powder was removed into three separate watch glasses, respectively and stained with one drop each of reagent. 1. With phloroglucinol, Sudan red III and concentrated HCl, pink-colour lignified cells were seen. 2. With ruthenium red, pink-colored mucilaginous cells were seen in the epidermis. 3. With Sudan red III, cuticle looks pink colour.

Powder when treated with dilute iodine solution, sulphuric acid shown in blue as hemicellulose was present. Powder on mixing with diluted iodine solution, it turned to blue as starch is present in endodermis.

Overall, 5mg of fine powder of bark was mounted in glycerine; stained with iodine, phlorogucinol, hydrochloric acid, and Sudan III.

The powder shows fragments of yellowish brown cork in the surface view, and the cells are thick-walled and polygonal in shape. Abundant clusters and rosette crystals of calcium oxalate and their broken fragments of various sizes are scattered as such and in parenchymatous cells of the phloem and cortex.

Fragments of septate, thick, and thin-walled lignified fibers are isolated or grouped, often getting detached at the septa. Fragments of parenchymatous tissues are filled with pinkish brown-colored matter. Simple and compound starch grains are scattered as such or embedded in parenchymatous cells.

Fragments of radically and tangentially longitudinally cut medullary rays are associated with crystal fibers. Groups of septate and aseptate fibers are seen.

Powder microscopy of Arjuna bark especially has a surface view of cork cells; cortical parenchyma has starch grains; parenchyma cells are filled with cluster and rosette crystals of calcium oxalate, starch grains, and reddish brown content; medullary rays are associated with starch grains and reddish brown content; and medullary rays are associated with crystal fibers. Starch grains, groups of rosette crystals, fragments of septate fibers, and groups of aseptate fibers etc are seen in the five samples.

Thin Layer Chromatography: Test Solution

Overall, 2 gm of powdered drug was extracted by using 100ml of methanol for 4h. The extract was used for thin layer chromatography (TLC).

Procedure

Overall, 5ml of test solution and 5ml of standard solution (toluene:ethyl acetate:formic acid in 5:5:1 ratio) were applied on pre-coated silica gel 60 F254 TLC plate thickness of 0.2 mm. The plate was developed in the solvent system in a chamber from a distance of 8 cm.

The color and retention factor (RF) values of the methanol extract of Arjuna bark five samples, and the bands are recorded in [Table 1] and [Figure 5].
Table 1: RF values and color bands in TLC of Arjuna methanol extract

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Figure 5: TLC of five samples of Arjuna

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  Discussion Top


Arjuna bark is broadly used in Ayurveda to treat a wide range of diseases; it is an important ingredient in many Ayurveda formulations, such as Asvagandharishtam, Ayaskriti, Parthadyarishtam, Aravindasavam, Devadarvyarishtam, Nyagrodhadi kashayam, Virataradi kashayam, Trikantakadi ghritam, Virataradi ghritam, Sarvamayantaka ghritam, Lakshaguggulu, Arimedadi tailam, Nagarjunabhra rasa, and Arjunaghritam.[10],[11] Hence, understanding its importance and wide usage, it is essential to study the authenticity of market samples and at the same time to find out adulterants in the market samples. Adulteration is an illegal practice of adding cheaper ingredients to good-quality products to increase their quantity. The deliberate material added to a drug reduces the quality of the drug; thus, if adulterated drugs are used to prepare any products, the finished products definitely possess a low quality and substandard products emerge. This study is much needed due to many Terminalia species available are adulterated. Earlier, a study with PCR method was developed to identify and discriminate T. arjuna from all Terminalia species. As the method has a great specificity and sensitivity, that can be utilized as quality control of raw drug material.[12]

Before procurement of any crude drugs from the market, it is very essential to have knowledge of the drug of the source plant, especially the officinal part of the plant in the terms of pharmacognostical standards. Keeping this in mind, in the present study, the source plant was identified, and bark samples and market samples from different south Indian states were collected. The five samples screened were compared and found same presentations with organoleptic characteristics, microscopic examination of bark, polorisation and fluorescence microscopic examination, powder microscopy, TLC of drug as details given, but the Hyderabad market sample (Sample 3) had less than 5% was found different to Arjuna bark. Later, by the features, it was found that the bark sample of Terminalia paniculata, T.S. of T. paniculata bark looks entirely different to Arjuna. Possibly, this adulterant mixing might happen by chance or deliberately by the traders. With this study, the author concludes that market sample of Arjuna is T. arjuna. Adulteration is almost negligent in all source samples. It is also found that Terminalia tomentosa is sold separately as a black variety.


  Conclusion Top


After comparing samples of Arjuna bark collected from source tree and market sample show same presentations. Arjuna bark of market samples can be acceptable. Terminalia paniculata was found as an adulterant in one market sample of Arjuna bark in negligible quantity.

Acknowledgement

The author would like to acknowledge the Vice Chancellor, SCSVMV Deemed University, Dr. M. Prasanna for their support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Neelam S, Singh VK. Efficacy and advancement of Terminalia Arjuna in Indian herbal drug research: A review. Trends Appl Sci Res 2019;14:233-42.  Back to cited text no. 1
    
2.
Vrindya Vaidyaka of Vrinda, commentary by Sadananda Khemaraja, Sri Krishnadasa Mumbai ed. 1967. p. 122.  Back to cited text no. 2
    
3.
Chaudhary SP, Kirtika , Singh AK, Ram B. An Ayurvedic review of Arjuna from Samhita. Int J Res Ayurveda Pharm2017;8(Suppl 2) :1-9.  Back to cited text no. 3
    
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Chopra RN, Chopra IC, Handa KL, Kapur LD, Chopra’s Indigenous Drugs of India. 1st ed. Calcutta: UN Dhur and Sons; 1958, . pp. 421-4.  Back to cited text no. 4
    
5.
Chopra RN, Chopra IC, Handa KL, Kapoor LD. Indigenous Drugs of India. 2nd ed. Calcutta: U.N. Dhur & Co.; 1958. p. 421-4.  Back to cited text no. 5
    
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Tewari RC, Sharma P, Sharma AK. Pharmacognosy and classical uses of Arjuna (Terminalia Arjuna Roxb.): A critical review world. J Pharm Res 2015;4:2684-94.  Back to cited text no. 6
    
7.
Johansen DA. Plant Micro Technique. 1st ed. New York: McGraw Hill Book Company; 1940. p. 182-203.  Back to cited text no. 7
    
8.
Gamble JS. Flora of the Presidency of Madras. Calcutta: Botanical Survey of India; 1957. p. 1-3.  Back to cited text no. 8
    
9.
Thammanna P, Rao KN, Madhava Chetty K. Angiospermic Wealth of Tirumala. Tirupati: T.T.D. Press; 1994. p. 6-7.  Back to cited text no. 9
    
10.
Sastry JLN. Dravyaguna Vijnana (Study of Essential Medicinal Plants of Ayurveda). Vol. 2, 2nd ed. Varanasi: Chaukhambha Orientalia; 2005. p. 496.  Back to cited text no. 10
    
11.
Govindadas . Bhaiṣajya Ratnavali. 11th ed. Vidyotini Hindi Commentary by Shastri AD. Varanasi: Chaukhamba Sanskrit Sansthan; 1993. p. 491  Back to cited text no. 11
    
12.
Sharma S, Shrivastava N. DNA-based simultaneous identification of three Terminalia species targeting adulteration. Pharmacogn Mag 2016;12:S379-83.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

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