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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 3  |  Page : 215-222

Macroscopic, microscopic, and preliminary analytical evaluation and quality assessment of the root of Samarakhadyam (Byttneria herbacea Roxb.): An extrapharmacopoeial drug of Ayurveda


1 Department of Dravyaguna Vigyana, JS Ayurved Mahavidyalaya, Nadiad, Gujarat, India
2 Department of Dravyaguna Vigyana, IPGT and RA, GAU, Jamnagar, Gujarat, India
3 Pharmacognosy Laboratory, GAU, Jamnagar, Gujarat, India
4 Pharmaceutical Chemistry Laboratory, IPGT and RA, GAU, Jamnagar, Gujarat, India

Date of Submission22-Oct-2019
Date of Decision03-Jan-2020
Date of Acceptance04-Mar-2020
Date of Web Publication27-Jul-2020

Correspondence Address:
Dr. Tarun Sharma
Department of Dravyaguna Vigyana, JS Ayurved Mahavidyalaya, Nadiad - 387 001, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijhas.IJHAS_94_19

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  Abstract 


BACKGROUND: Byttneria herbacea Roxb., an extrapharmacopoeial plant of Ayurveda, locally known as Samarakhai by tribal people of Odisha, belongs to the family Sterculiaceae. The roots of B. herbacea is traditionally claimed to be used in the management of wounds, fractures, swellings, gynecological disorders, diarrhea, cuts, ulcers, and others. Although the plant is used traditionally, for its pharmacognostical characteristics, no proper scientific evaluation has been reported.
AIMS: To establish and standardize the root of the plant for its microscopical and physicochemical characteristics along with different qualitative tests and to assess heavy metals, aflatoxins, and insecticide and pesticide residue.
MATERIALS AND METHODS: After proper authentication, the roots were exposed to macroscopy, microscopy and physicochemical, qualitative and high-performance thin-layer chromatography study as per the Ayurvedic Pharmacopoeia of India. Heavy metal analysis, aflatoxin analysis, and insecticide and pesticide residue analysis of the root were carried out following the standard methods.
RESULTS: Microscopic study, through transverse section of root shows outer multilayered elongated compactly arranged cork cells, multiseriate medullary rays embedded with brown content, starch grains and prismatic crystals, centrally located vascular bundle consist of xylem and phloem. Physicochemical parameters showed that water-soluble extractive value (11.995%) was more than that of methanol-soluble extractive (5.936%). Chromatography study exhibited 8 peaks at 254 nm and 4 peaks at 366 nm. Heavy metals and aflatoxins B2, G1, and G2 were found in the prescribed limit, whereas aflatoxin B1 was above the limit of quantification. Insecticide and pesticide residues were not detected in the sample.
CONCLUSION: Typical microscopical characteristics are multiseriate medullary rays, tannin contents, and prismatic crystals. Heavy metals and aflatoxins B2, G1, and G2 were found below the limit of quantification. The findings of the study may be helpful to identify, standardize, and for quality assessment of root of B. herbacea.

Keywords: Aflatoxins, heavy metals, high-performance thin-layer chromatography, insecticides, pesticides, Samarakhai


How to cite this article:
Sharma T, Acharya R, Harisha C R, Shukla V J. Macroscopic, microscopic, and preliminary analytical evaluation and quality assessment of the root of Samarakhadyam (Byttneria herbacea Roxb.): An extrapharmacopoeial drug of Ayurveda. Int J Health Allied Sci 2020;9:215-22

How to cite this URL:
Sharma T, Acharya R, Harisha C R, Shukla V J. Macroscopic, microscopic, and preliminary analytical evaluation and quality assessment of the root of Samarakhadyam (Byttneria herbacea Roxb.): An extrapharmacopoeial drug of Ayurveda. Int J Health Allied Sci [serial online] 2020 [cited 2020 Aug 6];9:215-22. Available from: http://www.ijhas.in/text.asp?2020/9/3/215/290723




  Introduction Top


India has rich floristic and ethnic diversity. Medicinal plants employed by traditional healers, however not recorded in classical texts of Ayurveda, are nominated as Anukta dravya and quoted as extrapharmacopoeial plants of Ayurveda.[1] These Anukta dravyas are the outcome of the studies published through numerous ethnomedicinal and anthropological survey studies.[2]Byttneria herbacea, family Sterculiaceae, an extrapharmacopoeial folklore medicinal plant, used as a healing herb, known and marketed as Samarakhai in Odisha, is frequently found in Peninsular India from Gujarat southward to Tamil Nadu and in Odisha and Bihar.[3],[4]

B. herbacea Roxb. is a branched herb, with ascending branches with a perennial woody rootstock. Leaves are distant, ovate-lanceolate, acuminate, glabrescent, toothed, paler below, base cordate or rounded, 3–5 nerved, and petiolate. It has small, pale purple flowers in short cyme and pale green capsule covered with soft subulate prickles.[3] Ethnobotanical studies report the uses of its root in the management of many diseases. Its roots are being employed in the management of wounds, fractures, sprain, swellings, cuts, ulcers, boils, body pain, asthma, cholera, diarrhea, seminal weakness, loss of libido, leukorrhea, and others.[5],[6] The roots are being cooked and eaten as vegetable in Odisha.[7] Antioxidant activity of aqueous extract of root has been reported.[8]

Traditional herbs and herbal products are thought about to be mild, nontoxic, and even harmless as a result of their natural origin. In fact, contamination of crude medicinal plants as well as their products has been reported.[9],[10] It is so imperative to confirm quality of the preparations derived from the traditional plants using modern techniques and applying appropriate standards. Standardization of raw drugs in herbal industry is a vital step toward quality control. Many analytical parameters such as physicochemical values, microorganism contamination, heavy metals, aflatoxins, and insecticide and pesticide residue ought to be dole out as a measure of quality check.

In spite of its high medicinal value, the pharmacognostical and preliminary phytochemical characteristics of its root are not reported till date. Hence, the present study has been carried out to establish its identification and standardization characteristics and quality assessment through estimation of microscopical, physicochemical, and phytochemical characteristics and analysis of heavy metal, aflatoxins, and pesticide and insecticide residues.


  Materials and Methods Top


Collection, authentication, and preservation of the sample

The whole plant of B. herbacea was collected and identified by the local taxonomist from its natural habitat Gandhamardan hill ranges of Paikmal, Bargarh district of Odisha, during October to November 2016. Then, the plant herbarium was authenticated from the Botanical Survey of India, Kolkata (Specimen No. CNH/2016/Tech. II/68). A specimen of the sample herbarium has been deposited in the Pharmacognosy Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar (Specimen No. Ph. M: 6200/16-17), for future reference. The roots were separated and washed with tap water and stored in a solution of 70% ethyl alcohol: glacial acetic acid: formalin in the ratio of 90:5:5 to utilize them for microscopic studies whenever needed.[11] The remaining parts of the roots were dried under the shade. Then, the dried roots were powdered by mechanical grinder and sieved through 60# for powder microscopy, physicochemical parameters, and qualitative tests. The root powder was stored in an air-tight glass container.

Preparation of plant extract

Five grams of powdered root was macerated with 100 ml methanol in a closed flask for 24 h, shaking frequently during 6 h, and allowed to stand for 18 h. After 24 h, samples were filtered and methanol extract was collected. This extract was used for preliminary phytochemical screening [12] and high-performance thin-layer chromatography (HPTLC) study.

Macroscopic study

Morphological characteristics such as shape, size, texture, and color of the root of B. herbacea Roxb. were studied systematically as per visual observation, following standard procedure of taxonomy and verified with existing floras and standard books for authentication.[4],[13]

Microscopic study

Microscopy of transverse sections

Free-hand thin transverse sections of root were taken. Sections were cleared with chloral hydrate and observed for the presence of any crystals and then were stained with phloroglucinol (20 mg/ml of alcohol) along with concentrated hydrochloric acid (6N) to notice the lignified elements such as fibers and vessels following the standard procedure.[14]

Organoleptic study

The color, odor, touch, and taste of B. herbacea root powder were recorded through visual and sensory observation.[15]

Powder microscopy

Root powder (60#) was cleared with chloral hydrate solution. Then, the sample was examined using distilled water and after staining with different suitable reagents, i.e., phloroglucinol (20 mg/ml of alcohol) along with hydrochloric acid (6N) and iodine solution (2 g iodine and 3 g potassium iodide in 100 ml water) under a compound microscope (QUASMO, India), and photographs were taken using Kodak EasyShare C140, 8.2 megapixels ×3 optical/×5 digital zoom HD camera.[15],[16]

Physicochemical analysis

Assessment of the parameters such as moisture content, foreign matter, ash value, acid-insoluble ash, water-soluble extractive, alcohol-soluble extractive, and pH was carried out by following the standard procedures recommended by the Ayurvedic Pharmacopoeia of India (API)[17] and other standard text.[18]

High-performance thin-layer chromatography study

Method

HPTLC study of the extract was carried out by precoated silica gel 60 F254 plates which possess standardized adsorption layers, at room temperature. All the solvent systems were selected by trial and error method. The chromatogram was developed in CAMAG twin-trough glass chambers on 10 × 10 cm plates till the mobile phase travelled up to the distance of 8 cm from the starting point. After development, the plate was dried at room temperature for 5–10 min. Scanning was performed by CAMAG HPTLC densitometer in absorbance mode at 254 nm and 366 nm and colour of the resolve bands, Rf values were recorded.[19]

High-performance thin-layer chromatography fingerprinting profile

One track as 10 μL of sample was applied on an E. Merck aluminum plate precoated with silica gel 60 F254 of 0.2 mm thickness using Linomat IV applicator.

Development of solvent system

A number of solvent systems were tried to find out the best mobile phase. The solvent system toluene: ethyl acetate: diethylamine (7:2:1) gave the best resolution and maximum number of spots.

Development of chromatogram

The chromatogram was developed in CAMAG twin-trough glass presaturated with mobile phase, i.e., toluene: ethyl acetate: diethylamine (7:2:1) up to the distance at 80 mm.

Scanning and detection of spots

The developed and air-dried chromatoplate was at 254 nm and 366 nm to obtain planer chromatogram. Scanning was performed by CAMAG HPTLC densitometer in absorbance mode at 254 nm and 366 nm, and color of the resolve bands and Rf values were noted.

Heavy metal analysis

Estimation of heavy metals such as lead (Pb), cadmium (Cd), mercury (Hg), and arsenic (As) were carried out at IIT Mumbai. Pb, Cd, Hg, and As were estimated by inductively coupled plasma atomic emission spectrometry method.[20]

Aflatoxin analysis

The aflatoxin analysis was carried out at Gujarat Laboratory, Ahmedabad. The method adopted for analysis is GL/SOP/l-049 method.[21]

Insecticide and pesticide residue analysis

The insecticide and pesticide residue analysis was carried out at Pollucon Laboratory, Surat. The method adopted for analysis is EPA method 3540-8151 A.[22]


  Results and Discussion Top


Macroscopic study

The root system consists of woody taproot and its lateral branches. Length and width of the root (cut pieces) range from 8.5 to 25.5 cm and 0.5 to 1.8 cm (diameter), respectively. Externally, the root is dark brown in color with exfoliating thick cork and striations devoid of lenticels. The inner part of the root is woody with light cream color. Fracture is short and slightly fibrous [Figure 1]a.
Figure 1: Macroscopy and microscopy of Byttneria herbacea root. (a) Measurement of fresh root cut pieces along with fractured surface. (b) Diagrammatic section of root. (c) Typical pattern of medullary rays. (d) Cork and peripheral tissues. (e) Detailed T.S. of central stellar region of root shows medullary rays and xylem and xylem parenchyma. (f) Detailed stained T.S. of root shows xylem and xylem parenchyma. (g) Detailed stained T.S. of root shows Tannin content. (h) Simple and compound starch grains

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Microscopic study

Transverse section

Schematic transverse section of the root is spherical in shape with centrally located vascular bundle and radially arranged medullary rays.

Detailed Transverse Section of the root shows tabular elongated compactly arranged cork cells in 12–14 layers filled with brown content, followed by cortex of 30–40 layers. Cortical cells often filled with brown content and simple and compound grains. Medullary rays emerging from center are broader and multiseriate, but when extending to the cortical zone, they become narrower and almost uni- or biseriate. Medullary rays are embedded with brown content, starch grains, and prismatic crystals.

Centrally located vascular bundle consists of xylem and phloem. At the zone of phloem region, alternatively, pericyclic fiber patches are embedded all over the transverse section [Figure 1]b, [Figure 1]d, [Figure 1]e, [Figure 1]f, [Figure 1]h.

Organoleptic characteristics

The root powder is light greyish coffee in color with characteristic slight aromatic odor, rough and fibrous in touch, and sweet and astringent in taste with mucilage [Figure 2]a.
Figure 2: Powder microscopy of Byttneria herbacea root powder. (a) Powder of root. (b) Fragment of cortical cells in tangential view. (c) Micro measurement of simple starch grain. (d) Micro measurement of compound starch grain. (e) Tannin content (brown content). (f) Fragment of unicellular trichome. (g) Fragment of bordered pitted and scalariform vessel. (h) Group of simple fibers

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Powder microscopy

Microscopy of the root powder shows the presence of ample amount of simple and compound starch grains, fragment of simple unicellular trichomes, cork cells in surface view filled with brown content, fragment of cortical cells in tangential view, fibers passing through medullary rays, fragment of group of simple fibers, and fragment of bordered pitted and scalariform vessels [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e, [Figure 2]f, [Figure 2]g, [Figure 2]h.

Preliminary physicochemical analysis

Root powder was found to be devoid of any foreign matter, which may be due to the good harvesting practice followed during the collection of the drug. Loss on drying was found to be 12.075% w/w. The loss on drying of any sample is directly related to its moisture content. An excess of water in medicinal plant materials will encourage microbial growth, the presence of fungi or insects, and may affect its preservation.[23] It is found that water-soluble extractive value (11.995% w/w) was more than that of methanol-soluble extractive value (5.936% w/w). Hence, suggested that root would be more soluble in water. Ash value was found to be 7.078% w/w. The ash value shows the presence of inorganic and salt materials in the sample.[23] Acid-insoluble ash value was 1.231% w/w. Acid-insoluble ash indicates the presence of more siliceous matter in the drug. pH value of water extract of the root was 6.5, which indicates its weak acidic nature. The detailed results of physicochemical analysis are given in [Table 1].
Table 1: Preliminary physicochemical analysis of Byttneria herbacea root powder

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Preliminary phytochemical analysis

Preliminary phytochemical test revealed the presence of saponins, steroids, tannins, alkaloids, flavonoids, and phenolic compounds in methanol extract of the root sample. The results of tests performed are depicted in [Table 2].
Table 2: Qualitative analysis of Byttneria herbacea root in methanol extract

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High-performance thin-layer chromatography

The methanol extract of the root showed 8 peaks and 4 peaks at UV range of 254 nm and 366 nm, respectively. Common Rf value 0.03, 0.15, 0.27, and 0.94 were found under UV range of 254 nm and 366 nm. The Rf values are mentioned in [Table 3]. The photos of HPTLC plate, 3d graphs, and peak display at UV ranges are portrayed in [Figure 3].
Table 3: Rf values obtained at short UV light (254 nm) and long UV light (366 nm) of Byttneria herbacea root powder

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Figure 3: High-performance thin-layer chromatography of Byttneria herbacea root. (a) High-performance thin-layer chromatography plate at 254 nm. (b) High-performance thin-layer chromatography plate at 366 nm. (c) Peak display at 254 nm. (d) Peak display at 366 nm

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Heavy metal analysis

Harmful heavy metals such as lead, cadmium, mercury, and arsenic compounds were found <0.01 ppm in root sample. Permissible limits of lead, mercury, cadmium, and arsenic are 10 ppm, 1 ppm, 0.3 ppm, and 3 ppm, respectively.[24] The term heavy metal refers to any metallic chemical element that encompasses a relatively high density and is harmful at low concentrations. Metals are broadly distributed throughout nature and occur freely in soil and water.[25] Among the heavy metals, lead, cadmium, mercury, and arsenic are harmful metals and have mutagenic effects even at terribly low concentration. Several cases of human disease, malfunction, and malformation of organs due to metal toxicity have been reported. Not only human beings, but also animals and plants are also affected by toxic levels of heavy metals.[26] The exact identification of metals is required for correct diagnosis due to considerable overlap between the clinical syndromes associated with heavy metal poisoning.[27]

Contamination of herbal materials with toxic substances is usually attributed to many causes. These embrace environmental pollution (i.e., contaminated emissions from factories and leaded petrol and contaminated water likewise as runoff water that finds its way into rivers, lakes, and also the sea), soil composition, and fertilizers. This contamination of the herbal material ends up in contamination of the products throughout varied stages of the manufacturing method. As toxicant substances are probably present in several foods, because of their abundance in nature, it is necessary to note that concomitant uptake of herbal products would augment the full concentration of toxicant metals consumed by people, though best practice guidelines are followed.[28]

Aflatoxin analysis

Aflatoxin B1 was found above the limit of quantification, whereas aflatoxins B2, G1, and G2 were below the limit of quantification [Table 4]. Permissible limit of aflatoxin B1 and G1 is 0.5 ppm and aflatoxin B2 and G2 is 0.1 ppm, as mentioned in API.[29] Aflatoxins (AFs) are secondary metabolites produced by fungi Aspergillus, particularly Aspergillus flavus and Aspergillus parasiticus,[30] which grow in soil, decaying vegetation, hay, and grains. Once contaminated food is processed, aflatoxins enter the general food supply where they have been found in each pet and human foods, furthermore as in feed stocks for agricultural animals.[31] There are four naturally occurring aflatoxins, designated B1, B2, G1, and G2, with aflatoxin B1 being the foremost common and toxic. Aflatoxin B1 received greater attention than any other mycotoxins as a result of its demonstrable carcinogenic effect in vulnerable animals and its acute toxic effects in human.[32] The carcinogenic, immunosuppressive, and mutagenic effects of aflatoxins on many animals are totally documented.[33]
Table 4: Results of aflatoxins content of Byttneria herbacea root

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Aflatoxins are extensively studied and are classified as Group 1 human carcinogens by the International Agency for Research on Cancer.[33] Experimental data gathered throughout the last three decades on the loss of productivity in farm animals overwhelming contaminated feeds and also the carcinogenicity in experimental animals provide sufficient evidence concerning the dangerous nature of aflatoxins.[34] The correlation statistics between the consumption of aflatoxin-contaminated foods and also the increase of the incidence of liver carcinoma in many populations in South East Asia and Africa suggests the threat expose to human health by aflatoxin.[35]

Mycotoxins are considered inevitable contaminants in foods and feed stuffs because agronomical technology has not yet advanced to level at which preharvest infection of vulnerable crops by fungi can be eliminated.[36] Beneath favorable conditions generally found in tropical and subtropical regions, including high temperatures and high humidness, these molds, usually found on dead and decaying vegetation, will invade food crops. Drought stress, insect harm, and poor storage may contribute to higher incidence of the molds in temperate regions. Food crops will become contaminated both before and after harvesting. Improper storage conditions that favor molds growth (warm and humid storage environments) can typically lead to levels of contamination much higher than those found within the field.[37] This drug was collected from Odisha, which is also a high temperature and high humidity zone; thus, due to climatic conditions and improper storage condition, aflatoxin B1 might be found above the quantification limit.

Insecticide and pesticide residue analysis

Insecticide and pesticide residue were not detected in the root sample. The results of insecticide and pesticide residue are depicted in [Table 5]. Minimum detection limit of method is 0.1 mg/kg. Pesticides are chemical compounds used to control or eradicate pests. According to their activity, they are classified as insecticides, fungicides, herbicides, nematicides, rodenticides, and others.[38] The residues of pesticides as well as metabolites and degradation products will remain in plants or within the soil that become a notable supply of contamination for herbal medicines.
Table 5: Results of various insecticide and pesticide residue in Byttneria herbacea root

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Medicinal plant materials might contain pesticide residues, which accumulate as a result of agricultural practices, such as spraying, treatment of soils throughout cultivation, and administration of fumigants during storage.[39] Pesticides, used throughout cultivation of crops, will stay as residues in foodstuffs, particularly vegetables and fruits. Exposure of the population to pesticide residues could also be harmful and might be reduced by washing the foodstuffs completely in running water or by peeling. Cooking and alternative processes can even reduce such residues.[40]


  Conclusion Top


The macroscopic characteristics such as long, thick, fleshy, tuberous root with exfoliating thick cork and striations devoid of lenticels and microscopic characteristics such as typical pattern of medullary rays, brown content, starch grains, and prismatic crystals in T. S. of root are identifying characteristic of B. herbacea. Physicochemical, qualitative test reports, and HPTLC will help in establishing standards in identity, degree of purity, and quality of the plant material and act as standards for quality assurance. B. herbacea are found to be safe as heavy metal, pesticide residues, and aflatoxin content were below the limit of quantification.

Acknowledgement

The authors are thankful to Director, IPGT & RA, Gujarat Ayurved University, Jamnagar and Ministry of AYUSH for providing financial support and other facilities to carry out the research work. We express our thankfulness to Mr. B. N. Hota, Rtd. DFO, Govt. of Odisha; Mr. Pareshwar Sahoo, Pharmacognosy expert; Dhala Bhai, plant collector and other traditional healers who helped us during drug identification and collection at Gandhamardan Hills, Paikmal, Bargarh, Odisha.

Financial support and sponsorship

The study was fully funded as a part of Ph.D. research project and IMR project by Institute for Post Graduate Teaching and Research in Ayurveda, GAU, Jamnagar and Ministry of AYUSH.

Conflicts of interest

There are no conflicts of interest.



 
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40.
Anonymous, Dietary Guidelines for Indians - A Manual. 2nd ed. Hyderabad: National Institute of Nutrition; 2011. p. 24.  Back to cited text no. 40
    


    Figures

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

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