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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 7  |  Issue : 2  |  Page : 119-132

DNA bar-coding, pharmacognostical, and phytochemical analysis of Atalantia monophylla DC. leaves


1 Department of Dravyaguna, ITRA, Jamnagar, Gujarat, India
2 Department of Pharmacognosy, ITRA, Jamnagar, Gujarat, India
3 Department of Pharmaceutical Laboratory, ITRA, Jamnagar, Gujarat, India
4 Dr. Prabhakar Kore Basic Science Research Centre, KLE Academy of Higher Education and Research, Belagavi, Karnataka, India

Date of Submission22-Jul-2021
Date of Acceptance06-Feb-2022
Date of Web Publication14-Sep-2022

Correspondence Address:
Das Ashokkumar Nathulal
Department of Dravyaguna, ITRA, Jamnagar, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdras.jdras_18_21

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  Abstract 

BACKGROUND: Leaves of Atalantia monophylla DC. (Syn. Limonia monophylla L.), family Rutaceae, are traditionally used to treat rheumatoid pain, hemiplegia, itch and cutaneous complaints, cough, and as an antidote to snake bite, etc. The present study reports the detailed morphology, anatomy, genetic barcoding, physiochemical, and phytochemical aspects of A. monophylla leaves. METHODS: The sample of leaves of the plant were collected from one of its natural habitat, Athagarh forest, Odisha, India. The detailed macroscopic observations were made with naked eyes, and microscopy characters of leaf, stomatal index, and powder microscopy were done as per standard protocol. Physicochemical and qualitative preliminary phytochemical analysis, high-performance thin layer chromatography (HPTLC), and DNA bar-coding studies were carried out as per Ayurvedic Pharmacopoeia of India methods and other standard guidelines. RESULTS: Leaves were opposite, unifoliate, ovate-elliptic, entire, emarginate, and glabrous. Transverse section (TS) through petiole showed epidermis, cortex, pericyclic fibers, and vascular bundles along with trichome. TS of lamina passing through midrib shows lamina with upper and lower epidermis, lignified elements, interrupted by stomata and unicellular trichome. Microscopic characteristics of the leaves powder show the presence of oil globule, rhomboidal crystal, prismatic crystal, and fragment of paracytic stomata. The result of loss on drying, total ash, acid-insoluble ash, water, methanol-soluble extractive, and the obtained pH values were about 10.47 ± 0.40%, 6.94 ± 0.95%, 1.00 ± 0.00%, 30.41 ± 2.26%, 7.64 ± 0.56%, and 6.6, respectively. Qualitative phytochemical analysis shows the presence of carbohydrates, glycosides, saponins, alkaloids, tannins, and flavonoids and the HPTLC study revealed 29 spots at both short (254 nm) and long (366 nm) ultraviolet rays. CONCLUSION: Epidermal cells, oil globule, rhomboidal crystal, prismatic crystal, paracytic stomata, and simple unicellular trichome are key characters for identification of its leaves. The results obtained from pharmacognostical, phytochemical, physicochemical, and DNA bar-coding studies may serve as the diagnostic tool of A. monophylla toward its authentication and identification and also in maintaining of quality, strength, purity, and efficacy of the leaves.

Keywords: Atalantia monophylla, DNA bar-coding, HPTLC, physicochemical


How to cite this article:
Ashokkumar Nathulal D, Acharya R, Rudrappa HC, Shukla VJ, Hegde S. DNA bar-coding, pharmacognostical, and phytochemical analysis of Atalantia monophylla DC. leaves. J Drug Res Ayurvedic Sci 2022;7:119-32

How to cite this URL:
Ashokkumar Nathulal D, Acharya R, Rudrappa HC, Shukla VJ, Hegde S. DNA bar-coding, pharmacognostical, and phytochemical analysis of Atalantia monophylla DC. leaves. J Drug Res Ayurvedic Sci [serial online] 2022 [cited 2022 Sep 25];7:119-32. Available from: http://www.jdrasccras.com/text.asp?2022/7/2/119/356051




  Introduction Top


Atalantia monophylla DC. (Syn. Limonia monophylla L.), family Rutaceae, is a large shrub or small tree with spine, alternate, emarginate leaves bearing petiole and flower white, short axillary or sub-terminal, sub-sessile, pubescent corymb.[1]

The ethno-medicinal claims of A. monophylla leaves has been reported in the management of rheumatoid pain, hemiplegia, antidote to snake bite, itch and cutaneous complaints, cough, etc.[2]

Authentication and standardization are the prerequisite steps, especially for herbal drugs and their formulation in the traditional system of medicine. The present investigation of A. monophylla leaves was taken up to establish pharmacognostical profile, which helps in identification and also in standardization of the drug in crude form.[3] So, in the present article, an attempt has been planned to study A. monophylla leaves for its morphology, anatomy, physiochemical parameters, and phytochemical screening including HPTLC and DNA bar-coding.


  Materials and Methods Top


Collection, authentication, and preservation

A. monophylla plant was identified with the help of different floras. The leaves sample were collected by the scholar from its natural habitats, Athagarh forest, Odisha, India (latitude: 20.463754, longitude: 85.406931), on December 2019 from the help of a local taxonomist. Leaves sample were well-preserved both in wet [ethyl alcohol (90): glacial acetic acid (5): formalin (5)] and dry conditions. Herbarium sheet with reference no. phm/6300/2021 was submitted to the pharmacognosy laboratory of ITRA for future references [Figure 1](G) and (H) and [Figure 2].
Figure 1: Morphology of A. monophylla plant. (A) Plant in natural habitat; (B) ventral part of leaves; (C) dorsal part of leaves; (D) leaf with spine; (E) flowering; (F) fruiting of plant; (G) herbarium (phm/6300/2021); (H) wet preserved sample

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Figure 2: Herbarium (phm/6300/2021)

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Processing and preservation of drug

Dried leaves shaded, grounded by a mechanical grinder and sieved through a 60# mesh, and powdered were kept in an air-tight glass jar for its pharmacognostical and analytical evaluation.

Macroscopic study

Various macroscopic features, i.e., shape, venations, margin, apex, base, surface, and texture of the fresh leaves, were studied referring various floras and texts.[4] Macroscopic observation was done with naked eyes and a centimeter scale was used to measure the leaf size.

Organoleptic study

Test drugs in fresh, dry, and powdered leaves were exposed to examine various organoleptic characters such as color, odor, taste, and touch.[5]

Microscopic study

Transverse sections

Free-hand thin transverse section (TS) of the leaf was taken and cleaned with choral hydrate to observe the anatomical structure and microscopical characters with the help of a Quasmo binocular compound microscope. For histochemical test, thin TSs of the leaves were exposed to phloroglucinol (2 g of 100 mL of 90% alcohol) + Conc. HCl (10% glacial HCl dissolved in 100 mL water), iodine, and ferric chloride 5% (w/v in 90% of alcohol) solution for observation of lignin, starch grains, and tannin, respectively.[6]

Powder microscopy

Microscopic features of the leaves powder were studied, and photographs (Kodak camera with 8.2 megapixel) were taken with the help of a Quasmo binocular compound microscope.

Surface study/quantitative microscopy study

The surface study of epidermis was done/conducted to define nature and distribution of stomata and epidermal cells. Quantitative microscopy study of leaves was done/conducted to determine the size of stomata and stomatal index. The study was done in triplicate, and data were presented in mean value.[7]

Stomatal index (SI) = 100/E + S

where S is the number of stomata per unit area and E the number of epidermal cells in the same unit area.

DNA bar-coding

The fresh tender leaves were subjected to genetic bar-coding, and the study was carried out at the Pharmacognosy Laboratory of KLE, Belagavi, Karnataka, India.

DNA isolation

Genomic DNA was extracted from the collected one leaf samples plant individually using the modified CTAB method.[8] The amount and purity of DNA was measured by using a bio-photometer (Eppendorf AG 22331 Hamburg model and serial no. 6132BG904470) and 1% agarose gel electrophoresis (GeNeiTM model and serial no. 02/19/F/166). Final dilution was made up to 50 ng/μL with TE buffer (10 mM Tris HCI, pH 8.0 and 0.1 mM EDTA, pH 8.0) and stored at -20°C temperature.

PCR amplifications of ITS2 region

Amplification of the ITS2 region was carried out in a Veriti Thermal Cycler (Applied Biosystems) and the 50 μL reaction mixture contained 50 ng genomic DNA, 10 μM primer, 25 μL of Taq Master Mix Red (2×) with 1.5 mM MgCl2 (AMPLIQON). For enhancing and facilitating the PCR amplification, dimethyl sulfoxide (100%) was used in each assay.[9]

Amplification conditions and primer details are given in [Table 1]. After confirming amplification in 1% agarose gel electrophoresis and purified using Mini Elute PCR Purification Kit (Qiagen), PCR products were sequenced.
Table 1: Primers used for ITS2 amplification and PCR amplification conditions

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Data analysis

The Bio-edit Sequence Alignment Editor Version 7.2.5 was used to view and trimmed the initial raw sequences.[11] MEGA version 7.0.14 was used to compare the highly similar sequences for nucleotide Basic Local Alignment Search (BLAST) and multiple sequence alignments.[12]

Physicochemical analysis and preliminary phytochemical screening

The powdered leaves was analyzed for physicochemical parameters such as moisture content, ash value, acid-insoluble ash, pH, water-soluble and alcohol-soluble extractive value as per standard procedures recommended by the Ayurvedic Pharmacopoeia of India (API).[13] For qualitative analysis, the methanolic and aqueous extract of the powdered leaves was analyzed for the presence or absence of the major/main chemical constituent as per standard method.[14]

HPTLC study

The Camag, Muttenz (Switzerland) HPTLC system consists of Linomat V auto-sprayer, which was connected to a nitrogen cylinder, a twin trough glass chamber (10 × 10 cm), and a plate heater. Precoating was performed by using TLC aluminum pre-coated pl Silica gel 60 GF254 (10 × 10 cm, layer thickness 0.2 mm [E. Merck KGaA, Darmstadt, Germany)], and toluene: ethyl acetate (9:1) V/V was used as mobile phase. Prior to sample application, TLC plates were two times pre-washed with 10 mL of methanol and activated at 80°C for 5 min. The maceration method was used for the preparation of leaf extract of A. monophylla and 1 mg/mL smeared on plate with the help of Linomat V applicator. The TLC plate was developed and done by Camag Twin Trough Glass Chamber with mobile phase for 30 min after saturating, and 10 min was the development time. The plate after removed from the chamber was air-dried at room temperature. The developed plate was scanned in a TLC Scanner (Camag Reprostar III) with D2 and W lamp, tungsten lamp as detectors at the absorption of short UV 254 nm and long UV 366 nm. Densitometry chromatogram and 3D graph analysis were done with the help of TLC scanner III with Win cat’s 4.02 software.[15]


  Results Top


Macroscopic study

Leaves are alternate, unifoliate, short petiole, coriaceous bright green, stipular scales subulate, axillary bud often spinous, leaflet ovate—elliptic or lanceolate, 2.5–7.5 × 1.2–3.5 cm, entire, notched or bilobed at tip emarginate, glabrous, finely nerved with 1–8 secondary nerves, base rounded, petiole 5–7 mm, pubescent [Figure 1(A]-F)].

Organoleptic characters: Fresh sample

The leaves were darkish green and smooth on the upper surface, whereas pale green on the lower surface, odor aromatic, and taste astringent and bitter.

Microscopic study

TS of petiole

Diagrammatic section oval- to ball-shaped in outline with epidermis and wide cortex vascular bundle reduced to pith. Epidermis is a single layer compactly arranged and covered with a thick cuticle. Some of the epidermal cells were interrupted by unicellular trichome. The cortex is made up of parenchyma cells loaded with prismatic and rhomboidal crystal and clear gland filled with oil. In the cortical region, three to four lysigenous cavities are also observed; inner to cortex have circularly arranged discontinuous pericyclic fibers, lignified two to four layers. Single-layer endodermis leads to vascular bundles. Vascular bundles are circularly and radially arranged, phloem toward cortex, xylem toward the pith region. Being centrally located reduced pith made up of parenchyma cells [Figure 3](A-F).
Figure 3: (A) Macro-measurement of leaflet and petiole; (B) TS with epidermis (Ep), cortex (Co), pericyclic fiber, vascular bundle (Vb) along with trichome (tr); (C) cortical region with lysigenous cavity (Ly ca) and prismatic crystal (Pr cr); (D) Pericyclic fiber, vascular bundle (Vb), central pith (Cr pi); (E) lignified pericyclic fibers (Pr fb), phloem (Ph), xylem (Xy); (F) cortical cell, prismatic crystal (Pr cr)

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TS of lamina passing through midrib

The diagrammatic section is somewhat boat-shaped and shows centrally located vascular bundles of lamina. Lamina differentiated into upper and lower epidermis, both epidermal cells covered with thick cuticle are compactly arranged whereas some of the lower epidermal cells were interrupted by stomata and unicellular trichome present in both epidermis, and stomata is present only at the lower epidermis. Below the upper epidermis, single-layer spongy parenchyma consists of chlorophyll pigment. Toward the lower epidermis, multi-layer spongy parenchyma is present. Through midrib both epidermis layers followed by several layers of collenchyma cells and few layers of round tissue parenchymatous consist of crystal and oil globules. Vascular bundles are located at the center within bundle sheath. Pericyclic fibers formed two to three layered continuous and also discontinuous ring. Main vascular bundles found toward lower epidermis, whereas peristyle near upper epidermis. The main vascular bundle consists of phloem toward lower epidermis, xylem toward the center, whereas peristyle consists of a few elements of xylem and phloem [Figure 4](A-F).
Figure 4: (A) Macro-measurement of leaves; (B) TS through midrib (Mb) with upper epidermis (Up ep) and lower epidermis (Lo ep) centrally located vascular bundle; (C) upper palisade parenchyma (Up pl pr) and lower spongy parenchyma (Lo sp pr); (D) TS through midrib (Mb) after staining; (E) phloem (Ph) and xylem (Xy); (F) pericyclic fibers (Pr fb)

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Surface study or quantitative microscopy of epidermis

Paracytic stomata are present in the lower epidermis, whereas stomata are absent in the upper epidermis. [Figure 5](A). Quantitative microscopy of leaves was done to evaluate the number of epidermal cells, stomatal numbers, stomatal index, and size of the stomata [Figure 5](B) and (C).
Figure 5: Surface study of leaf. (A) Paracytic stomata at lower surface; (B-C) Micro-measurement of stomata

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The stomatal number, stomatal index, stomatal size were calculated by trial and error method (mean value was taken into consideration in the study). Results are tabularized in [Table 2] [Figure 5(A-C)].
Table 2: Quantitative microscopy of A. monophylla leaves

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Powder microscopy

Leaf powder was parrot greenish in color with slightly aromatic odor, astringent and bitter taste, smooth fibrous texture [Figure 6]A. Diagnostic powder characters of leaf show the presence of epidermal cells, oil globule, group of septate fibers, rhomboidal crystal, prismatic crystal, fragment of spongy parenchyma and palisade cells, fragment of paracytic stomata, annular vessel, and simple unicellular trichome [Figure 6](B-E).
Figure 6: Powder microscopy of A. monophylla leaf. (A) Dried leaves powder; (B) epidermal cells; (C) oil globules; (D) group of septate fibers; (E) rhomboidal crystal; (F) prismatic crystal; (G) fragment of spongy parenchyma and palisade cells; (H) fragment of paracytic stomata; (I) annular and spiral vessel; (J) simple unicellular trichome

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Histochemical test

Thin TS of leaves was exposed to various tests for the confirmation of the presence and absence of chemical constituents. Lignified cells were present, whereas starch and tannins cells were absent in the leaves of A. monophylla [Figure 6(F-H)]. Result are tabulated in [Table 3].
Table 3: Result of histochemical study of A. monophylla leaves

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Physicochemical parameters

The values of physicochemical parameters of leaves are expressed in percentage. The details result of physicochemical analysis has been depicted in [Table 4].
Table 4: Physicochemical parameters of A. monophylla leaves

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Preliminary phytochemical parameters

A. monophylla leaves were carried out for preliminary phytochemical screening by means of methanolic and aqueous extracts to identify the major/main chemical constituents. The result revealed the presence of carbohydrates, glycosides, alkaloids, and flavonoids both in aqueous and methanolic extracts, whereas saponins and tannins are revealed only in aqueous extract. The details results are quoted in [Table 5].
Table 5: Preliminary phytochemical investigation of A. monophylla leaves

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HPTLC study

The study of HPTLC methanolic leaf extract was scanned under 254 and 366 nm, and the obtained detailed Rf values are described in [Table 6]. The HPTLC plate, 3D graphs, and peak displays are depicted in [Figure 7](A-E).
Table 6: Rf values obtained at short UV light (254 nm) and long UV light (366 nm) of A. monophylla leaves

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Figure 7: High-performance thin layer chromatography (HPTLC) of A. monophylla leaf. (A) Peak display at 254 nm; (B) 3D graph at 254 nm; (C) peak display at 366 nm; (D) 3D graph at 366 nm; (E) 3D graph at UV–Vis range 254 and 366 nm

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In spectral comparison while demonstrating, 20 similar spectra were obtained at Rf values of 0.03, 0.10, 0.13, 0.18, 0.22, 0.27, 0.30, 0.32, 0.34, 0.37, 0.40, 0.51, 0.57, 0.59, 0.61, 0.63, 0.65, 0.67, 0.74 and 0.79 among the 29 spots at 254 nm and 29 spots at 366 nm.

DNA bar-coding

A. monophylla DC. DNA sequences were aligned and taxonomically identified and were queried for highly similar sequences from NCBI using nucleotide BLAST Tool in NCBI GenBank, and it revealed that the queried sample sequences were best matching or similar to A. monophylla at NCBI GenBank; our sequence found 98% query coverage with GenBank accession number GQ225867.1 (rbcL region) with 0.0 E-value and 99.45% identity and 92% query coverage with GenBank accession number AB456047.1 for other A. monophylla species. The difference might be because of the region selected for sequencing/edaphic factor/genetic variation and therefore, the sequence derived confirms its identification as A. monophylla and also the final sequence derived was deposited in NCBI GenBank with accession number MW828334.1 in the study. Details are given in [Table 7] and [Figure 8] and [Figure 9].
Table 7: Sequences producing significant alignments through BLAST analysis

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Figure 8: Phylogenetic tree of A. monophylla

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Figure 9: NCBI BLAST of A. monophylla

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  Discussion Top


Established standards can serve as an essential tool in correct identity and quality of the crude drug. The major information on identity, purity, and quality can be obtained from its microscopy and physicochemical parameters of the plant material.[3]

A macroscopic study technique, based on its morphological similarities, can be useful to differentiate the desired species or part of the plant. Macroscopic and microscopic characteristics are the first step to establish the identity and purity of medicinal plant material.[16]A. monophylla, a large shrub or a small tree, is having spines and the leaves are ovate-elliptic, entire, emarginate, and glabrous. These are the key features which may be helpful for the macroscopic identification of the plant.[17]

TS through petiole showed epidermis, cortex, pericyclic fibers, and vascular bundles along with trichome. TS of lamina passing through midrib shows lamina with upper and lower epidermis, lignified elements, interrupted by stomata and unicellular trichome. Microscopic leaves powder shows the presence of oil globule, rhomboidal crystal, prismatic crystal, and fragment of paracytic stomata. Microscopic examination is essential for the identification of broken or powdered materials of herbal resources. Microscopic characters which determine the histological characteristic of leaves can aid as diagnostic parameters in the primary identification and proper authentication of the botanical species and also will be helpful to establish specific standards to minimize the adulteration of the plant drug.[16]

The shape of epidermal cells and stomatal index are constant within a taxonomy; however, these specific characters vary among species and can be useful for demarcation of species.[18] Paracytic stomata apparatus of A. monophylla appears to be a constant characteristic, i.e., the stomatal index is stable within a species but differs between species and can be useful in helping to distinguish the species of A. monophylla. Simple unicellular trichome structure in the powdered leaves shows much diversity which serves main criteria tools in the identification of the plant.[19]

The DNA bar-coding results revealed that the queried sample sequences were best matched with A. monophylla isolate and therefore, the sequence derived confirms its identification as A. monophylla in the study. DNA bar-coding was used to achieve maximum efficiency for medicinal material identification and to systematize the material and also serve as matching analytical methods to report the important legal issue related to quality control of herbal material.[20]

The results of loss on drying of A. monophylla leaves were obtained to be 10.47 ± 0.40%, which indicate that the drying processes were efficient and the main parameter for measuring the efficiency of plant drying process indicates the stability during the storage period of the drug and also denotes the phytoconstituents present in the plant. This can be retained and can be stored for a longer duration which will not encourage the growth of decay process caused by microorganisms. So, it is essential to evaluate the physical constants as they help in identifying adulterants or improper handling of the plant material.[21]

The objective behind an ash value is to remove or eliminate all traces of organic matter present, which may sometimes interfere in an analytical determination and reflects the care taken in its preparation. Acid-insoluble ash residue can be obtained after boiling the total ash, which measures the amount or quantity of silica present, mainly as sand and siliceous earth.[22] The values found for total ash were 6.94 ± 0.95% and those of acid-insoluble ash were 1.00 ± 0.00%.

Extractive values in a specific solvent provide an indication about the purity of the crude drug, which indirectly helps to identify exhausted or adulterant drug. The extractive values provide clue regarding its solubility in a particular solvent and also indicate the presence of nature and number of chemical constituents in the crude drug.[21] The water and methanol extractive values were observed to be 30.41 ± 2.26% and 7.64 ± 0.56%, respectively. This shows that constituents of the drug are more extracted and soluble in water than methanol. The pH value of water extraction of leaf was 6.6, which indicates weak acidic in nature.

Preliminary qualitative phytochemical analysis screening of A. monophylla leaves shows the presence of carbohydrates, glycosides, saponins, alkaloid, tannins, and flavonoids. Preliminary screening of phytochemicals serves as a valuable step or as a tool, in the detection of the bioactive principles present in medicinal plants, which may lead to/help in drug discovery and development by pharmaceutical companies, and plant extract could be explored for its highest therapeutic efficacy in order to develop safe drugs for various diseases/disorders.[23] Flavonoids are one of the most powerful water-soluble antioxidants and free radical scavengers, which help in preventing oxidative cell damage and exhibit anti-cancer effect. Saponins are commonly used in hyperglycemia, antioxidant, anticancer, and anti-inflammatory conditions.[24]

Fingerprinting analysis of HPTLC is one of the diagnostic tools to link botanical identity to the chemical constituent profile of the plant and used to distinguish adulteration in raw materials and also be employed for quantitative purpose of marker compound.[25]

HPTLC of methanol extract of powdered A. monophylla leaves was evaluated using the toluene: ethyl acetate (9:1 v/v) solvent system and while demonstrating in spectral comparison, 20 similar spectra were obtained at Rf values of 0.03, 0.10, 0.13, 0.18, 0.22, 0.27, 0.30, 0.32, 0.34, 0.37, 0.40, 0.51, 0.57, 0.59, 0.61, 0.63, 0.65, 0.67, 0.74 and 0.79, among the 29 spots at 254 nm and 29 spots at 366 nm.

The preliminary qualitative phytochemical screening of the leaves of A. monophylla provides its usefulness in various pharmacological activities; these phytoconstituents are responsible for anti-inflammatory,[26] immunomodulatory,[27] antioxidant, antifungal,[28] anthelmintic,[29] antibacterial,[30] antimicrobial,[31] and anticancer[32] activities.


  Conclusion Top


A. monophylla is a large shrub or a small tree with spines. Its leaves are ovate-elliptic, entire, emarginate, and glabrous. The presence of epidermal cells, oil globule, group of septate fibers, rhomboidal crystal, prismatic crystal, fragment of spongy parenchyma and palisade cells, fragment of paracytic stomata, annular vessel, and simple unicellular trichome are specific microscopic characters of leaf powder. The apparatus of paracytic stomata appears to be a constant character, i.e., the stomatal index of the leaf has lower surface 33.33 mm2, which helps in the identification of its leaves. DNA bar-coding is used to systematize the material and maximize efficiency for the identification of plant material. The phytochemical screening, parameters of physicochemical characterization, and HPTLC study can serve as a powerful tool/criteria for the evaluation of identity/uniqueness and validity of the plant which can be used to detect adulteration in raw materials. The studies provide information about the identification, chemical constituents, and physicochemical characters which serve as diagnostic tools for standardization of the plant.

Acknowledgement

The authors are thankful to the Director, Pharmacognostic, Pharmaceutical Laboratory, ITRA, Jamnagar and pharmacognosy laboratory of KLE, Belagavi Karnataka for providing facilities to carry out the research work.

Financial support and sponsorship

Institute of Teaching and Research in Ayurveda Jamnagar, Gujarat.

Conflicts of interest

There are no conflicts of interest.





 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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