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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 4  |  Issue : 2  |  Page : 67-77

Standardization of Crateva religiosa G. Forst. leaf by pharmacognostical studies


1 Department of Pharmacognosy, SCRI, Chennai, Tamil Nadu, India
2 Department of Pharmacognosy, College of Pharmacy, Madurai Medial College, Madurai, Tamil Nadu, India

Date of Submission07-Apr-2022
Date of Acceptance05-Nov-2022
Date of Web Publication23-Jan-2023

Correspondence Address:
Ms. Ramu Mohanapriya
Department of Pharmacognosy, SCRI, Chennai – 600 106, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrsm.jrsm_12_22

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  Abstract 

Background: Crateva religiosa belongs to the family Capparaceae or Capparidaceae commonly known as Caper family. Leaves are used as stomachic, tonic, effective counter irritant, antirheumatic, and rubefacient.
Objective: The present study aimed to investigate the pharmacognostical and phytochemical parameters of C. religiosa G. Forst. fresh leaves. Materials and Methods: Pharmacognostical, physicochemical, and phytochemical parameters were determined according to the standard procedure.
Results: Macroscopical evaluation showed that the leaves were ovate or broadly elliptical with acute base and exstipulate with the entire margin. Leaves are smooth, with densely reticulate venation. The quantitative constants such as epidermal number, stomatal number and index, palisade ratio, vein islet, and termination number were determined. The powder microscopic investigations were carried out, and the characters were studied and documented. Phytochemical studies included physicochemical analysis, preliminary phytochemical analysis, UV–Vis spectroscopy, and high-performance thin-layer chromatography.
Conclusion: The identified parameters will be useful for authentication and quality control of the drug.

Keywords: Crateva religiosa, HPTLC, macroscopy, microscopy, physicochemical, quercetin


How to cite this article:
Mohanapriya R, Venkata Ratina Kumar T, Sunil Kumar KN. Standardization of Crateva religiosa G. Forst. leaf by pharmacognostical studies. J Res Siddha Med 2021;4:67-77

How to cite this URL:
Mohanapriya R, Venkata Ratina Kumar T, Sunil Kumar KN. Standardization of Crateva religiosa G. Forst. leaf by pharmacognostical studies. J Res Siddha Med [serial online] 2021 [cited 2023 Feb 6];4:67-77. Available from: http://www.jrsm.in/text.asp?2021/4/2/67/368438


  Introduction Top


Crateva religiosa is a moderate-sized, ornamental, deciduous, much branched tree, which is 15 m (50 ft) tall and 9 m (30 ft) wide. It is a perennial, small spreading tree with trifoliate leaves that emit pungent smell when bruised. This herb is in use since Vedic era. It grows well in sun or partial shade and normally prefers moist, rich, neutral-to-acid soil. It is often found in periodically inundated forests, usually below 100 m altitude, but also occurring up to 700 m altitude. In India and Polynesia, it is often planted around temples.[1]C. religiosa has various medicinal uses. The most common use of C. religiosa is lithotripsic. The bark is used in urinary disorders including kidney and bladder stones and also in calculous affection. It is also being used as antifungal, anti-inflammatory, antimicrobial, analgesic, anti-arthritic, anti-trypanosomal, and insecticidal, as a cure for various kinds of urinary disorders including benign prostate hyperplasia. The traditional uses of C. religiosa included for conditions such as restless leg syndrome, obesity, bone problems, urination and excretion, heart problems, convulsions, swelling and burning sensation in the soles of feet, hepatitis, proper growth, and neurologic pains. It is also used as astringent, cholagogue, anti-arthritic, antiemetic, antidote in snakebite; it improves digestion and increases appetite and biliary secretion. It is also used as a laxative, antipyretic, anthelminthic, demulcent in blood and chest diseases, bitter tonic, rubefacient, counter irritant, and vesicant.[2] The present study has been designed to prepare a complete monograph for C. religiosa leaves.


  Materials and Methods Top


C. religiosa leaves were collected from areas in and around Madurai (Alagar hills and Madurai Medical College campus) during September 2020 and was authenticated by Dr Stephen, Department of Botany, The American College, Madurai, Tamil Nadu, India.

Macro-/microscopic analysis

Macroscopic characters of fresh leaves were keenly observed under naked eyes to record the specific botanical characters. C. religiosa leaves were preserved in FAA (5% formalin—5 mL + 5% acetic acid—5 mL +70% ethyl alcohol—90 mL) for more than 48 h.[3] The preserved specimens were cut into thin transverse section using a sharp blade, and the sections were stained with safranin.[4],[5] Transverse sections were photographed using a Nikon ECLIPSE E200 trinocular microscope attached with a Zeiss Axio Cam ERc 5s digital camera under bright field light. Magnifications were indicated by a scale bar.

The quantitative microscopical parameters such as vein islet and vein termination number, stomatal number, stomatal index, and palisade ratio were determined on fresh leaves by using the standard procedure.[6],[7]

A pinch of the powdered sample passed through 80 pore size was mounted on a microscopic slide with a drop of 50% glycerol.[8] Photomicrographs of diagnostic characters were observed using a Nikon ECLIPSE E200 trinocular microscope attached with a Zeiss ERc 5s digital camera under bright field light and captured. Magnifications were indicated by a scale bar.

Physicochemical parameters

The powder is subjected for physicochemical evaluation parameters such as loss on drying, extractive value with different solvents such as petroleum ether, chloroform, ethyl acetate, benzene, ethanol, and water in increasing order of polarity,[9] and ash value (total, acid-insoluble, and water-soluble ash) as per the standard procedure.[10]

Extraction

Dried leaf material was defatted with petroleum ether and then the ethanolic extract of the leaves was prepared by cold maceration techniques.

Preliminary phytoconstituents screening

As per the standard procedure, preliminary phytoconstituents screening was done to detect the presence of active constituents in the ethanolic extract of C. religiosa leaves (EECRL).[11],[12]

Quantitative estimation of phytoconstituents

The quantity of some phytoconstituents present in the EECRL was estimated by using the standard methods such as the Folin–Ciocâlteu method for the total phenolic content, aluminum chloride method for the total flavonoid content,[13] and Folin–Denis method for the total tannin content.[13]

UV spectral studies for ethanolic extract and quercetin

The extract and the standard quercetin were dissolved in ethanol and transferred to a cuvette (quartz) and scanned under the UV range from 200 to 600 nm in the UV–Vis spectrophotometer.[14],[15]

High-performance thin layer chromatography (HPTLC)

The plates used for HPTLC were silica gel 60 F254 (10 × 5 cm) with 0.2 mm. About 0.1 mg/mL of the standard quercetin solution and 1 mg/mL of the EECRL solution were made. The plate was developed using toluene: ethyl acetate: formic acid: methanol (5.5:3:1:0.5) as mobile phase in CAMAG HPTLC with winCATS 1.4.3 software. Then 2 µL of the sample and standard were applied in the form of band (8 mm) using LINOMAT 5—a sample applicator in the pre-coated plate (Track 1: Extract, Track 2: Standard quercetin). Then the chromatograph was developed for 15 minutes, dried at room temperature, and scanned at 254 and 520 nm in Densitometry TLC Scanner 3.[16]


  Results Top


Macroscopical studies

The mature leaf of C. religiosa is dark green in color, whereas the tender leaf is light green in color, with characteristic odor and bitter taste. The leaf is ovate, lanceolate, or broadly elliptic in shape with 10–20 cm long and 3.5–6 cm wide, glabrous on the dorsal side and coriaceous in the ventral side. The leaves were arranged in a trifoliate manner with acute or acuminate apex, subacute base, and entire margin. There were about 9–12 pairs of lateral nerves in a leaf with densely reticulate venation [Figure 1].
Figure 1: Macroscopy of C. religiosa G. Forst. leaves

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Microscopical studies

TS of rachis

Rachis has an urn-shaped outline with lateral wings [Figure 2](a). Epidermis is single-layered which is covered by a thick-walled cuticle followed by the cortex; collenchyma cells are present in the lateral wing region. Cortex consists of two layers: the first layer is composed of 1–2 layers of collenchyma cells which is followed by 5–12 layers of parenchyma cells [Figure 2](b). Abundant starch grains are distributed in parenchyma cells [Figure 2](e). A “U”-shaped ring of collateral vascular bundle is seen at the center [Figure 2](c). Phloem is present toward adaxial side which is formed by the thin-walled group of cells. Xylem is present toward the abaxial side which consists of xylem vessels, xylem parenchyma, and fibers. Pericycle covers the vascular bundle which is observed as discontinuous patches of thick-walled fibers with some calcium oxalate crystals. Vascular bundle also consists of prismatic crystals and numerous starch grains [Figure 2](e). Two to three seriate ray strands separate the vascular bundles from each other.
Figure 2: TS of C. religiosa-rachis. (a) Diagrammatic view of TS of rachis. (b) Upper region enlarged. (c) Lower region enlarged. (d) Xylem vessels. (e) Prismatic crystal and starch grains. Ct: cortex, E: epidermis, Per: prismatic crystal, Per: pericycle, Ph: phloem, SG: starch gains, Vb: vascular bundle, Ve: vessel

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TS of petiole

TS of petiole is circular–oval in outline, which is semicircular with thick short lateral adaxial wings and median wide short hump [Figure 3](a). Petiole is made up of prominent epidermal layer, parenchymatous ground tissue, and vascular bundle of many segments in deep arc. Epidermis is single-layered formed of thick-walled cells coated with cuticle which is wavy in nature [Figure 3](b); cells are radially oblong. Cortex consists of two-layered cells which is made up of four to five layers of collenchyma cells followed by three to four layers of collenchyma and parenchyma cells [Figure 3](b). Parenchyma is homogeneous. About 9–13 radial oblong elliptical collateral vascular bundles are present [Figure 3](c). Each bundle has two or three radial rows of vessels. Vessels are circular to elliptical and are 20 µm wide with thick vessel walls and wide lumen [Figure 3](e). In the vascular bundle, xylem is facing toward the center, which has distinct metaxylem and protoxylem. Phloem facing toward the outside consists of few thin-walled cells. About two to three seriate medullary rays are passing between the bundles. Pith is made up of parenchyma cells [Figure 3](d).
Figure 3: TS of C. religiosa-petiole. (a) Diagrammatic view of TS of petiole. (b) Upper region enlarged. (c) Middle portion enlarged. (d) Lower region enlarged. (e) Vascular bundle enlarged. Col: collenchyma, Ct: cortex, Cu: cuticle, E: epidermis, MR: medullary ray, Pa: parenchyma, Ph: phloem, Pi: pith, Vb: vascular bundle, Ve: vessel

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TS of lamina through midrib

Midrib

Adaxial side of the midrib is broadly conical, whereas abaxial side of the midrib is broadly semicircular [Figure 4](a). The layer of the upper epidermis above the midrib is somewhat triangular in outline and it flattens toward lamina region. Upper epidermal cells are wide, whereas lower epidermal cells are narrow and elongated. Both layers are covered by striated cuticle. The cortex in the midrib region consists of two to four layers of collenchyma cells, which are followed by four to seven layers of parenchyma cells [Figure 4](b). Midrib has a wide arc of several discrete segments of the collateral vascular bundle. The vessels are wide circular or ovate with thick-walled [Figure 4](c). Starch grains are present [Figure 4](d). About two to three seriate medullary rays are seen between each vascular bundle [Figure 4](c). The vascular bundle consists of xylem and phloem. Protoxylem is directed toward the adaxial side, whereas the phloem is directed toward the abaxial side. Metaxylem vessels are 30 µm wide. Phloem cells are large semicircular which are angular and thick-walled [Figure 4](e).
Figure 4: TS of C. religiosa-leaflet (lamina through midrib). (a) Diagrammatic view of TS of lamina through midrib. (b) Upper region of midrib. (c) Middle region of midrib. (d) Lower region of midrib. (e) Phloem region of midrib. (f) TS of lamina. (g) TS of leaf margin. (h) Upper region of lamina. (i) Lower region of lamina. Col: collenchyma, Cu: cuticle, LE: lower epidermis, Me: mesophyll cells, Pa: parenchyma, Pal: palisade, Ph: phloem, SG: starch gain, SP: spongy parenchyma, UE: upper epidermis, Ve: vessel, XvP: xylem parenchyma

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Lamina

Lamina is smooth and even on both surface and dorsiventral. Adaxial epidermis is fairly thick, rectangular, and has squarish cells [Figure 4(f)]. Lamina shows upper and lower epidermis and mesophyll cells. Upper epidermis is devoid of stomata, whereas lower epidermis contains a number of anomocytic stomata [Figure 4(g)]. Both the epidermides are covered by striated cuticle [[Figure 4(h)] and (i)]. Lamina consists of three to four layers of palisade cells and four to five layers of loosely arranged spongy parenchyma with intercellular spaces [[Figure 4(h)].

Quantitative microscopy

Quantitative parameters such as vein islet number, vein termination number, stomatal number, stomatal index, and palisade ratio were determined [Table 1].
Table 1: Quantitative microscopy of C. religiosa G. Forst. leaf

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Powder microscopy

Powder microscopy showed the presence of epidermal cells covered with striated cuticle, epidermal cells showing anomocytic stomata, palisade tissues, spongy parenchyma cells, spiral xylem vessels, starch grains embedded in the parenchyma cells, starch grains, and prismatic crystals [Figure 5].
Figure 5: Powder microscopy of C.religiosa G. Forst. leaves

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Physicochemical studies

Physicochemical parameters such as LOD, extractive value for various solvents, and ash values were expressed in % w/w [Table 2].
Table 2: Physicochemical constants of C. religiosa G. Forst. leaf

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Preliminary phytochemical screening of ethanolic extract

Preliminary phytochemical analysis revealed the presence of alkaloids, carbohydrates, sterols, protein, amino acids, terpenoids, tannins, and coumarin.

Quantitative estimation of phytoconstituents

The total phenol, flavonoid, and tannin content were found to be 64.6, 85.65, and 45.2 mg/g, respectively [Table 3][Table 4][Table 5] and [Figure 6][Figure 7][Figure 8].
Table 3: Total phenolic content in ethanolic extract of C. religiosa leaves

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Table 4: Total flavonoid content in ethanolic extract of C. religiosa leaves

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Table 5: Total tannin content in ethanolic extract of C. religiosa leaves

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Figure 6: Calibration curve of standard gallic acid

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Figure 7: Calibration curve of standard quercetin

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Figure 8: Calibration curve of standard tannic acid

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UV spectral studies

The quercetin shows the absorbance maxima 0.7500 and 1.608 (λmax) at 256 and 250 nm in standard quercetin and ethanolic extract, respectively, which show the presence of quercetin in the extract. UV spectra and the corresponding wavelength are shown in [Table 6].
Table 6: UV spectra and peaks observed for standard quercetin and ethanolic extract of C. religiosa leaves

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Screening of quercetin by the HPTLC method in ethanolic extract

HPTLC analysis was carried out for the ethanolic extract of C. religiosa leaves. Then the Rf value and the peak area of the standard quercetin and extract were noted at 254 and 520 nm. At 254 nm, the sample shows 10 peaks and quercetin shows a corresponding single peak nearing the Rf 0.45–0.47, whereas in 520 nm, the sample shows nine peaks and quercetin shows a corresponding peak at the same Rf. The results are shown in [Table 7] and [Table 8], and the comparative value is shown in [Table 9]. The amount of quercetin present in the sample was calculated by using the area of the standard peak and the corresponding sample peak with respect to the concentration of the standard and the sample. From this calculation, the amount of quercetin present in the ethanolic extract was found to be 2.79–3.1 mg/g.
Table 7: HPTLC studies for quercetin and ethanolic extract of C. religiosa leaves at 254 nm

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Table 8: HPTLC studies for quercetin and ethanolic extract of C. religiosa leaves at 520 nm

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Table 9: Rf value and area for standard quercetin and ethanolic extract of C. religiosa leaves at different wavelengths

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  Discussion Top


Macro-/microscopic studies along with the physicochemical analysis and HPTLC were done for quality control of the drug. Pharmacognostical studies revealed diagnostic microscopic features of C. religiosa leaves. The general anatomical characters of the Capparidaceae family are as follows: lower epidermis is papillose, stomata are ranunculaceous in the mature leaf, generally present on both surface in isobilateral leaves but usually confined to the lower surface in dorsiventral leaves. This study revealed that C. religiosa leaves are dorsiventral and have ranunculaceous stomata presented on the lower epidermis alone.[17] The quantitative constants were determined and are considered as significant, especially for the evaluation of this plant. The findings of the present study correlated with previous reports.[18] Characterization of physiochemical properties attained strong interests in the pharmaceutical research area and is now a standard method. Physicochemical parameters such as loss on drying were used to find the deterioration time of the drug, and the total ash value represents inorganic residue; acid-insoluble ash represents the siliceous substance, whereas water-soluble ash represents the inorganic contents. Extractive values are being used to choose the effective solvent to extract the phytoconstituents. Preliminary phytochemical screening is a valuable step in the detection of the bioactive principles present in the medicinal plants which leads to drug discovery and development. As medicinal plants have been used in the treatment of different ailments, the therapeutic activity of the plant is related to the secondary metabolites present in the plant. The preliminary screening of the extract indicates the presence of various secondary metabolites, and the quantitative estimation of the existing secondary metabolites leads a way for the detection of the active principle. The quantity of that active principle present in the medicinal plant plays a very important role with regard to their potential pharmacological effect. Spectral data obtained by UV estimation show absorbance of the quercetin in the standard and sample. Thus, by generating this kind of spectral data for many herbal drugs in an appropriate manner using a UV–Vis spectrophotometer, it will be useful in the quality control of the herbal drug. HPTLC is one of the many sophisticated, flexible, robust, and cost-effective separation techniques used in the discovery, development, and analysis of new drugs. In the present study, the presence of quercetin in the extract was determined which will be useful for further pharmacological studies on the C. religiosa leaves as quercetin possesses many activities like inhibiting angiogenesis-mediated human prostate tumor growth,[19] angiogenesis-mediated human retinoblastoma growth by targeting vascular endothelial growth factor receptor.[20] It has beneficial effect of lowering blood pressure and inflammation[21]; it also possesses antioxidant and anti-inflammatory properties[22] and acts as a chemo-preventive agent for prostate cancer.[23]


  Conclusion Top


Macro-/microscopic observations, physicochemical analysis, preliminary phytochemical screening, estimation, UV, and HPTLC fingerprinting could be utilized as reference limits for the quality control standards to study the leaves of C. religiosa. The data obtained from the phytochemical study can be used to study the therapeutic efficacy of the plant leaf on the pharmacological activity.

Acknowledgements

The pharmacognostical study was carried out at the SCRI-Department of Pharmacognosy, Chennai, and the phytochemical studies were carried out in Madurai Medical College. We wish to express our sincere thanks to friends and professors who encouraged and helped us to do this work.

Authors’ contribution

MPR carried out all studies with the guidance of TVRK. KNSK revised the manuscript to publishable format.

Financial support and sponsorship

Nil.

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]
 
 
    Tables

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



 

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