Pharmacognostic and phytochemical investigation of the leaves of Malvastrum coromandelianum (L.) Garcke.

BACKGROUND: Malvastrum coromandelianum belongs to the family Malvaceae, commonly known as false mallow. Ethnobotanical survey revealed that it is used to treat various disorders. Pharmacological screening revealed that the plant possess antinoceceptive, anti-inflammatory, analgesic, and antibacterial activities. Lack of standardization parameters for herbal raw material is a great hindrance in ensuring the purity of M. coromandelianum. The present work was taken up to with a focus to set standardization parameters for M. coromandelianum.
MATERIALS AND METHODS: The plant was subjected to macroscopic and microscopic studies. Physicochemical parameters such as ash value and extractive value were determined by standard procedures. Different extracts were screened for the presence of secondary metabolites. Phenolic and flavonoid contents were estimated. Plant was subjected for high performance thin layer chromatography (HPTLC) analysis using standard chromatographic procedure. RESULT: The microscopic characteristics showed the dorsiventral nature of leaf. Two types of trichomes were observed: Covering, unicellular, uniseriate, and bi-cellular head sessile glandular. Vascular bundle was surrounded by spongy parenchyma. Phytochemical screening revealed the presence alkaloids, tannins, amino acid proteins, and carbohydrates. The phenolic and flavonoid content estimation revealed the presence of appreciable amount of these constituents, while HPTLC analysis showed the presence of β-sitosterol in petroleum ether extract.
CONCLUSION: These findings will be useful for the establishment of standardization parameters for M. coromandelianum.

INTRODUCTION

Malvastrum coromandelianum (L.) Garcke (family Malvaceae), commonly known as false mallow, broom weed, and clock plant. Various parts of this plant are used by numerous tribal populations throughout the world. Mexican Kickapoo Indians use the crushed leaves of this herb along with salt or alcohol to cure ringworm infection.[1] Bhil tribes of Rajasthan use this plant in the form of decoction to cure jaundice.[2] In Mexico leaf infusion of this plant is used to cure diabetes.[3] In traditional Indian system of medicine the plant is reported as an anti-inflammatory, analgesic, and antidysenteric.[456] Pharmacological screening showed various activities for this plant like antinociceptive,[7] anti-inflammatory, and analgesic activity,[8] and antimicrobial activity.[910] Since no data on its pharmacognostical and phytochemical aspects have been reported so far, the present study was undertaken to establish the standardization parameters for M. coromandelianum.

MATERIALS AND METHODS

Plant was collected from Tumkur District, Karnataka, India. It was washed thoroughly under running tap water to remove dirt and adhering matter. Plant was authenticated by Dr. Gopalkrishna Bhat, Retd. Professor, Department of Botany, Poorna Prajna College, Udupi, Karnataka. A voucher specimen PP598 was deposited in the Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal, India. Fresh plant material was used for the microscopical study, while some part of the leaves was dried and subjected for 60#[11] powdering for the determination of ash values and extractive values.

Pharmacognostical studies

For detailed microscopical observations, freehand thin transverse sections passing through the midrib were taken. Sections were cleaned with chloral hydrate and observed as such for the presence of any crystals, followed by staining with phloroglucinol and hydrochloric acid. Slides were observed under Olympus System Microscope, Model BX41TF, Olympus Corporation, Japan fitted with Olympus DP20 camera. Images were captured with the help of Cell A software, Olympus Corporation, Japan. Powdered leaf was also standardized for the determination of total ash, water soluble ash, acid insoluble ash, and extractive values.[12] Fluorescence analysis of leaf powder was performed as per an earlier reported procedure.[13]

Phytochemical screening

For phytochemical screening, 250 g of powdered leaf was extracted successively with analytical grade petroleum ether (60-80°), chloroform, acetone, and methanol using Soxhlet extractor. After each solvent extraction, plant material was dried at 45°C. Mark remaining after methanol extraction was macerated with water for 24 h. All the extracts were concentrated under reduced pressure in Buchi R-210 Rotavapor. All these extracts were screened for the presence of different secondary metabolites by following the standard procedures.[14]

Determination of total phenolic content

For total phenolic content estimation in different leaf extracts, calibration curve was prepared by mixing methanolic solution of gallic acid as a standard (1 ml; 10-100 μg/ml) with ten times diluted 5 ml Folin-Ciocalteu reagent and 4 ml sodium carbonate (0.7 M). After 2 h, absorbance was measured at 765 nm with a Shimadzu UV-1650PC Spectrophotometer, Japan. Same experiment was performed with different plant extracts (100 μg/ml). All determinations were carried out thrice. Total phenolic content in the extract in mg/g gallic acid equivalents (GAE) was calculated by the following formula:[15]

T = C.V/M

where T = total phenolic content (mg/g) of plant extract, in GAE; C = concentration of gallic acid established from the calibration curve (mg/ml); V = volume of extract (ml) M = weight of plant extract (g).

Determination of flavonoid content

The aluminum chloride colorimetric method was used to determine the total flavonoid content. Different concentrations (10-100 μg/ml) of quercetin as standard were used to plot calibration curve. Reaction mixture consisted of 0.5 ml of standard, 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm with a Shimadzu UV-1650PC spectrophotometer. Blank was emptied of 10% aluminum chloride and was substituted by the same amount of distilled water. Same procedure was repeated by replacing 0.5 ml of standard with 0.5 ml extract (100 μg/ml). Content of total flavonoid was calculated using the same formula used for the calculation of total phenolic content. Results were expressed as mg/g quercetin equivalent.[16]

High performance thin layer chromatography (HPTLC) analysis of petroleum ether extract

Presence of β-sitosterol in the petroleum ether extract was confirmed by HPTLC using β-sitosterol (fluka analytical 40% pure) as the standard marker (100 μg/ml). Petroleum ether extract was dissolved in methanol to prepare a concentration of 1000 μg/ml. Camag HPTLC system equipped with Linomat 5 sample applicator and Camag thin layer chromatography Scanner 3 was used. HPTLC plates coated with silica gel 60 F254 was used as stationary phase, while mobile phase consisted of benzene and ethyl acetate in the ratio of 9.5:0.5. After development, the plate was sprayed with 10% sulfuric acid in methanol and heated at 105°C for 10 min. and later scanned at 366 nm. Data was analyzed using winCATS software version 1.2.6 (Camag HPTLC System, Switzerland).

RESULTS AND DISCUSSION

Macroscopic characters of leaf

M. coromandelianum is a strong-stemmed, woody-rooted herb, grows up to 1 m in height. Leaves are ovate or ovate-elliptic, 4.5 cm long, 3.5 cm wide, with sharp or blunt apex, prominent midrib, margins serrated, three-nerved from base. Leaf stalks are 1.5-4 cm long [Figure 1a and b].

Figure 1

(a, b) Malvastrum coromandelianum leaf morphology

Microscopical characters

The histology of the leaf can be best studied as lamina and midrib region [Figure 2a]. The transverse section of leaf lamina is dorsiventral with single layered lower and upper epidermis, compactly arranged and cuticulized [Figure 2b]. The epidermis showed two types of modifications, i.e., trichomes and stomata. The two types of trichomes that are unicellular, uniseriate, lignified covering trichomes which are more on lower epidermis than upper one; [Figure 2c] while bi-cellular head, sessile, nonlignified glandular trichomes are found on both epidermis [Figure 2a, Figure 2d]. The three celled unequal anisocytic type stomata are well distributed in lamina region [Figure 2e]. The spongy parenchyma of mesophyll shows the absence of ergastic cell content.

Figure 2

(a) Transverse section of leaf passing through midrib. (b) Transverse section of leaf lamina. (c) Transverse section of midrib showing collenchyma covering trichome. (d) Surface view of leaf for glandular trichome. (e) Surface view of leaf for stomata. (f) Transverse section of leaf passing through midrib Ct: Cuticle, T1: Covering trichome, T2: Bi-cellular sessile glandular trichome, St: Stomata, Xy: Xylem, Ph: Phloem, Sp: Spongy parenchyma, Co: Collenchyma

The midrib region shows a similar type of epidermis which is devoid of stomata. Dorsal surface of midrib shows concave shape. Below and above the upper and lower epidermis the thick cellulosic cell walled compactly arranged two to three layered collenchyma is present; which is responsible for giving mechanical support and expansion of the lamina. Vascular bundle is present at the center of the midrib responsible for conduction of food and nutrient; surrounded by spongy parenchyma. The vascular bundle is arc shaped, bi-collateral type; the phloem surrounds the xylem. The phloem shows the presence of sieve tubes and companion tubes; phloem fibers are absent. The xylem shows well developed spiral xylem vessels responsible for conduction of water [Figure 2f].

Powder microscopy

Powder microscopy of the leaf showed the presence of unicellular, lignified covering trichome with smooth cuticle [Figure 3a and b], spiral xylem vessels slightly lignified [Figure 3c] and anisocytic stomata [Figure 3d].

Figure 3

(a-d) Powder microscopy of leaf. T: Unicellular, lignified, covering trichome, XyV: Xylem vessel, St: Anisocytic stomata

Physical constants

Ash value determination is a very important tool to access the quality of herbal raw material since higher ash value is an indication of adulteration and or improper processing of raw material. The percentage variation of the weight of ash in certain drugs from sample to sample is very small and any marked difference indicates a change in quality. Percentages of total ash, acid-insoluble ash and water-soluble ash are listed in Table 1. Extractive value determination is also very important and using this, the quality of the raw material can be judged. This is because already exhausted raw material will result in lower extractive values.[17] Results of water soluble and alcohol soluble extractive values are shown in Table 1.

Table 1

Ash values and extractive values of Malvastrum coromandelianum

Dried leaf powder of M. coromandelianum was extracted successively by Soxhlet apparatus with solvents of increasing polarity like petroleum ether, chloroform, acetone and methanol followed by maceration with water. The percentage yield of the extract was found to be more in aqueous and methanolic extract and minimum in acetone extract [Table 2]. Results of chemical tests indicate the presence of various secondary metabolites like alkaloids, fixed oils, saponins, phenolic, tannins, carbohydrates, and proteins. The results are shown in Table 3 and the results of fluorescence analysis are shown in Table 4.

Table 2

Percentage yield of different extracts by successive solvent extraction of Malvastrum coromandelianum leaf

Table 3

Qualitative chemical analysis of Malvastrum coromandelianum

Table 4

Fluorescence analysis of leaf powder of Malvastrum coromandelianum

Total phenolic and flavonoid content estimation

Plant phenolics are well-known for their antioxidant properties.[1518] In the present study, methanolic extract contains the maximum amount of phenolic compounds [Table 5]. Flavonoids are well known for their wide spectrum of biological activities and hence estimation of total flavonoid content can be a quality control parameter for the herbal raw material. In the present study, chloroform extract contained the maximum flavonoid content followed by methanolic extract [Table 6].

Table 5

Total phenolic content in different extracts of Malvastrum coromandelianum

Table 6

Total flavonoid content in different extracts of Malvastrum coromandelianum

High performance thin layer chromatography analysis

HPTLC is a flexible tool for identification and quantification of secondary plant metabolites. When properly employed, HPTLC provides a visual display of compounds present in the test materials. Identification and quantification of β-sitosterol in the petroleum ether extract was confirmed by the HPTLC analysis of developed chromatogram at 366 nm. β-sitosterol in sample [Figure 4] (Rf = 0.26, area under curve (AUC) = 459.8) was identified by comparing the Rf value of standard [Figure 5](0.26, AUC = 1068.1). It was further confirmed by overlay spectral analysis [Figure 6]. Image of developed plate after derivatization with 10% sulfuric acid in methanol under 366 nm is shown in Figure 7. The percentage yield of β-sitosterol was found to be 1.7% w/w. β-sitosterol is one of the phytosterols, which is reported for its analgesic, anthelminthic and antimutagenic,[19] anti-inflammatory[20] and antihepatotoxic activities.[21]

Figure 4

High performance thin layer chromatography chromatogram of petroleum ether extract of leaf

Figure 5

High performance thin layer chromatography chromatogram of standard β-sitosterol

Figure 6

Overlay spectra of β-sitosterol in standard and petroleum ether extract

Figure 7

Image of thin layer chromatography plate at 366 nm after derivatization

CONCLUSION

Evaluation of crude drug involves the determination of identity, purity and quality. Purity is the absence of extraneous matter, while the amount of active constituents present in the crude drug is referred to as quality. Macorscopic and microscopic evaluation is an important parameter in accessing the identity of herbal raw material. At the same time, qualitative and quantitative screening of secondary metabolites focuses on quality of raw material. Moreover, therapeutic potential of plant is solely dependent on the nature and amount of phytoconstituents in them. Hence, accessing the quality of herbal raw material in terms of their chemical composition becomes very imperative. To conclude, various macroscopic, microscopic, physical and phytochemical aspects/parameters listed here for M. coromandelianum in the present work can be used with respect to its identification, authentication and standardization