Pharmacognostical evaluation of aerial parts of Graptophyllum pictum (L.) Griff. (Syn: Justicia picta Linn.): A well-known folklore medicinal plant.

OBJECTIVE: Graptophyllum pictum (L.) Griff. (Family-Acanthaceae) occupies a key role in traditional system of medicine. Since an extensive literature survey did not provide any information about studies on its standardization. Therefore, we designed the current study to establish the quality control parameters of G. pictum aerial parts.
MATERIALS AND METHODS: The investigation included determination of various standardization parameters such as macroscopic and microscopic studies, physicochemical parameters as well as phytochemical analysis of the crude drug.
RESULTS: The microscopy study of aerial parts revealed that stem shows typical dicotyledonous characters with prismatic crystals of calcium oxalate in the cortical region and dorsiventral leaf. Physicochemical constants such as moisture content, ash values, fluorescence analysis, and extractive values were established. Preliminary phytochemical analysis confirmed the presence of alkaloids, flavonoids, saponins, tannins, etc.
CONCLUSION: The present study suggests establishing the parameters for pharmacopoeial standardization of G. pictum.

INTRODUCTION

In spite of great advances observed in modern medicine in recent decades, plants still are important and conducive to health care system. It is estimated that about 25% of all modern medicines are directly or indirectly derived from higher plants.[1] In Ayurveda, the drugs of plant, mineral and animals origins are exploited due to their safety and efficacy in the treatment of diseases either as a single drug or as polyherbal formulations.[2] Standardization is a prime requirement to maintain the quality and safety of polyherbal formulations to attain the desired therapeutic effect.[3] Standardization assures safety, efficacy, quality, and acceptability of the polyherbal formulations by reducing batch to batch variation.[4] These standards are based on pharmacognostical, physicochemical, phytochemical, and other biological parameters.

Graptophyllum pictum (L.) Griff. (Family-Acanthaceae) commonly known as Joseph's coat in English, is a tropical evergreen shrub which has oval to elliptic leaves (up to 6 inches long) that are deep green, varyingly blotched with cream along the midveins [Figure 1].[5] The Caricature plant is native of New Guinea, neighboring Islands, and widely distributed in India, Mexico, United States, Ghana, and Bolivia.[67] Traditionally, G. pictum was used as folklore medicine in enhancing fertility, poultice on cuts, wounds, and all kinds of swellings and for the treatment of ulcers, abscesses, hemorrhoids,[8] constipation, rheumatism, urinary infections, scabies, hepatomegaly, and ear diseases.[9] It also has anti-inflammatory,[10] anti-plaque,[11] and antidiabetic activities.[12] However, this medicinal plant has not been studied pharmacognosticaly. The current study reports the pharmacognostical and phytochemical investigations of aerial parts of G. pictum (L.).

Figure 1

(a) Graptophyllum pictum (L.) Griff. Plant. (b) TS of stem of G. pictum at 10X showed single layered epidermal cells (Ep) covered with cuticle (CT), hypodermis (Hp), collenchyma (CCh), endodermis (Ed), pericycle (PC), parenchymatous cortex (Co), vascular bundle (VB), medullary rays (MR), and pith (PT). (c) TS of stem of G. pictum at 10X showed single layered epidermal cells (Ep) with trichome (Tr) and cystolith (Cyst), hypodermis (Hp), collenchyma (CCh), parenchymatous cortex (Co), xylem (Xy) and phloem (Ph), medullary rays (MR), and pith (PT). (d) TS of stem of G. pictum at 10X showed conjoint, collateral, and open vascular bundles (VB) consists of endarch Xylem (Xy) and Phloem (Ph), cortex (Co), endodermis (Ed), pericycle (PC), medullary rays (MR), and parenchymatous pith (PT). (e) TS of stem of G. pictum viewed at 40X showed vascular bundles (VB) consists of Xylem (Xy) and Phloem (Ph) along with sclerenchymatous cells (Sc). (f) TS of stem of G. pictum viewed at 40X showed epidermal cells (Ep), cuticle (Cu), hypodermis (Hp), unicellular covering trichomes (Tr), and parenchymatous cortex (CO)

MATERIALS AND METHODS

Plant collection and authentication

The fresh plant was collected from Krishnendra Nursery, near Lalbagh, Bengaluru, Karnataka, India. The plant material was taxonomically identified by Dr. D.C. Saini, Senior Scientist, Palaeobotany, Birbal Sahni Institute of Palaeobotany (BSIP), Lucknow, Uttar Pradesh, India. The voucher specimen (BSIP02) was preserved in BSIP herbarium and the crude drug sample was deposited in the Department of Pharmacognosy, Teerthanker Mahaveer College of Pharmacy, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India for future reference.

Pharmacognostical studies

Macroscopic evaluation

Macroscopic study included the determination of various organoleptic characters viz., color, odor, taste, texture, fracture, and shape of aerial parts of G. pictum.[131415]

Microscopic evaluation

Freehand sections of stem and leaves were taken and stained using safranin. The sections were observed under a compound microscope. Powder microscopy was also performed and the diagnostic characters were noted.[131415]

Determination of physicochemical parameters

Foreign organic matter

The drug sample to be examined was weighed and spread on a white tile uniformly without overlapping. The foreign matter was separated manually and examined in daylight with unaided eye. The suspected particles were transferred into a Petri dish. After complete separation, the weight of the foreign matter was taken, and the percentage (%) (w/w) was determined.[1314]

Moisture content

About 10 g of drug (without preliminary drying) after accurately weighing was placed in a tared evaporating dish and was dried at 105°C. The drying and weighing were continued at 1 h intervals until the difference between two successive weighings was not more than 0.25%. A constant weight was supposed to have reached when two consecutive weighing after drying for 30 min and cooling for 30 min in a desiccator, showed not more than 0.01 g difference.[131415161718]

Swelling index

Specified quantity of plant material of required fineness was introduced into a 25 ml glass-stoppered measuring cylinder. 25 ml of water was added, and the mixture was shaken thoroughly every 10 min for 1 h. The crude drug was allowed to stand for 3 h at room temperature. The volume occupied by the plant material was measured in milliliter including any sticky mucilage. The mean value of the individual determination was finally calculated relative to 1 g of plant material.[1319]

Foaming index

About 1 g of the plant material was reduced to a coarse powder, weighed accurately, and transferred to a 500 ml conical flask containing 100 ml of boiling water. It was maintained at moderate boiling for 30 min, cooled, and filtered into a 100 ml volumetric flask. Sufficient water was added through the filter to dilute the filtrate to make up the volume. The decoction was poured into 10 stoppered test tubes in successive portions, that is, 1 ml, 2 ml, 3 ml, etc., and the volume of liquid in each test tube was adjusted with water to 10 ml. The tubes were stoppered and shaken in a lengthwise motion for 15 s at the rate of two shakes per second and then allowed to stand for 15 min. The height of the foam was measured, and the result was calculated.[19]

Ash values

Total ash

About 2.0 g of the crude drug was accurately weighed and incinerated in a silica crucible at a temperature not exceeding 450°C until free from carbon. The resulting ash was then cooled and weighed. The procedure was repeated to obtain a constant weight. The percentage of total ash with reference to the air-dried drug was finally calculated.[131415161718]

Acid insoluble ash

To the crucible containing total ash, 25 ml of dilute hydrochloric acid was added. The insoluble matter was collected on an ashless filter paper. It was then washed with hot water until it became neutral and ignited to a constant weight. The residue was allowed to cool in a suitable desiccator for 30 min, and it was immediately weighed. The procedure was repeated to obtain a constant weight. The percentage of acid-insoluble ash with reference to the air-dried drug was finally calculated.[131415161718]

Water soluble ash

To the crucible containing total ash, 25 ml of water was added and boiled for 5 min. The insoluble matter was collected on an ashless filter paper. It was then washed with hot water and ignited for 15 min at a temperature not exceeding 450°C. The procedure was repeated to obtain a constant weight. The difference in the weight of ash and weight of insoluble matter was calculated. The percentage of water-soluble ash with reference to the air-dried drug was finally determined.[131415161718]

Sulfated ash

Silica crucible was heated to redness for 10 min, and it was allowed to cool in a desiccator and weighed. 1-2 g of the crude drug was accurately weighed and was put into the crucible. It was ignited gently at first so that it could thoroughly charred, then cooled and moistened with 1 ml of sulfuric acid. The sample was heated gently at first until white fumes ceased off and later on ignited at a temperature of 800 ± 25°C until all black disappeared. The crucible was then allowed to cool and again few drops of sulfuric acid were added and heated again. The steps were repeated until two successive weighings did not have a difference of weight more than 0.5 mg.[131415161718]

Cold extractive values

Extractive values for the plant drug sample were determined using different solvents, that is, petroleum ether, chloroform, ethyl acetate, ethanol, and water.[131415161718]

Alcohol soluble extractive

About 5 g of air-dried drug was first coarsely powdered and macerated with 100 ml of alcohol of specified strength in a closed flask for 24 h. The flask was shaken frequently for 6 h and allowed to stand for 18 h. The extract was filtered rapidly and was evaporated up to 25 ml rapidly to dryness in a china dish. Further, it was dried to obtain a constant weight. The percentage yield of alcohol-soluble extractive with reference to air-dried drug was finally calculated. Similar procedures were followed with petroleum ether, chloroform, ethyl acetate, ethanol, and water to find out their respective extractive values.

Hot extractive values

The air-dried aerial parts of G. pictum were made into a coarse powder, which was further extracted with Soxhlet apparatus using petroleum ether (60–80°C) and ethanol. The extracts were concentrated under reduced pressure and evaporated to dryness on a water bath. The extracts were stored in a desiccator for phytochemical analysis.

Fluorescence analysis

A small quantity of dried and finely powdered crude drug was placed on a grease-free clean microscopic slide and the same was treated with 1–2 drops of the freshly prepared reagent solutions separately, that is, 1 N sodium hydroxide in methanol, 1 N sodium hydroxide in water, 50% sulfuric acid, 50% nitric acid, and 1N HCl. The added reagents were mixed by gentle tilting the slides and waited for 1–2 min. Then each slide was placed inside the UV chamber and viewed in natural and ultraviolet lights. All extracts obtained were subjected to fluorescence analysis. The colors observed by application of different reagents were recorded.[2021]

Preliminary phytochemical investigation

Determination of organic phytoconstituents

Petroleum ether extract and ethanolic extracts were subjected to preliminary phytochemical analysis to identify the various organic phytoconstituents such as alkaloids, glycosides, saponins, flavonoids, tannins, and steroids.[131422]

Determination of inorganic constituents

Ash of drug material was treated with 50% v/v HCl, kept for 1 h, and filtered. The filtrate was used for qualitative determination of various inorganic constituents such as calcium, magnesium, potassium, iron, etc.[1314]

RESULTS

Macroscopic evaluation

Macroscopic characters of aerial parts of G. pictum are as shown in Table 1.

Table 1

Organoleptic evaluation of Graptophyllum pictum aerial parts

Microscopic evaluation

Transverse section of stem

Transverse section of the stem, as shown in Figure 1a–f, reveals the presence of single layered rectangular cells with thick and smooth cuticle. Epidermis possesses unicellular covering trichomes and cystolith. Below the epidermis, the hypodermis is represented by few layers of compactly arranged collenchyma cells without any intercellular spaces. The cortex is composed of many layers of compactly arranged parenchyma cells without intercellular spaces. Endodermis is the innermost layer of the cortex and conspicuous outside the patch of sclerenchyma. Pericyclic region occurring as a ring is made up of 3–4 layers of compactly arranged, lignified sclerenchymatous cells. Vascular bundles located in a group, are conjoint, collateral, and open. Xylem is described as endarch with radially arranged medullary rays present in between vascular bundles. Pith comprises large, thin-walled, lignified parenchyma cells with intercellular spaces.

Powder microscopy of stem

Diagnostic characters of powder microscopy of stem shows the presence of prismatic calcium oxalate crystals; starch grains rounded, minute, simple, and compound (2–4); thick walled sclerenchymatous cells; elongated phloem fiber, and spirally arranged xylem vessels [Figure 2a–d].

Figure 2

(a) Powdered characteristics of stem of Graptophyllum pictum revealed the presence of Phloem fiber (PFi) and starch grain (SG). (b) Powdered characteristics of stem of G. pictum showed the presence of spiral Xylem vessels (Xy) and starch grain (SG). (c) Powdered characteristics of stem of G. pictum showed the presence of sclerenchyma cells (Sc) and starch grain (SG). (d) Powdered characteristics of stem of G. pictum showed the presence of Calcium oxalate crystal (CC)

Transverse section of leaf

Transverse section of leaf passing through midrib region, as shown in Figure 3a–h, reveals single layered adaxial epidermis composed of rectangular cells and covered with thin cuticle. Abaxial epidermis consists of bluntly angled cells. Glandular trichomes and cystolith are present on both the surfaces. Diacytic (caryophyllus) stomata are present on the abaxial epidermal surface but absent on adaxial epidermal surface. Mesophyll cells are differentiated between two: Radially elongated and compactly arranged palisade cells in two layers just beneath adaxial epidermis followed by multilayered, loosely arranged spongy parenchyma cells. Vascular bundles are arc-shaped, collateral, and open.

Figure 3

(a) TS of leaf of Graptophyllum pictum at 10X showed adaxial epidermis (AdE) with cystolith (Cyst), followed by double layer of palisade cells (Pal), abaxial epidermis (AbE), glandular trichome (GT), spongy parenchyma (SP), and vascular bundles (VB). (b) TS of lamina of G. pictum at 10X showed adaxial epidermis (AdE) with thin cuticle followed by double layer of palisade cells (Pal), abaxial epidermis (AbE), and spongy parenchyma (SP). (c) TS of lamina of G. pictum at 40X showed adaxial epidermis (AdE) with thin cuticle (CT) followed by double layer of palisade cells (Pal), cystolith (Cyst), and spongy parenchyma (SP). (d) TS of lamina of G. pictum at 40X showed adaxial epidermis (AdE) with thin cuticle and glandular trichome (GT) followed by double layer of palisade cells (Pal) and spongy parenchyma (SP). (e) TS of lamina of G. pictum at 40X showed abaxial epidermis (AbE) and spongy parenchyma (SP). (f) TS of midrib region of leaf of G. pictum at 40X showed arc shaped collateral and open vascular bundle. (g) Adaxial surface view of leaf of G. pictum at 40X showed glandular trichomes (GT) and upper epidermal cells (AdE). (h) Abaxial surface view of leaf of G. pictum at 40X showed diacytic (caryophyllous) stomata (St), glandular trichomes (GT), and lower epidermal cells (AbE)

Powder microscopy of leaf

Powder characteristics of G. pictum leaves as shown in Figure 4a–c reveal rectangular epidermal cells, annular xylem vessels, and rosette type calcium oxalate crystals.

Figure 4

(a) Powdered characteristics of leaves of Graptophyllum pictum revealed the presence of rectangular epidermal cells (EC). (b) Powdered characteristics of leaves of G. pictum revealed the presence of annular xylem vessel (XV). (c) Powdered characteristics of leaves of G. pictum revealed the presence of rosette-like calcium oxalate crystal (Cr)

Physicochemical evaluation

The results of physicochemical parameters are summarized in Table 2.

Table 2

Physicochemical values of Graptophyllum pictum aerial parts

Foreign organic content

The foreign organic content was found to be 1% w/w.

Moisture content of aerial parts of G. pictum was found to be 14.56% w/w.

Swelling and foaming index

The swelling index was observed as nil while the foaming index was <100.

Ash values

Ash value gives an idea about inorganic content as well as extraneous matter (e.g. soil and sand) clinging to the surface of the plant. Total ash, acid insoluble ash, water soluble ash, and sulfated ash of the aerial parts of G. pictum were recorded as 10, 1.7, 8.2, and 19.1% w/w, respectively.

Extractive values

Cold extractive values for solvents, that is, petroleum ether, chloroform, ethyl acetate, ethanol, and water were noted as 5, 7.5, 7, 10, and 14.2% w/w, respectively. Extractive values using soxhlet method for solvents such as petroleum ether and ethanol in successive order were recorded as 6.4% and 12.6% w/w, respectively.

The results of fluorescence analysis are given in Table 3.

Table 3

Fluorescence analysis of Graptophyllum pictum aerial parts

Phytochemical analysis

Preliminary phytochemical screening of petroleum ether and ethanolic extract showed the presence of alkaloids, flavonoids, tannins, glycosides, saponins, and steroids as organic phytoconstituents [Table 4a]. Qualitative analysis of various inorganic elements revealed the presence of calcium, iron, sulfate, phosphates, and chlorides [Table 4b].

Table 4a

Organic phytoconstituents of Graptophyllum pictum aerial parts

Table 4b

Inorganic elements of Graptophyllum pictum aerial parts

DISCUSSION

In the present study, various pharmacognostical and physicochemical parameters such as ash values, extractive values, and moisture content of aerial parts of G. pictum were established. These parameters are important tools for determination of the identity, quality, and purity of the drug. Macroscopic identity of any medicinal plant material is based on size, shape, color, odor, texture, fracture, etc. Transverse section of stem confirmed the presence of epidermis, hypodermis, wide cortex, vascular bundles, and large parenchymatous pith while the leaf shows the presence of epidermis, diacytic stomata, mesophyll and collateral, open vascular bundles. Moisture in conjunction with a suitable temperature leads to the activation of enzymes and provides a suitable condition to the proliferation of micro-organisms which is a major factor responsible for the deterioration of the drugs and formulations.[18] Swelling index is the property of medicinal plant containing gums, mucilage, pectin or hemicelluloses, which may be responsible for specific therapeutic or pharmaceutical utility while foaming index determines the saponin content of the drug.[14] Total ash indicates the presence of inorganic salts such as phosphates, carbonates, and silicates of sodium, potassium, magnesium, calcium, etc. Acid insoluble ash is the residue obtained after boiling the ash with hydrochloric acid and igniting the insoluble portion. It gives a measure of sand and other siliceous matter. Water soluble extractive value shows the presence of water soluble inorganic salts. Sulfated ash value gives more consistent data by treatment of ash with sulfuric acid, which converts all the oxides and carbonates to sulfates.[1418] Phytochemical standardization encompasses all possible information regarding the chemical constituents present in an herbal drug. Preliminary phytochemical analysis showed the presence of glycosides, steroids in petroleum ether extract and alkaloids, glycosides, saponins, flavonoids in ethanolic extract of aerial parts of G. pictum.

CONCLUSION

Different quality control parameters including pharmacognostical, physicochemical, and phytochemical have been established for G. pictum. These parameters have been determined to be of future use in identifying authentic G. pictum drug and setting the pharmacopoeial standards for further studies. The investigations on this plant would be continued in the thrust areas of phytochemistry and pharmacology.