Pharmacognostical evaluation of Cardiospermum halicacabum Linn. leaf and stem.

BACKGROUND: Cardiospermum halicacabum Linn (Sapindaceae) is an important medicinal plant in the traditional system of medicine, known as karṇasphoṭa. The root of it is officially included in Ayurvedic Pharmacopoeia for its therapeutic uses such as jvara, kuṣṭha, pāṇḍu, kṣaya and sandhivāta etc. As no detailed analysis of macroscopy, microscopy characters of the plant, except root, have been carried out till date, it was thought worth to carry out the detailed macroscopic and microscopic study of leaves and stem, following standard pharmacognostical procedures.
MATERIALS AND METHODS: Pharmacognostic studies of C. halicacabum were carried out, and in this, the macroscopic, microscopic, physicochemical, fluorescence and phytochemical analyses were done. Physicochemical parameters such as total ash, moisture content, extractive values were determined by World Health Organization guidelines. The microscopic features of leaf and stem components were observed.
RESULTS: Macroscopically the leaves are bi-ternate, ovate-lanceolate in shape with dentate margin. Microscopically, leaf shows prominent midrib and thin dorsiventral lamina. The midrib shows the presence of epidermal layers, angular collenchyma, palisade cells and vascular strands comprised of thin walled xylem and thick walled phloem elements. The lamina shows prominent, narrow and cylindrical upper epidermis. The upper epidermal cells are large and contain mucilage, whereas lower epidermis possesses thin, small and elliptical epidermal cells. The mesophyll was differentiated into two zones upper and lower. The upper zones show narrow cylindrical palisade cells and lower zone shows 2-3 layers of loosely arranged spongy parenchyma cells. In the Paradermal section of the lamina we observe anomocytic stomata. The transverse section of stem shows a pentagonal appearance with five short blunt ridges and prominent cuticle. Parenchymatous cells, cortical sclerenchyma, lignified xylem fibers, phloem and pit were also found. In the powder microscopy of whole plant, glandular trichomes, non-glandular trichomes, fragments of lamina, xylem elements, parenchyma cells and fibers are observed. Phytochemical screening reveals that the C. halicacabum extract contains glycosides, carbohydrates, flavonoids, phytosterols, phenolic compounds and saponin.
CONCLUSION: Various pharmacognostic characters observed in this study help in identification, quality, purity and standardization of C. halicacabum.

INTRODUCTION

Cardiospermum halicacabum Linn. (Sapindaceae), English name: Balloon Vine, is an annual or sometimes perennial climber, commonly found as a weed throughout India. The tender, young shoots are used as a vegetable, fodder, diuretic, stomachic and rubefacient. It is used in conditions like rheumatism, lumbago, nervous diseases, and as a demulcent in orchitis and in dropsy. In Sri Lanka, it is used for the treatment of skeletal fractures. The juice of the herb is used to cure ear ache and to reduce hardened tumors. It exhibits significant analgesic, anti-inflammatory activity and vasodepressant activity, which is transient in nature. In vitro studies have revealed its antispasmodic and curate-like actions. It is for this reason perhaps that the plant finds its use in Ayurvedic medicine.[1] The leaf of this plant mixed with castor oil is administered internally to treat rheumatism and to check lumbago.[2] Two glasses of a 12 h maceration of aerial parts of the plant are consumed or used for bathing in the treatment of hyperthermia in a few places and in some others, water extracts of the seed are used.[3]

Previous studies which have reported phytochemical investigations of the plant revealed the presence of flavones, aglycones, triterpenoids, glycosides, carbohydrates, fatty acids and volatile esters in the different extracts of the plant.[4567] Apart from this, pharmacological evaluation of C. halicacabum extracts showed that the plant possesses antimalarial,[8] antifilarial,[9] antiparasitic,[10] antipyretic,[11] anti-inflammatory,[12] antianxiety[13] and nephroprotective activity.[14] Furthermore, the plant showed antiulcer activity,[15] antihyperglycemic activity against streptozotocin-induced diabetes in rats and antidiarrheal potential in mice.[7]

C. halicacabum is a plant of interest in traditional medicine. In order to ensure the quality of its supply, and prevent adulteration and substitution prevailing on the crude drug markets of India, the present study deals with a detailed pharmacognostical evaluation of the leaf and stem of C. halicacabum. No extensive pharmacognostic study on leaf and stem are reported till date. Therefore, the present study includes macro- and microscopic characters, fluorescence analysis of powder, physico-chemical values, preliminary phytochemical screening of different extractives.

MATERIALS AND METHODS

Plant material

C. halicacabum was collected from Tirupati, Andhra Pradesh, India in the month of October 2009. The plant was identified and authenticated by Professor K. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India under the reference letter number SVU/SC/12/55/09-10.

Macroscopic evaluation

The macroscopic characters of fresh leaves and stem of C. halicacabum such as size and shape, color, surface, venation, apex, margin, lamina, texture, odor and taste were recorded.

Physicochemical and phytochemical analysis

Physicochemical parameters such as ash values and extractive values were determined according to the well-established procedures.[1617] Preliminary phytochemical screening was carried out using the standard procedure.[18]

Fluorescence analysis

Powdered material was treated with various chemical reagents and exposed to visible, ultraviolet light (Short UV) and long UV to study their behaviour under fluorescence.[1920] The changes in appearance and color were observed and recorded.

Microscopic evaluation

In microscopic evaluation, studies were conducted qualitatively and quantitatively.

Qualitative microscopy

Care was taken to select healthy and normal leaf and stem material. The required samples of stem and leaves were cut and removed from the plant and fixed in a solution consisting of formalin 5 ml + acetic acid 5 ml + 70% ethyl alcohol 90 ml. After 24 h of fixing, the specimens of leaves and stem were dehydrated with graded series of tertiary-Butyl alcohol as per the schedule given by Sass, 1940.[21] Infiltration of the specimens of leaves and stems were carried by gradual addition of paraffin wax (mp 58-60°C) until tertiary Butyl alcohol solution attained super saturation. The specimens of leaves and stems were cast into paraffin blocks.

The paraffin embedded specimens of leaves and stems were sectioned with the help of rotary microtome. The thickness of the sections was 10-12 μm. Dewaxing of the sections was done using the customary procedure.[22] The sections were stained with toluidine blue as per the method published by O’Brien et al.[23] Toluidine blue is a polychromatic stain. The staining results were remarkably good and some cytochemical reactions were also obtained. The dye rendered the cellulose walls pink, lignified cells blue, suberin dark green, mucilage violet and protein bodies blue etc., Wherever necessary, sections of leaf and stem were also stained with safranin and fast green and potassium iodate (for starch).

To study the stomatal morphology, venation pattern and trichome distribution, paradermal sections (sections taken parallel to the surface of the leaf) as well as clearing of leaf with 5% of sodium hydroxide or epidermal peeling by partial maceration employing Jeffery's maceration fluid[21] were prepared. Glycerine mounted temporary preparations were made for macerated/cleared leaf.

Microscopic descriptions of tissues are supplemented with micrographs wherever necessary. Photographs of different magnifications were taken with Nikon lab photo-2 microscopic unit. For the study of crystals, starch grains and lignified cells, polarized light was employed. Since these structures have birefringent property, under polarized light they appear bright against the dark background. Magnifications of the figures are indicated by the scale bars. Descriptive terms of the anatomical features are as given in the standard anatomy books.[2425]

Powder microscopy

For powder microscopy, the dried aerial part material was powdered. Powdered material was cleared with sodium hydroxide and mounted in glycerine medium after staining. Different staining reagents such as toluidine blue, safranin, fast green and iodine were used. Different cell components were studied and measured using photomicrography.

Quantitative microscopy

In quantitative microscopy determination of stomatal number, stomatal index, vein islet, vein termination number and palisade ratio were carried out.[26]

RESULTS

Macroscopy of stem and leaves

The plant is a herbaceous vine. The leaves are bi-ternate. Leaflets are ovate-lanceolate, having glabrous texture, smooth surface and dentate margins. Length of the leaf is 3-5 cm and breadth 1.5-2 cm. Stems having width 0.2-0.3 cm and green in color. Petiole is 1.5-2.5 cm. Leaf has a bitter taste and characteristic leafy odor. Flowers tetramerous irregular, 2 + 2 sepals, 2 + 2 unequal petals. Stamens-8, unequal. Ovary is tricarpellary with one ovule in each carpel. Stigma is trifid. Fruits are globose, winged bloated capsule.

Physicochemical analysis of aerial part plant powder viz. ash values, extractive values and moisture content are presented in Table 1. The preliminary phytochemical analysis of the extract of the aerial part shows the presence of glycoside, carbohydrate, flavonoid, phytosterols, phenolic compounds and saponin.

Table 1

Physicochemical evaluation C. halicacabum powder

The fluorescence analysis of aerial part plant powder material under day and UV (short 254 nm) light is recorded in Table 2.

Table 2

Fluorescence analysis of whole plant powder of C. halicacabum

Microscopy evaluation

Microscopy of leaf

The leaf has prominent, adaxially and abaxially projecting midrib and thin dorsiventral lamina [Figure 1a]. The adaxial part of the midrib is thick and pyramid like and the abaxial part is semicircular with undulate outline. The midrib is 350 μm thick. The adaxial cone is 150 μm wide at the base and the abaxial part is 300 μm wide. The midrib possesses thick, distinct epidermal layers of fairly large squarish, thick walled cells. The inner part of the adaxial cone includes a cluster of angular collenchyma cells. The palisade cells extend up-to the shoulders of the adaxial cone. The lower part of the midrib consists of fairly thick walled, angular cells. The vascular strand is fairly prominent. It is triangular and comprises a cluster of wide circular thin walled xylem elements and a thick band of phloem elements [Figure 1a].

Figure 1

(a) Transverse section of leaf through midrib. (b) Transverse section of lamina. AbP: Abaxial part; AdC: Adaxial cone; Ep: Epi-dermis; GT: Ground tissue; La: Lamina; Ph: Phloem; X: Xylem; AdE: Adaxial epidermis; Mu: Mucilage; PM: Palisade mesophyll; SM: Spongy mesophyll

The lamina is 60-70 μm thick. The adaxial (upper) epidermis is prominent, narrow and cylindrical. Some of the upper epidermal cells are dilated and possess dense mucilage. The abaxial epidermal cells are thin, the cells being small and elliptical. The mesophyll is differentiated into upper band of narrow cylindrical palisade cells and lower zone of two or three layers of lobed loosely arranged spongy parenchyma cells [Figure 1b].

As seen in paradermal section of the lamina, the epidermal cells are wide and possess thin highly wavy anticlinal walls [Figure 2a]. The cells assume amoeboid outline. The surfaces of the cells are smooth and no cuticular markings are evident. The stomata are dense and diffuse in distribution. They were of anomocytic type. No specific subsidiary cells are seen. The guard cells are elliptical and the stomatal pores are slit like. The guard cells are 50 × 80 μm in size [Figure 2b].

Figure 2

(a) Paradermal section of the lamina showing epidermal cells and stomata in surface view. (b) Paradermal section of the lamina show-ing epidermal cells and stomata in surface view. AW: Anticlinal walls; EC: Epidermal cells; St: Stomata; EC: Epidermal cells; St: Stomata

The lateral veins are conspicuous. They are straight and uniformly thin. The vein-islets are distinct and they had distinct thin straight vein boundaries. The islets are fairly wide and their shape may be rectangular, squarish or polygonal [Figure 3a].

Figure 3

(a) Venation of the lamina by leaf cleared at low magnifica-tion. (b)Venation of the lamina by leaf cleared at enlarged magnifica-tion. VI: Vein islet; VT: Vein termination

Vein-terminations are present in all islets. They are unbranched or branched once/twice and spread within the islets [Figure 3b].

Microscopy of stem

The stem is pentagonal in sectional view, having five short blunt ridges [Figure 4a]. It consists of a continuous layer of epidermis which possesses small spindle shape cells with prominent cuticle [Figure 4b]. Inner to the epidermis is a layer of small darkly strained cells followed by one or two layers of hyaline parenchyma cells. Within the ridges, the parenchyma tissue is wider and the cells are enlarged [Figure 5c].

Figure 4

(a) Entire view of transverse section of stem. (b)En-larged view of transverse section of stem. Co: Cortex; Ri: Ridges; SC: Sclerenchyma; VC: Vascular cylinder; Ep: Epidermis; Ph: Phloem; Pi: Pith; SC: Sclerenchyma; Ve: Vessels; X: Xylem

Figure 5

(a) Xylem strand with wide vessels in transverse section of stem. (b) Xylem strand with narrow vessels in transverse section of stem. (c)Cortex with sclerenchyma in transverse section of stem. Co: Cortex; SC: Sclerenchyma; Ph: Phloem; Ve: Vessels; XFi: Xylem fibres

There as a fairly thick continuous cylinder of cortical sclerenchyma, running all around the stem. The cylinder is 4-cells thick. In the region of the ridges, the sclerenchyma cylinder becomes much thicker having several layers of small cells [Figures 4b and 5c].

The vascular cylinder is angular in outline. It possesses outer wide cylinder of phloem enclosing inner xylem cylinder. Xylem includes a circle of solitary wide and narrow vessels associated with xylem fibers [Figure 5a and b]. The fibers are thick walled, lignified and have wide lumen. The vessels are circular and thin walled; the smallest vessel is 20 μm wide; the largest vessel is 80 μm wide. The pith consists of angular compact thin walled parenchyma cells.

Powder microscopy

Whole plant powder material exhibited the following inclusions when examined under the microscope.

Glandular trichomes

Capitate type glandular trichomes are common in the powder [Figure 6a]. They have thick, one celled stalk and a glandular body comprising two upper cells and two lower cells. The body cells have dense cytoplasm and prominent nucleus [Figure 6b]. The gland is 50 × 80 μm in size.

Figure 6

(a) Isolated capitate type of glandular trichomes seen in the powder. (b) Isolated capitate type of glandular trichomes seen in the powder. (c) Non glandular covering type of trichomes seen in the powder. (d) Veins of the lamina and marginal trichomes. (e) Fibres (wide and narrow) and parenchyma cells seen in powder. (f) Wide fibres seen in powder microscopy. (g) Narrow fibres and xylem elements seen in powder microscopy. (h) Fibres and vessel elements seen in powder microscopy. (i) Fibres and vessel elements seen in powder microscopy. (j) Short, wide vessel elements seen in powder microscopy. GTr: Glandular trichome; BC: Body cells; St: Stalk; NGTr: Non glandular trichomes; MTr: Marginal trichomes; MV: Marginal veins; VT: Vein termination; NFi: Narrow fibres; Pa: Parenchyma; WFi: Wide fibres; XF: Xylem fibres; Fi: Fibres; VE: Vessel elements; LWP: Lateral wall pits; PP: Perforation plate

Non glandular trichomes

Non glandular trichomes are more abundant than the glandular type. The non-glandular trichomes are unicellular, unbranched, and straight or curved, broad at the base, pointed at the tip. They are 15 × 50 μm in size [Figure 6c and d].

Lamina in powder

Fragmentary lamina is frequently seen in the powder, wherein vein-terminations are visible. The veins are thick and straight. The vein terminations are either unbranched or branched once or twice [Figure 6d].

Xylem elements

Different types of xylem elements are seen spread in the powder. Xylem parenchyma, xylem fibers and vessel elements are common inclusions.

Parenchyma cells

Long, scale like, thin-walled cells are seen mixed with other xylem elements. These parenchyma cells seem to serve the function of storage. The cells are up to 260 μm long and 30 μm wide [Figure 6e].

Fibers

Two types of fibers are seen in the powder [Figure 6e-g]. Some of the fibers have wide lumen and thin walls. Their ends taper abruptly and the middle part is spindle shaped. The wide fibers are 480 μm long and 30 μm wide in the middle.

The second type of fibers include narrow fibers. They have thick walls and narrow lumen [Figure 6g]. The cells are gradually tapering at the ends into pointed tips. The narrow fibers are 630 μm long and 15 μm wide.

Vessel elements

Vessel elements of primary xylem and secondary xylem are seen. Primary xylem vessels have close spiral lateral wall thickenings [Figure 6g]. The secondary xylem vessel elements have dense, multiseriate lateral wall pits [Figure 6h]. The vessel elements are long and cylindrical or short, wide and drum shaped [Figure 6i and j]. The vessel elements have wide, simple horizontal perforations. The lateral wall pits are either elliptical or circular. The vessel elements are 550-750 μm long.

Quantitative microscopy

Anomocytic type of stomata is present on the upper surface of leaves. The stomatal numbers of upper surface and lower surface were found as 19 and 15 respectively. The stomatal indexes of upper surface and lower surface were found 12.2 and 10.7 respectively. The vein islet and vein termination were calculated as 21 and 12 respectively. The palisade ratio was found to be 7.5.

DISCUSSION AND CONCLUSION

To establish the identity, purity, safety and quality of herbal drugs, standardization is an important tool. In order to standardize a drug, various macroscopic, microscopic, fluorescence analyses are done. Microscopic analysis is one of the economical and the simplest methods to begin with establishing the correct identification of the source material.[27]

The macroscopy and microscopy of leaves and stem of C. halicacabum have been carried out for the first time in this study. Macroscopic and microscopic studies of the leaf and stem will help identify the crude drug. The quantitative determination of some pharmacognostical parameters is useful for setting standards for crude drugs. Stomatal number, stomatal index value and palisade ratio, vein islet and vein termination value determination are equally important in the evaluation of crude drugs. These values help in the evaluation of purity of drugs.[28]

The information obtained from preliminary phytochemical screening will be useful in finding out the quality of the drug. Fluorescence is an important phenomenon exhibited by various chemical constituents show fluorescence in the visible range in day light. UV light produces fluorescence in many natural products (e.g. alkaloids like berberine) which do not visibly fluoresce in day light. If the substances themselves are not fluorescent, they may often be converted into fluorescent derivatives by applying different reagents. Some crude drugs are often assessed qualitatively in this way and it is an important parameter of pharmacognostical evaluation.[29]

These studies will be of help to the manufacturers for identification and selection of the raw materials for drug production.

Based on the above study it can be concluded that the parameters which are reported here can be considered to be distinct enough to identify and decide the authenticity of this drug in herbal industry/trade and this can be included as microscopic standards in Indian Herbal Pharmacopeia.