Pharmacognostic and Phytochemical studies of Azanza lampas (Cav.) Alef.: An Ethnomedicinally important root drug of Malvaceae

 

Partha Ghosh1, Chowdhury Habibur Rahaman2*

1Research Scholar, Department of Botany, Visva-Bharati University, Santiniketan-731235

2Associate Professor, Department of Botany, Visva-Bharati University, Santiniketan-731235

*Corresponding Author E-mail: habibur_cr@rediffmail.com, habibur_cr@yahoo.co.in

 

ABSTRACT:

Present study highlights the pharmacognostic characters and phytochemical profile of the crude drugs obtained from the leaf and root part of Azanza lampas (Cav.) Alef. (Malvaceae), an important ethnomedicinal plant. Different parts of this plant are traditionally used in curing gonorrhea, syphilis, jaundice, kidney problems, wounds, etc. Pharmacognostic study revealed that epidermal cells of upper and lower leaf surfaces are irregular in shape and their anticlinal walls are wavy in upper surface and straight to wavy in lower surface. In upper surface of leaf, stomata were strictly of anisocytic type and anomocytic type of stomata mixed with anisocytic type observed in lower surface. Stomatal indices of upper and lower surfaces were 8.11 and 16.70, respectively. Palisade ratio was 3.86. Glandular and non-glandular types of trichome were observed in both surfaces of the leaf. Trichome indices were 21.2 and 18.45 in upper and lower surfaces of the leaf, respectively. Phytochemical groups namely alkaloids, saponins, tannins, anthraquinones and glycosides have been detected in leaf and root extracts of this plant. Physical constants like moisture content, total ash, acid insoluble ash, water soluble ash have been determined for both parts of the plant. Moisture content was found higher in root(10.7%) than leaf(6.75%). Total ash value was higher in root (17.72%) than the leaf (9.54%). In leaf, contents of total phenolics and flavonoids were 154.42mg of GAE /g and 47.55mg of CE/g which are higher than that of root part.But, the contents of total alkaloids (158.86 mg of PE /g), tannins (78.89 mg of TAE /g) and ascorbic acid (274.34µg/100g ) were found higher in root than the leaf part. Considering the good contents of therapeutically important phytochemical groups, both leaf and root parts of this plant are found medicinally very potent. Present study provides some pharmacognostic features by which the crude drugs of this ethnomedicinal plant can properly be identified.

 

KEYWORDS: Azanza lampas, Foliar micromorphology, Pharmacognostic study, Phytochemical screening, Physicochemical analysis.

 

 

 

 


 

 

 

 

 

INTRODUCTION:

Medicinal plants are known as important ingredient of remedies employed in almost all traditional systems of medicine throughout the world. Even today, majority of the world population depends on herbal medicine for their primary healthcare practices1. The herbal medical practices provide safe and affordable primary healthcare to the people2. Modern synthetic drugs often produce undesirable side effects on the patients. This vital reason basically prompted the people to opt the alternative and traditional systems of medicine. Herbal based drugs are found safer, cheaper and more efficacious than that of the synthetic drugs3. Now, the use of natural medicines or herbal drugs has gained momentum and the demand for herbal raw drugs and other products is increasing steadily. However, the great constraint in promoting the use of herbal drugs is the lack of scientific evaluation and standardization. Further, confusion in the identification of medicinal plants and their substitutes, adulteration, lack of valid and reliable scientific information for their therapeutic efficacy are some of the main problems concerned. Standardization of herbal medicines and its quality control are the most important challenges in bringing the full acceptance of the people concerned with use of the herbal drugs4. WHO has brought out the guidelines for the evaluation of quality, safety and efficacy of crude drugs. Pharmacognosy is a simple and reliable tool which provides complete information about the crude drugs for its proper identification. Scientific studies in the field of pharmacognosy have been preceded on various lines covering morpho-anatomical characterization of plant parts used as crude drug, their physico–chemical parameters, phytochemical screening, biological assay and on many other diverse approaches. So, preparation of the pharmacognostic standards for proper identification of the crude drugs and detection of adulteration is considered as an essential step towards natural product research. Pharmacognostic standardization including physicochemical analysis and phytochemical screening of a number of medicinal plants have been carried out to develop the pharmacopoeial standards which are successfully employed in identification and authentication of the crude drugs obtained from the respective plant sources5,6,7,8,9. However, no such work has been done earlier to study the pharmacognostic standards of the crude drugs obtained from different parts of the plant Azanza lampas. For this reason present study has been undertaken to evaluate the pharmacognostic as well as phytochemical and physicochemical properties of A. lampas which may serve as standard reference for proper identification of its crude drug materials.

 

 

 

 

MATERIAL AND METHODS:

Material:

 

Fig. 1- A plant of Azanza lampas (Cav.) Alef. with a flower and fruit (inset)

Scientific name: Azanza lampas (Cav.) Alef.

Synonyms: Thespesia lampas (Cav.) Dalz. and Gibs.; Hibiscus lampas Cav.

Vernacular names:

Common Mallow (English); Ban Kapas (Bengali); Jangli Bhendi (Hindi); Ram Bhendi (Marathi); Jangli Paras-piplo (Gujrathi); Kondapratti (Telegu and Tamil); Kattuparatii (Malayalam); Turuve  (Kannada).

 

Tribal name: Ban-Kapasi.

 

Botanical description:

A large shrub, 1.5-2 m high. Leaves 3-lobed, 7-14 cm long; lobes triangular, middle lobe acuminate, two side lobes acute, margin entire or slightly undulate and ciliate, pubescent above and tomentose beneath; stipule subulate. Flowers on 7-14 cm long axillary peduncle, showy and 10 cm in diameter; pedicel 1-2 cm long, hairy; bracts 5, subulate, 1-1.3 cm long. Calyx copular; lobes free above with 5 subulate teeth, 1 cm long. Corolla yellow with crimson centre. Capsule 2.5 cm long, pilose, 4-5 valved, ovoid. Seeds small, turbinate, black, naked, except for a minute tuft of brown hair at the tip.

 

Flowering and fruiting time:

September to December.

 

Collection sites:

Illambazar forest areas (GPS locations-23035/40.25// N and 87026/55.6// E,23036/3.2// N and 87024/1.5// E, 23036/31.6// N and  87026/29.3//).

 

Occurrence:

The plant is found in humid as well as seasonally dry regions with an average annual rainfall of 1500-1700 mm. It is a light-loving plant grown in grass fields, teak forest and dry deciduous forests. In Java, it occurs in relatively dry areas at the altitudes up to 300 m. In Philippines, the plant is observed in open locations at low and medium altitudes. In Laos, it grows spontaneously on alluvial soils near watercourses or ponds. The plant thrives on fertile clayey or loamy soils, but will grow on less favourable soils if sufficient moisture is available.

 

Geographical distribution:

South and South-east Asia, Tropical East Africa. In India, it is distributed in different parts of western India, Deccan, North east and in Himalayas up to1200 m.

 

Medicinal uses:

Root:

It is used to treat gonorrhea and syphilis. The paste of root is used as a cure for jaundice, to heal scratches and wounds. The root juice is used as a health tonic. Root extract is given for jaundice, stomachache, kidney stones, swelling and severe body pain10,11,12.

 

Stem:

It is used in the treatment of inflammation, hyperacidity, epistaxis, bronchitis, cough, dysentery, fever, sun stroke, carbuncles and worms10.

 

Flower: It is used for skin diseases10.

 

Methods:

Collection of plant materials:

The fresh, well grown and matured leaf and root parts of A. lampas were collected from the Illambazar forest, Birbhum district, West Bengal, India. The plant sample has been identified and authenticated with the help of different standard flora13,14. The voucher specimen has been deposited at the Visva-Bharati Herbarium, Department of Botany, Visva-Bharati, Santiniketan, West Bengal for future reference.

 

Study of foliar micromorphology:

Leaf samples were cleared following the Bokhari’s method15. The cleared leaf samples were then mounted on the slide with a drop of 10% glycerine and 1% aqueous safranin and observed under compound light microscope (ZEISS, AXIO STAR plus, 176045).

 

Vegetative anatomy (petiole and root):

For this study, free hand sections of the petiole and root of the selected plant were made, stained suitably following safranin-light green staining schedule16  and studied under Compound Light microscope (ZEISS, AXIO STAR plus, 176045). Photographs of the suitable sections were taken with the help of photographic system attached with the said microscope.

 

Xylem elements study:

The root pieces (1 cm) were macerated following the standard method16. Boiled stem samples were then washed in distilled water for several times and observed under compound light microscope (ZEISS, AXIO STAR plus, 176045) for xylem elements study.

 

 

Organoleptic study:

This study of powdered crude drugs was done with the help of sensory organs following the standard methods17 which includes external morphology, colour, odour, taste, etc. of the crude drug.

 

Physicochemical evaluation:

Physicochemical parameters of the powdered plant samples like moisture content, ash value (total ash, acid insoluble ash, water soluble ash and sulphated ash), extractive value, swelling index and foaming index were determined as per guidelines of Indian Pharmacopoeia and WHO18,19. The fluorescence characters of the powdered plant samples treated with different reagents were observed under visible and long UV light (366 nm)20.

 

Powder microscopy:

Shade dried plant parts were powdered with the help of an electric grinder till a fine powder was obtained. This fine powder of plant parts were subjected to powder microscopy as per standard procedure18, 21.

 

Phytochemical study:

Preliminary phytochemical screening:

Aqueous, ethanolic, ethyl acetate, chloroform and hexane extracts were used for different chemical colour reaction tests with the help of different reagents to detect different phytochemical groups present in the powdered samples following standard methods21,22,23.

 

Estimation of total alkaloid content:

The total alkaloid contents in different parts of this plant sample were measured using 1,10-phenanthroline method described by Singh et al. (2004)24 with slight modifications. 100 mg plant powder was extracted in 10 ml 80% ethanol. This was filtered through filter paper and centrifuged at 5000rpm for 10 min. Supernatant obtained was used for the further estimation total alkaloids. The reaction mixture contained 1ml plant extract, 1ml of 0.025 M FeCl3 in 0.5M HCl and 1ml of 0.05 M of 1,10- phenanthroline in ethanol. The mixture was incubated for 30 minutes in hot water bath with maintained temperature of 70 ± 20C. The absorbance of red coloured complex was measured at 510 nm against reagent blank. Alkaloid contents were estimated and it was calculated with the help of standard curve of pilocarpine (0.1mg/mL, 10mg dissolved in 10ml ethanol and diluted to 100mL with distilled water). The values were expressed as mg/g Pilocarpine equivalent.

 

Estimation of total phenolic content:

Total phenolic content was estimated by standard method25. Plant sample of 0.5 g was homogenized in 5 ml of 80% ethanol. Homogenates was centrifuged at 10,000 rpm for 20 min. Supernatant was collected and then dried. Residue was dissolved in 5 ml of distilled water. 0.5 ml of aliquot, distilled water and folin- ciocalteau reagent were mixed in a test tube. After 3 minutes, 20% sodium carbonate was added to the test tube and mixed it thoroughly. Test tubes were placed on boiling water bath for 1 min and cooled it at room temperature. Then absorbance was measured at 650 nm wave length against a blank.

 

Estimation of total flavonoid content:

It was estimated employing the aluminium chloride method26. Plant sample of 0.5 g was homogenized in 5 ml of 90% methanol. Homogenates was centrifuged at 10,000 rpm for 20 min. Supernatant was collected and volume was adjusted to 10ml with methanol. Then 0.5ml of sample extract was taken in a test tube, subsequently 1.5 ml methanol, 0.1ml of 10% aluminium chloride solution, 0.1ml of 1M potassium acetate solution and 2.8 ml distilled water were added to the test tube and mixed it thoroughly. Absorbance was taken at 415 nm against the suitable blank using Shimadzu UV-1800 double beam spectrophotometer.

 

Estimation of total tannin content:

Method of Afify et al., (2012) 27 with slight modification was employed. The powdered plant sample of 500mg and 75ml distilled water were taken in a conical flask. It was then boiled for 30 minutes. After cooling, the boiled plant sample was centrifuged at 2000 rpm for 20 minutes. The residue was discarded and the volume of supernatant was adjusted to 100 ml with distilled water. Then the extract was used for the estimation of the tannins. 1 mL of the plant extract was taken in a volumetric flask containing 75mL distilled water. Then 5ml of Folin-Denis reagent and 10ml of sodium carbonate solution were added to the flask and volume adjusted to 100 ml with distilled water. Content in the flasks was thoroughly mixed, kept 30 minutes and absorbance was measured at 700nm on Shimadzu UV-1800 double beam spectrophotometer. A blank was prepared with distilled water instead of the sample. Tannins were estimated and calculated with the help of standard curve of tannic acid (0.1mg/mL) and expressed as mg of TAE/g.

 

Estimation of ascorbic acid:

Plant sample of 1g was homogenized in 25 ml of 4% oxalic acid. Homogenates were filtered. 10 ml of the filtrate was transferred to a conical flask and bromine water was added to the flask drop wise until the extract turns orange or yellow. Finally, the volume was made up to 25 ml with 4% oxalic acid. From this stock solution, 2 ml of sample was pipetted out and volume was made up to 3ml with distilled water. 1 ml of DNPH reagent followed by 1-2 drops of thiourea was added to the test tube. Test tube content was mixed thoroughly and incubated at 37o C for 3 hr. After incubation, 7 ml of 80% sulphuric acid was added to the test tube and absorbance was measured at 540 nm against a blank28.

 

RESULTS:

Foliar micromorphology:

Descriptions of the epidermal cells of the leaf, stomata and trichomes along with their measurements are given below.

 

Epidermis:

Leaf epidermal cells are irregular in shape with wavy anticlinal cell walls in upper surface and cells with straight to wavy anticlinal cell walls in the lower surface. Size of the epidermal cells in the upper surface is 88.42± 1.549 μm × 39.14± 0.812 μm and it is 77.19 ± 1.427 μm × 35.04 ± 0.897 μm in the lower leaf surface. Frequency of the epidermal cells is 148.49± 2.237/ mm2 on the upper surface and it is 397.21± 3.03/ mm2 on the lower surface. Palisade ratio is 3.86± 0.053(Figs. 2 and 3).

 

Stomatal complex:

Stomata are present in both surfaces of the leaves, i.e. are amphistomatic. Adaxially stomata are confined to margins of the midrib and exclusively anisocytic type whereas few stomata of anomocytic type are mixed with anisocytic type in the lower surface of leaf. Size of the stomata is 25.09± 0.284 μm × 14.06± 0.163 μm in the upper surface and it is 28.49 ± 0.325 μm × 16.17 ± 0.271 μm in the lower surface. Stomatal frequency is 13.03± 0.836/mm2 and stomatal index is 8.11±0.16 in the upper surface. Stomatal frequency and stomatal index in the lower surface are 98.45 ± 2.082/mm2 and 16.70 ± 0.545, respectively (Figs. 2 and 3).

 

 

Fig.2- A portion of upper epidermis with stomata (ST)

 

 

Fig.3- A portion of lower epidermis with stomata (ST)

 

Trichomes:

Both non-glandular and glandular types of trichomes are observed in both the leaf surfaces. Non-glandular trichomes areof two types – unicellular trichome with pointed apex, sometime apex is curved; other type is stellate, 5-7 armed trichome. Size of the unicellular trichome is 404.64 ± 5.242µm × 5.65 ± 0.243µm in the upper surface and 408.37 ± 13.54 µm × 6.16 ± 0.298 µm in the lower surface. Frequency of the non-glandular unicellular trichome is 5.45 ± 0.091 /mm2 in the upper surface and 4.56 ± 0.089 /mm2 in the lower surface.  Size of the each arm of stellate trichome is 415.65 ± 13.322 µm × 6.23 ± 0.272 µm and 459.56 ± 4.004 µm × 6.87 ± 0.177 µm in upper and lower surface, respectively.  Frequency of the stellate trichome is 8.79 ± 0.303 /mm2 in the upper surface and 7.33 ± 0.336 /mm2 in the lower surface. Glandular trichomes are with quadri-cellular globose head and a bi-celled stalk observed in both the leaf surfaces. The size of the glandular trichome is 28.75 ± 0.357µm × 21.43 ± 0.354 µm and frequency is 6.97 ± 0.714 /mm2 in the upper surface. The size of glandular trichomes in lower surface is 29.13 ± 0.342 µm × 22.97 ± 0.336 and frequency is 6.56 ± 0.437 /mm2. Trichome indices for glandular trichome are 2.63±0.481 and 1.62±0.107 in upper and lower surfaces, respectively. Trichome index of unicellular one is 3.59±0.548 in upper surface and it is 1.13±0.074 in lower surface. Trichome indices for stellate trichomes are 5.66±0.213 and 2.15± 0.036 in upper and lower surfaces, respectively (Figs. 4, 5, 6 and 7).

 

Fig.4- Unicellular non-glandular trichome

 

 

Fig.5- Stellate trichome (side view)

 

 

Fig.6- Stellate trichome (top view)

 

 

Fig.7- A Glandular trichome

 

 

4. Crystals:

Star shaped crystals of calcium oxalate (sphaeraphides) are present on both surfaces of the leaf. Diameter of the crystals present in the upper epidermis is 31.36± 0.024 μm and it is 32.76±0.026μm in the lower epidermis (Fig. 8).

 

 

Fig.8- Crystals (CY)

 

Vegetative anatomy:

A.    Petiole anatomy:

Outline of the petiole is circular in transverse section and petiole consists of the following tissue organization (Fig. 9).

 

 

 

Fig.9- T.S. of petiole

 

Epidermis:

It consists of single layered compactly arranged tubular parenchymatous cells with thick cuticle on the outer wall. Epidermis is 21.41 µm in thickness.

 

Hypodermis:

Beneath the epidermis 2-3 layers of collenchyma cells are present which constitute the hypodermis.  It is 28.54µm in thickness.

 

Middle cortex:

Below the hypodermis, 10-12 layers of parenchyma cells with profuse intercellular spaces forming the middle cortex zone which is 156.97-178.38 µm thick. Cortex is terminated by a single layer of larger cells known as starch sheath.

 

Sclerenchyma tissue:

Patches of sclerenchyma cells forming a discontinuous ring just above the phloem zone.

 

Vascular bundles:

A continuous cylinder of vascular tissue present just below the cortex zone. It is of 114.85-224.58 µm thick. Vascular cylinder is composed of xylem tissue present inner side of it and phloem tissue outer side.  Phloem is scanty.

 

Pith:

The massive pith is comprised of 12-15 layers of parenchyma cells with profuse intercellular spaces. Thickness of the pith is 214.1-228.4µm.

 

 

 

B.    Root anatomy:

The transverse section of the root is circular in outline and following tissue organization is observed in the root (Fig.-10-A, B and C).

 

 

 

 

Fig.10-A-T.S. of root, B and C-Magnified view.

 

Epiblema:

It consists of single layered, compactly arranged parenchymatous cells; 14.27 µm in thickness.

 

Cortex:

The cortex is massive, parenchymatous, 27-29 cell layered, 288 .72 – 313.94 µm in thickness.  Cells are polygonal, thin walled.

 

Vascular tissue:

Vascular cylinder present at centre of the root and it is of 234.54 µm in thickness. Primary xylem is scanty. It occupies the centre of the root with four small projecting strands, reflecting its tetrarch nature in primary body of the root. Secondary xylem is massive, surrounding the centrally located primary xylem. Vessel elements in secondary xylem are circular in outline, broader in diameter and present in cluster. Many tyloses present in the vessel lumen. From the centre, four strands of parenchyma radiate through the secondary xylem mass separating it into four large patches. Parenchyma cells of the radiating strand are mostly rectangular in shape, radially arranged and dividing through anticlinal as well as periclinal plane.  Cells of the radiating strands enlarge the girth of the root and increase the bulk of parenchyma tissue by adding more tissue to it.  Parenchyma tissue in root mainly performs the storage function.

 

Cambium is not a continuous layer. It is localized on the outer side of secondary xylem patches. External to the cambium phloem is present. It is many-celled thick. In phloem proportionately more parenchyma cells are present that perform storage function mainly.

 

Pith: The pith is absent.

 

Xylem elements:

General description and measurement of the xylem elements of the macerated root part of the investigated plant have been represented bellow.

Vessel element:

Perforation plates of the vessel elements are simple. On the basis of their size, vessel elements are of 3 types. Type 1: vessel element is very large with obliquely placed perforation pate, tail short, pits simple arranged in diagonal rows. Size of the element is 214.5± 1.708 μm × 42.67± 2.434 µm and frequency is 8.33±0.603 /mm2 (Fig. 11). Type 2: vessel element is smaller in size with transversely placed perforation plate, pits are bordered and arranged in horizontal rows, tail absent. Size of the element is 75.66± 0.842 µm × 49.41 ±0.416  µm and frequency is 3.5±0.544 /mm2 (Fig. 12). Type 3: vessel element is moderate in size with transversely placed perforation plate, pits are bordered and arranged in horizontal rows, tail absent. Size of the element is 125.14 ± 1.024 µm × 59.21 ± 0.456 µm and frequency is 3.5±0.544 /mm2(Fig. 13).

 

 

Fig.11- A vessel element (Type-1)

 

 

Fig.12- A vessel element (Type-2)

 

 

Fig.13- A vessel element (Type -3)

 

 

Fig.14- A portion of tracheid

 

Tracheids:

Tracheids are very long with spiral side wall thickening. Diameter is 21.18± 0.306 µm and frequency is 3.83± 0.393/ mm2(Fig. 14).

 

Fibres:

Fibres are typically libriform type and aseptate. On the basis of their size and structure fibres are of 3 types. Type 1- moderate in size, middle broad, with abruptly tapering pointed tips. Size is 562.74 ± 13.985µm × 18.078 ± 0.661 µm and frequency is 19.26 ± 0.386 / mm2 (Fig. 15). Type 2- shorter in length, wider diameter, ends tapered and pointed. Size is 365.35± 6.514 µm × 32.37± 1.152 µm and frequency is 5.99± 0.188/mm (Fig. 16). Type 3- very long, narrow width, with regularly tapering pointed ends. Size is 686.18± 8.944µm × 14.27 ± 1.036 µm and frequency is 12.20± 0.516/mm (Fig. 17).

 

 

Fig.15- A fibre (Type-1)

 

 

Fig.16- A fibre (Type-2)

 

 

Fig.17- A fibre (Type-3)

 

 

Organoleptic features of the powdered drug:

The colour, odour, taste and texture of the leaf and root of the investigated plant have been presented in the table below (Table- 1; Figs. 18 and 19).

 

Table-1: Organoleptic features of different parts of the investigated plant

Organoleptic features

Leaf

Root

Colour

Greenish yellow

Reddish brown

Odour

No specific odour

No specific odour

Taste

Acrid

Bitter

Texture

Herbaceous

Fibrous

 

 

Fig.18- Powdered root part

 

 

Fig.19- Powdered leaf part

 

Physicochemical analysis:

For physicochemical characterization of the powdered plant samples the moisture content, total ash content, acid insoluble ash, water soluble ash, sulphated ash, extractive value, swelling index and foaming index were determined and results were presented (Table-2).

 


 

 

Table-2: Physicochemical analysis

Sl. No.

Physicochemical parameters

Average value (root part)

Average value   (leaf part)

1.

Moisture content (% w/w)

10.70±0.17

6.75±0.12

2.

Ash value (% w/w)

a. Total ash

 

17.72±0.53

 

9.54±0.32

b. Acid insoluble ash

1.97±0.017

2.57±0.014

c. Water soluble ash

1.79±0.12

2.19 ± 0.122

d. Sulphated ash

5.67±0.33

6.17±0.253

3.

Extractive value (% w/w)a. Water soluble extract

17.44±2.62

14.54±1.22

b. Ethanol soluble extract

8.53±0.16

5.83±0.46

c. Ethyl acetate soluble extract

5.83 ± 0.44

5.33 ± 0.244

 d. Chloroform extract

2.5±0.50

2.66±0.45

e. Hexane extract

1.66±0.66

1.66±0.66

4.

Foaming index

250± 0.16

300± 0.16

5.

Swelling index

6.67±0.166

6.16±0.136

 

 

Table -3: UV fluorescence nature of the crude drug powder

Materials and treatments

Root powder

Leaf powder

Under visible light

Under UV light (365nm)

Under visible light

Under UV light (365nm)

Powder as such

Reddish brown

Light green

Greyish green

Fluorescent green

Paper with powder

Pale brown

Blue

Light green

Light yellow

Treated with 50% HNO3

Brick red

Olive green

Olive green

Orange

Treated with Methanol

Light brown

Pink

Yellowish green

Ashy green

Treated with 5% KOH

Dark coffee

Maroon

Yellowish green

Pinkish brown

Treated with Ethanol

Light brown

Pink

Olive green

Orange

Treated with Acetone

Pinkish brown

Pinkish red

Yellowish green

Ashy green

Treated with 1N HCl

Chocolate brown

Dark maroon

Dark green

Dark green

Treated with 1N  NaOH

Coffee colour

Maroon

Blackish green

Dark green

Treated with Antimony trichloride

Pale brown

Green

Olive green

Orange

Treated with 80% H2SO4

Dark coffee

Olive green

Yellowish green

Ashy green

 


 

 

Fluorescence analysis:

In this study it was observed that drug powder treated with different chemical reagents gives characteristic colours when seen under UV light and it is compared with the colours observed under visible light. In some cases marked differences in colours were observed (Table-3; Figs. 20 and 21).

 

 

Fig.20- Spot test for root powder under Visible light

 

 

Fig.21- Spot test for root powder under UV light

 

Powder microscopy:

The microscopy of root powder showed presence of small patches of cork cells, portion of tracheid, brachysclereids, calcium oxalate crystals, starch grains, tanniniferous cells and portion of broken fibers which are characteristic features of the root powder of the investigated plant (Figs. 22, 23, 24, 25, 26 and 27).

 

 

Fig.22- A patch of cork cells

 

 

Fig.23- Portion of a tracheary element

 

 

Fig.24- A brachysclereid

 

 

Fig.25- Starch grains

 

 

Fig.26- Tanniniferous cells

 

Fig.27- A portion of fibre

 

Preliminary phytochemical screening of the powdered drug:

Phytochemical screening of different solvent extracts of leaf and root parts of the investigated plant showed presence of different phytochemical groups in varying degrees (Table-4; Figs. 28 and 29).

 

 

 


Table -4: Microchemical colour reaction tests of different solvent extracts of the plant parts

Chemical groups

Test/ Reagent

Nature of change

Different solvent extracts of the root part

Different solvent extracts of the leaf part

Aq

Et

EA

He

Ch

Aq

Et

EA

He

Ch

Alkaloids

Mayer’s reagent

White cream ppt.

+

+

+

-

+

+

+

+

-

+

Wagner’s reagent

Orange brown ppt.

+

+

+

-

+

+

+

+

-

+

Dragendorff’s reagent

Orange brown ppt.

+

+

+

+

+

+

+

+

+

+

Reducing sugars

Fehling’s reagent

Brick red

+

+

+

-

-

+

+

+

-

-

Benedict’s reagent

Brick red

+

+

+

-

-

+

+

+

-

-

Carbohydrate

Molish’s test

Violet ring

+

+

+

-

-

+

+

+

-

-

Anthraquinones

Borntrager’s test

Pink colour

-

+

-

-

-

-

+

-

-

-

Saponins

1% Lead acetate solution

White ppt.

+

+

+

+

+

+

+

+

+

+

Proteins

Millon’s reagent

White ppt.

+

+

+

-

+

+

+

+

+

+

Lugol’s reagent

Faint yellow colour

+

+

+

-

-

+

+

+

-

-

Amino acids

Ninhydrin reagent

Purple colour

+

+

+

-

-

+

+

+

-

-

Flavonoids

Shinoda’s test

Magenta colour

+

+

+

-

-

+

+

+

-

-

Tannins

10% Ammonium hydroxide solution

Yellowish white ppt.

+

+

+

-

-

+

+

+

-

-

10% Lead acetate solution

Yellow ppt.

+

+

+

+

+

+

+

+

+

+

5% Ferric chloride solution

Greenish black

+

+

+

+

+

+

+

+

+

+

Steroids

Salkowski test

Reddish blue and green fluorescence

_

+

_

+

+

_

+

_

+

-

Lignin

Phloroglucinol+HCl

Red colour

-

+

-

-

-

-

+

-

-

-

Glycosides

10% NaOH solution

Yellow fluorescence

+

+

+

-

-

+

+

+

-

-

- = Absent;               + = Present (Different solvent extracts: Aq= Aqueous, Et=Ethanol, EA=Ethyl Acetate, He=Hexane, Ch=Chloroform)

 

 

Fig.28- Chemical colour reaction test for ethanolic extract of leaf

 

 

 

Fig.29- Chemical colour reaction test for ethanolic extract of root

 

 

Table 5: Phytochemical profile of the investigated plant

Plant parts

Total alkaloids

(mg of PE /g)

Total phenolics

(mg of GAE /g)

Total flavonoids

(mg of CE/g)

Total tannins

(mg of TAE /g)

Ascorbic acid (µg/100g tissue)

Leaf

125.43±0.836

154.42 ± 0.251

47.55±0.976

65.55± 0.686

216.54 ± 6.241

Root

158.86±0.428

143.61 ±0.258

27.14± 0.633

78.89  ± 0.722

274.34 ± 6.621

 

 

 

 


Total alkaloid content:

Alkaloids are known to be one of the important phytochemical groups that show a wide range of therapeutic properties. In root, content of alkaloids was 158.86 mg of PE/g which is higher than the leaf (125.43 mg of PE/g) part of the plant (Table-5).

 

Total phenolic content:

Phenolics are one of the major groups of antioxidant compounds reported to be involved in free radical scavenging activity and also responsible for curing a wide range of ailments. Total phenolic contents in leaf and root were 154.42 mg of GAE/g tissue and 143.61mg of GAE/g tissue, respectively. Here, content of phenolics is slightly higher than the content estimated in leaf (Table-5).

 

Total flavonoid content:

Flavonoids are very important group of phenolics that show a wide range of therapeutic properties. Total flavonoid contents in leaf and root were 47.55 mg of CE/g tissue and 27.14mg of CE/g tissue, respectively. Flavonoid content is also significantly higher in leaf than the root (Table-5).

 

Total tannin content:

High content of tannins was observed in root part (78.89 mg of TAE /g) in compare to the leaf part of the investigated plant (65.55mg of TAE /g) (Table-5).

 

Ascorbic acid content:

Ascorbic acidis one of important phytochemical that show a wide range of therapeutic properties. Ascorbic acid contents in leaf and root were 216.54 µg/g tissue and 274.34 µg/g tissue, respectively. Ascorbic acid content is significantly high in the root than the leaf (Table-5).

 

DISCUSSION:

Some of the pharmacognostic and phytochemical characters obtained from leaf and root of the plant A. lampas are found very distinct which can be used as marker character for identification of the respective leaf and root drugs in its fresh as well as dried form. Foliar micromorphology does play significant role in plant identification and also in authentication of leaf drugs5,6,7,8,9. Shape and size of the epidermal cells are found very distinctive which help in proper identification of specific plant29,30. In present study epidermal cells of the leaf are found irregular in shape with wavy anticlinal cell walls in upper surface and straight to wavy anticlinal cell walls in the lower surface. In previous studies, almost similar type of epidermal cells were observed in different members of the family Malvaceae where cells are polygonal, isodiametric or irregular in shape with straight to wavy and sinuous walls31,32. All these features of leaf epidermal cells provide uniqueness to certain extent which will help in identification of leaf part of this medicinal plant. Palisade ratio of the leaf is considered one of the diagnostic characters for identification of leaf drug. Palisade ratio here is 3.86 which is also specific to this medicinal species. Type and structure of stomata have great taxonomic as well as pharmacognostic value in proper identification of different plant taxa including medicinal plants32,34. Anomocytic, anisocytic, paracytic and tetracytic types of stomata are common in different members of Malvaceae31,32.  Here, in A.lampas, stomata are mainly of anisocytic type distributed in both upper and lower surfaces of the leaf. In upper surface, anisocytic stomata are confined to the laminar portions along both sides of the midrib which shows a specific distribution pattern of stomata in this plant. Anomocytic type of stomata also found mixed with anisocytic type in lower surface of the leaf. The observation made on stomatal type of this plant conforms the main types of stomata (Anomocytic, anisocytic, paracytic and tetracytic type) observed by previous workers in different members of the family Malvaceae31,32. Stomatal index is also used as marker for identification of plant species as well as the leaf drugs obtained from respective species35,5,6,7,9. Stomatal index value is 8.11in the upper surface and it is 16.70 in the lower surface of leaf which make this taxon distinct among the other members of the family Malvaceae. Trichome features are also very important in proper identification of the plants and considered as one of the valuable taxonomic markers now31. In present study, 3 types of trichomes were found on both surfaces of the leaf. Non-glandular trichomes are of unicellular and stellate types and glandular trichomes are with quadricellular globose head and a short stalk. The difference in the values of trichome index between upper (21.2) and lower (18.45) surfaces of the leaf is very distinct which provides a diagnostic marker for identification of leaf of this medicinal plant.

 

In Pharmacognosy, the physicochemical characters play a vital role in setting fingerprint for a crude drug and are successfully employed in detection of adulterants and improper handling of the crude drug18,19,35. Moisture content of a crude drug is an important parameter in respect of its shelf life because insufficient drying favours the growth of molds and microorganisms which ultimately spoil the biomass and active principles of the crude drugs19. So, moisture content is directly related to maintain the stability and quality of crude drugs. In this study, a noticeable difference was observed between moisture contents of leaf (6.75%) and root(10.7%) parts of this plant. Among the physical constants, ash value is considered as an important tool in appraisement of purity and identity of a crude drug and it also highlights the inorganic matter present in the crude drug18. In this study, it is noticed that total ash content is greater in root (17.72%) than the ash content of leaf (9.54%) which indicates that root of this medicinal plant contains more amounts of inorganic matters like carbonate, oxalate, phosphate including silica and siliceous earthy substances. Values for various parameters of ash observed in leaf and root of this plant are different and distinct from one another which can be used as identifying marker in authentication of the drug samples obtained from leaf and root of the plant A.lampas and also be used for quality control of those crude drugs.

 

In pharmacognostic evaluation of the crude drugs, extractive value is considered as one of the diagnostic features and is used in proper identification of the crude drugs. It also determines the amount of chemical constituents extracted by the particular solvent from certain amount of crude drug8. Extractive value also indicates the nature of chemical constituents present in the drug and also useful in estimation of specific constituents soluble in a particular solvent. Values of the extractable matters vary according to the polarity of solvent and purity of the crude drug. Here in this study, water was found to be the best extractive solvent among five solvents used, as it extracted out highest amounts of the chemical constituents from both leaf and root of this investigated plant. Ethanolic extractive value was found second best among the extractive values of five solvents and it was noticed that ethanolic extractive value of root (8.53%) is greater than the same solvent extractive of leaf (5.83%). The other solvents like, ethyl acetate, chloroform and hexane showed very low extractive values which indicate that comparatively lesser number and amount of extractable phytochemical groups have been leached out from both parts of the plant. So, it can be concluded that water solvent is the best option among the solvents taken here for extraction of phytochemicals and aqueous extractive values can be used as marker for identification of the crude drug samples of both leaf and root of this medicinal plant.

 

Fluorescence analysis of the crude drug powder produces characteristic colour changes when the drug samples treated with specific chemical reagents are exposed to UV light. Sometimes this change in colour of the crude drug is very unique and it is used as a finger print for proper identification of crude drugs when other physical and chemical parameters are found inadequate19. The same drug powder treated with various chemical reagents appears with different colours when seen under different wavelength of light. Here, 50 % HNO3 and SbCl3 treated powdered samples of leaf and root showed characteristic colour changes when illuminated under UV light which are quite distinct from its colour observed under visible light. Under UV light, the changes in colour of the drug samples were found very distinct and they are specific to the respective crude drugs obtained from the investigated parts of this medicinal plant.

 

 

Plants possess numerous phytochemical constituents and many of them are known to exhibit a wide range of biological activity. Chemical analysis and biological assay are two very important aspects employed for   pharmacognostic evaluation of crude drugs obtained from medicinal plants. Preliminary phytochemical screening is useful in prediction of the nature of crude drugs and also valuable for detection of chemical constituents present in it. The important phytochemical groups detected in the leaf and root of this investigated plant are alkaloids, anthraquinones, phenolics, saponins, tannins, glycosides, etc. Presence of such important phytochemical groups in both the parts of this medicinal shrub clearly indicates their therapeutic properties and also highlights the scientific basis of various ethnomedicinal uses of this investigated medicinal plant.

Phytochemical studies of both leaf and root of the plant A.lampas reveals that contents of total phenolics and flavonoids were found higher in leaf than the root part. But, total alkaloids, tannins and ascorbic acid contents were noticed in higher amounts in root than the leaf. Considering their phytochemical content, both leaf and root of this medicinal plant are found quite rich in significant amounts of therapeutically potent phytochemical groups like phenolics, flavonoids, tannins, alkaloids and ascorbic acid. Further scientific investigations on both leaf and root of this ethnomedicinal plant are recommended for development of noble natural products through extensive phytochemical and different biological activity studies.

 

ACKNOWLEDGEMENTS:

We are thankful to the Head, Department of Botany, Visva-Bharati, for providing the necessary laboratory facilities. Financial assistance sanctioned by the UGC in the form a major research project [Ref. No.-42-972/2013(SR)] is sincerely acknowledged.

 

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Received on 19.08.2018          Modified on 28.09.2018

Accepted on 10.10.2018  ©A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2018; 10(4): 259-271.

DOI: 10.5958/0975-4385.2018.00042.0