INTRODUCTION:

Medicinal plants have been playing a significant role in primary health care of human beings and their domesticated animals from the very beginning of human civilization. There is a long-standing history regarding the uses of plants as a natural source of treatment and therapies for human health in many indigenous societies¹. The World Health Organization reported that 80 % of the world populations rely chiefly on indigenous medicine and that the majority of traditional therapies involve in the use of plant extracts or of their active constituents². Over 25% of modern medicines that are commonly used worldwide contain compounds extracted from medicinal plants³. Herbal medicines are playing important role in providing health care to the large section of population, especially in developing countries and also play a vital role in drug development programme in the pharmaceutical industries⁴. It has been noticed that the synthetic modern drugs prescribed by the allopathic doctors are often found expensive, inadequate for the treatment of many chronic diseases and sometimes with anarchic side effects also⁵. Now- a -days, application of herbal medicines is gradually being increased at a high rate globally because of their - a) efficacy in curing different chronic and incurable diseases, b) cost effectiveness, c) easy availability in the locality, and d) very low or no side effects. World Health Organization (WHO) has rightly taken the initiative to encourage and promote the uses of herbal remedies in national health care programs of different countries in the world. It is also observed that, in the last few decades, there has been an exponential growth in the field of herbal medicine and bioactive phytochemicals due to failure of modern medicine in providing effective treatment to many of the chronic diseases and in combating the emergence of multidrug resistant microorganisms.

Scientists all over the world are now engaged in medicinal plant research to investigate the herbal medicines for screening their bioactive chemicals and evaluating its biological activity. In the process of drug development several important aspects like, pharmacology, clinical aspects, stereo chemistry of the drug molecule, its designing, drug delivery, dosage, etc. are also taken into consideration^6,7. For successful development of new drugs from natural products, the first and most vital step is the collection and identification of the genuine plant materials. Appropriate knowledge of such plant materials (crude drugs) is very important in preparation, safety and efficacy of the herbal products. Pharmacognosy is a simple and reliable tool which provides complete information about the crude drugs for its proper identification^4,8. The WHO emphasized the need to ensure quality control of medicinal plant products by using modern techniques and suitable standards⁹. 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 sources^9,10,11,12. However, no such work has been done earlier to study the pharmacognostic standards of the crude drugs obtained from different parts of C. scarabaeoides. In this context, present study has been undertaken to evaluate the pharmacognostic as well as phytochemical and physicochemical properties of Cajanus scarabaeoides (L.) Thouars which may serve as standard reference for proper identification of its crude drug materials.

MATERIAL AND METHODS:

Material

Scientific name: Cajanus scarabaeoides (L.) Thouars

Synonyms: Atylosia scarabaeoides (L.) Benth. ; Cantharospermum scarabaeoides (L.) Baill.

Local names: Ban kurti; Lodha name- Ban kulatha; Mundari name- Bir- hore.

Common English name: Wild cajanus.

Parts used: Stem, leaf, whole plant, root, fruits and seeds.

Botanical characters

Annual or sometimes biennial, twining herb. Leaves alternate, 3-foliolate; leaflets entire, acute or subacute, pubescent, base obtuse, 3-nerved; terminal leaflet 2.5-4 cm long, obovate-lanceolate or rhomboid; petiolule 1-2 mm, pubescent; stipules 1 mm, filiform. Flowers small,yellow, 1-6, on short densely pubescent peduncles; pedicel 4-6 mm long. Calyx 5-7 mm long; teeth linear, acute. Corolla papilionaceous, standard 8-9 mm long. Stamen diadelphous. Pods 1.7-2.5 cm long, distinctly jointed, with dense brown hairs, 4-6 seeded (Fig.- 1).

Flowering and fruiting season- August to February.

Habitat: It is commonly growing in the forests, bushes, village shrubberies and on the hedges. Occasionally found along the roadsides.

Distribution: The plant is grown in Tropical Asia, Australia, Madagascar and West Africa. It is found throughout India, up to 1800m in the Western Himalayas.

Medicinal uses:

Whole plant- it is used for swelling and pain in leg during pregnancy, night fever, renal stone, eye diseases, dropsy, anaemia, hemiplegia, burns, wounds, small-pox, syphilis, gonorrhoea, spermatorrhoea, gravel, cholera, dysentery, snake-bite and rinderpest^13,14,15.

Leaves- Leaf paste is used to cure swelling of the body, sores, venereal diseases and pain due to inflammation¹³.

Seeds- Crushed seeds are used to cure tapeworm^13,14.

Ethnoveterinary uses:

Whole plant- It is also used for curing diarrhea and dysentery in cattle. Plant decoction is given to cows as a remedy for tongue and mouth sores^{14, 16}.

Leaves- Leaf paste is used to cure swelling of throat in cattle^16,13,15.

Mode of administration:

In Ayurveda: 1)Whole plant decoction mixed with honey taken orally as tonic after delivery of a woman; 2) A decoction of the whole plant mixed with black pepper given in diarrhea and dysentery; 3) The paste of the leaves administered orally to cure swelling of the body; 4) Fresh leaf paste applied topically to get relief from pain in rheumatism; 5) Paste of fresh stem and leaves applied on sores and in venereal diseases; 6) Powder of root taken orally to improve digestion power and to cure stomachache and abdominal gas; 7) Crushed roots extract used as ear drop in deafness; 10) Plant decoction given to cows as a remedy of tongue and mouth sores¹⁵.

In Tribal medicine: 1) Mundas prescribe crushed seeds to take orally as cure for tapeworm¹⁶.

In Veterinary medicine: 1) Whole plant extract and decoction of stem bark of Kumbhi (Caryeya arborea) (3:1) is used to treat dysentery in cattle; 2) Leaf paste is applied topically to cure swelling of the throat in cattle; 3) Plants are taken orally to cure dysentery and diarrhea of cattle¹⁵^{, 16}.

Methods

The fresh, well grown and matured plants were collected from the road side of Muluk, Birbhum, West Bengal, India in the month of July, 2016. The plant species has been identified and authenticated with the help of different standard floras. The collected fresh stem, leaves and fruits were washed thoroughly under tap water, shade dried, ground them into powder and finally kept the powdered samples in airtight containers separately for future use. The fresh plant materials were used for the study of macro and micro- morphological, anatomical and xylem maceration studies. The dried plant powders were used for powder microscopy, physicochemical analysis and preliminary phytochemical studies.

Study of foliar micromorphology: Leaf samples were cleared following the Bokhari’s method¹⁷. 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.

Vegetative anatomy (stem and petiole)- For this study, free hand sections of the stem and petiole of the selected plant were made, stained suitably following safranin-light green staining schedule¹⁸ and studied under Compound light microscope (ZEISS, AXIOSTAR plus, 176045).

Photographs of the suitable sections were taken with the help of photographic system attached with the said microscope.

Xylem elements study: The stem pieces (1 cm) were macerated following the standard method¹⁸. Boiled stem samples were then washed in distilled water for several times and observed under compound light microscope for xylem elements study.

Organoleptic study: This study of crude drugs was done with the help of sensory organs following the standard methods ¹⁹ which includes external morphology, colour, odour, taste, etc. of the crude drug.

Physicochemical evaluation:

Physicochemical parameters like moisture content, ash value (total ash, acid insoluble ash, water soluble ash and sulphated ash), extractive value, swelling index and foaming index of the powdered plant samples were determined as per guidelines of Indian Pharmacopoeia and WHO ^20,21.

Moisture content study- About 5 gm of leaf sample were weighed and dried for few days. Then the sample was incubated at 80°- 90°C temperature for one hour. Final weight of the sample was taken and calculated the percentage of moisture content ²².

Fluorescence analysis- For this study, the powdered plant samples were treated with different chemical reagents and observed the change in colour of the treated plant powder when seen under visible and UV light (365 nm). It is done to confirm the purity of the drug ⁹.

Histochemical study: Transverse sections of the stem and leaf petiole were kept in several glass slides; then one to two drops of specific reagents (Wagner’s, Dragendroff’s, Mayer’s, Lugol’s, Millon’s, 1% lead acetate, Phloroglucinol, Ferric chloride, etc.) added to the sections and kept for few minutes to allow the specific reaction between reagents and phytochemicals present in the cells. Sections were then observed under the compound light microscope to detect different phytochemical groups localized in different tissue zones in the respective sections^23,24,25.

Extraction: Each dried plant part of 10 gm was ground and extracted with a particular solvent of 100ml for 48 hours in a continuous shaking at room temperature. The extract was filtered and then it was dried by using a rotary evaporator under vacuum at a temperature of 45°C.

Determination of extractive value: 10 gm of powdered sample of each plant part was extracted successively in a 100ml conical flask with the solvents ethanol, ethyl acetate, chloroform and hexane separately. The respective solvent extracts were then allowed to dry at room temperature. After drying, weight of each solvent extract was noted and extractive value was determined by the following formula²⁶.

Weight of residue obtained

Extractive value (%) =-------------------------- X 100

Weight of the plant material taken

Phytochemistry:

Preliminary phytochemical screening: Ethanol, ethyl acetate, chloroform and hexane extracts of leaf, stem and fruit powders 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 methods ^9,11.

Estimation of total phenolic content: Total phenolic content was estimated by standard method ²⁷. 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 method ²⁸. Stock solution of each plant part extract was prepared by dissolving 100 mg of extract in 5ml methanol and the volume was made 10ml with methanol. Then 0.5ml of sample extract was taken in a test tube, subsequently 1.5ml 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 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) with slight modifications ²⁹. 100mg bark powder was extracted in 10ml 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 0.025M FeCl3 in 0.5M HCl and 1ml of 0.05M 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 510nm 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 tannin content: Method of Afify et al., (2012) with slight modification was employed ³⁰. 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. One 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 gallic acid (0.1mg/mL) and expressed as mg of GAE/g.

RESULTS:

Foliar micromorphology: General description along with measurements of the epidermal cells, stomata, trichomes are given below.

Epidermis- Cells are irregular in shape in both upper and lower surfaces of the leaf. Cell walls are sinuous in outline on the upper surface and it is wavy in lower surface. Size of the epidermal cells on upper surface is 224.97±1.86 μm × 72.00±3.30 μm and it is 160.14±2.19μm × 55.21±4.18μm on the lower leaf surface. Frequency of the epidermal cells is 75.33/mm² on the upper surface and it is 114.5/mm² on the lower surface. Palisade ratio is 3.97 (Fig. 2 and 3).

Stomatal complex- Leaves are amphistomatic; stomata are present on both the epidermal surfaces. Stomata are strictly of paracytic type. Size of the stomata is 90.51 μm × 50.60 μm on the upper surface and 101.25 μm × 82.02 μm on the lower surface. Stomatal frequency is 8.82±0.66/mm2 and 58.97 / mm² on the upper and lower surfaces, respectively. Stomatal index is 12.06 on upper surface and it is 37.60 on lower surface (Fig.- 2 and 3).

Trichomes- Trichomes are non-glandular, unicellular with pointed tips and present on both surfaces of the leaf. Size of the trichomes of adaxial surface is 72.17 µm × 2.17 µm and it is 69.18 µm × 1.96 μm in case of abaxial surface. Frequency of trichomes is 1.85 /mm² and 61.68 /mm² for adaxial and abaxial surfaces, respectively (Fig. 4).

Vegetative anatomy

Stem anatomy: Cross-section of the stem is almost circular in outline. The epidermis is uniseriate, cuticularised and many non-glandular unicellular trichomes are present on it. Cortex is differentiated into 3 distinct zones i.e., hypodermis is of 1-2 cell layers of collenchyma; middle cortex made of 3-4 layers of large parenchyma cells and single layer of starch sheath is made of single layer large barrel shaped cells. Just above the phloem zone, 3-4 layers of discontinuous patches of sclerenchyma cells are present. Discontinuous sclerenchymatous patches present just above the phloem layer. Vascular bundles are collateral, conjoint and open type with scanty phloem and massive xylem tissues. At the centre of the stem massive, parenchymatous, 12-14 cell layered pith is present. (Fig. 5).

Petiole anatomy: In transverse section, outline of the petiole is concavo-convex, semilunar, penta-angular with 2 distinct wings. Epidermis is single layered with many trichomes. Cuticle is thin. Beneath the epidermis 2-4 layers of hypodermis followed by 2-3 layers of sclerenchyma are present. Vascular bundle is 5 in number and situated beneath each ridge of petiole. At the centre, ground tissue is composed of large parenchyma cells with intercellular spaces (Fig-6).

Xylem elements: General description along with measurements of the xylem elements of stem has been presented below.

Vessel elements: Perforation plates of the vessel elements are simple and obliquely placed. Pits on the side wall of the elements are simple and arranged in horizontal rows. Tails are absent. Size of the vessel element is 11μm × 5.4μm and frequency is 19.2/mm² (Fig-7).

Tracheids: They are very long and with spiral side wall thickening. The diameter of tracheid is 27.64μm and frequency is 9.07/mm² (Fig -9).

Fibres: Fibres are typically libriform type with pointed ends. Septa and pits are totally absent. Size of the fibre is 32.14μm × 1.5μm and frequency is 21/mm² (Fig -8).

Histochemical study: Histochemical study has been carried out to detect various phytochemicals groups localized in different tissue zones of the stem. Different phytochemical groups like tannins, proteins, alkaloids, lignin, saponins, etc. have found localized in different tissue zones of the stem. It has also been observed that vascular bundles and cortical zone are the main active sites for synthesis of different phytochemical groups.

Organoleptic features of the powdered plant samples: The colour, odour, taste and texture of the three parts of the investigated plant have been presented in the table below.

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

Organoleptic features	Leaf	Stem	Fruit
Colour	Green	Yellowish green	Brownish
Odour	Aromatic	Aromatic	Aromatic
Taste	Acrid	Acrid	Slightly acrid and salty
Texture	Smooth	Fibrous	Powdery

Moisture content and Ash value: Moisture content, ash value of the leaf and bark powder drugs are given in tabular form (Table-2). Moisture contents of leaf, stem and fruit powder are 10.75%, 7.5% and 9%, respectively. Ash value is 14.29% for leaf, 7.30% for stem and it is 4.40% for fruit powder. In leaf, percentage of acid insoluble and water soluble ash is 2.4% and 3.2%, respectively. Percentage of acid insoluble and water soluble ash in stem is 1.2% and 1.6%, respectively. Whereas the percentage value of acid insoluble and water soluble ash in fruit is 1.02% and 1.54%, respectively.

Extractive value: Percentage yield of individual solvent extracts of the plant parts (extractive values) varies according to the nature of the solvent. It was found that extractive value of ethanolic extract is highest among the four solvent extracts for this plant. In three different parts of the investigated plant, the extractive values for polar solvents (i.e. Ethanol and ethyl acetate) were much higher than that of non polar solvents (i.e. Chloroform, hexane).

Preliminary phytochemical screening of the powdered plant samples: Phytochemical screening of different solvent extracts of leaf, stem and fruit parts of the investigated plant showed presence of different phytochemical groups in varying degrees (Table-4).

Table-2. Moisture content and ash value of different parts of the investigated plant

Powdered plant sample	Moisture content (%)	Total ash (%)	Water soluble ash (%)	Acid insoluble ash (%)
Leaf powder	10.75	14.29	3.2	2.4
Stem powder	7.5	7.30	1.6	1.2
Fruit powder	9	4.40	1.54	1.02

Table-3. Extractive value of different parts of the investigated plant

Plant parts	Extractive value (%)
Plant parts	Ethanol	Ethyl acetate	Chloroform	Hexane
Leaf	2.4	1.9	1.7	1.3
Stem	4	1.7	1	1.1
Fruit	2.3	0.9	0.7	1

Table- 4. Microchemical colour reaction tests of different solvent extracts of the investigated plant

Chemical groups

Tests

Colour change

Different parts of the plant

Leaf

Stem

Fruit

Alkaloids

Mayer’s reagent

White/ Cream ppt.

+++

Wegner’s reagent

Orange brown ppt.

+++

Dragendroff’s reagent

Orange brown ppt.

Reducing sugars

Fehling’s reagent

Brick red ppt.

Benedict’s reagent

Brick red ppt.

Steroids

Salkowaski test

Reddish-blue and green fluorescence

Anthraquinones

Bontrager’s test

Pink colour

Proteins

Lugol’s reagent

Faint yellow colour

Millon’s reagent

White ppt.

Saponins

1% Lead acetate solution

White ppt.

Amino acids

Ninhydrin reagent

Purple colour

Lignin

Phloroglucinol + HCl

Red

Tannins

10% NH₄OH solution

Yellow

10% lead acetate solution

White

5% FeCl₃ solution

Blackish-green colour

Flavonoids

Shinoda test

Magenta colour

+++

10% NaOH solution

Yellow colour

+ = Present ; - = Absent

Table 5. UV fluorescence nature of the powder samples of the investigated plant

Materials and treatment	Leaf powder		Stem powder		Fruit powder
Materials and treatment	In visible light	In UV light	In visible light	In UV light	In visible light	In UV light
Powder as such	Yellowish-green	Brownish-green	Yellowish-green	Chartreuse	Brownish	Maroon
Paper stretches with powder	Green	Lemon	Greyish pale green	Chartreuse	Brownish	Maroon
Treated with 1N NaOH	Brown	Dark green	Brown	Maroon	Light green	Dark green
Treated with 1N HCl	Lemon	Maroon	Olive	Blackish green	Brown	Coffee
Treated with 80% H₂SO₄	Coffee	Chocolate	Coffee	Blackish green	Blackish green	Dark green
Treated with Antimony trichloride	Cream	Violet	Olive	Turquoise	Lemon	Rose red
Treated with 50% HNO₃	Orange	Dark maroon	Orange	Dark brown	Orange	Maroon
Treated with 5% KOH	Brown	Dark green	Brown	Lemon	Lemon	Maroon
Treated with Methanol	Olive	Ruby	Olive	Ruby	Olive	Sky blue
Ethanol	Lemon	Rose red	Lemon	Rose red	Olive	Violet
Treated with Acetone	Lemon	Magenta	Lemon	Magenta	Lemon	Purple

Table- 6. Phytochemical profiles of different parts of the investigated plant

Plant parts	Total phenolics (mg of GAE /g)	Total flavonoids (mg of CE/g)	Total tannins (mg of GAE /g)	Total alkaloids (mg of PE/g)
Leaf	176.92	31.98	51.71	5.86
Stem	194.57	41.00	43.21	5.11
Fruit	142.57	9.78	63.81	5.043

DISCUSSION:

Present investigation reveals some of the characters obtained from the pharmacognostic, physicochemical and phytochemical studies, are found very distinct and they can be used as marker for the identification of the herb Cajanus scarabaeoides (L.) Thouars in its fresh as well as dried from. The information generated in this study will enrich the data base of Indian Pharmacopoeia by incorporating the pharmacognostic information of this ethnomedicinally important twining herb. Foliar micromorphology does have immense importance in plant identification and also in authentication of leaf drugs^31,32,33. Epidermal cell shape and size are sometimes found as very distinctive ones which help in proper identification of specific plants^{34, 35, 36, 37}. Here in this study, it has been observed that epidermal cells are irregular in shape but cell wall outline of the upper epidermis is sinuous and it is wavy for the cells of lower epidermis. Studies of stomata can have a great taxonomic as well as pharmacognostic value in proper identification of different plant taxa including medicinal plants. Here, strictly paracytic type of stomata is found in both the surfaces of the leaf. Stomatal frequency of a species may vary with different environmental factors, but the stomatal index is considered as distinct character for identification of the plant species. In this investigation, stomatal indexes of Cajanus scarabaeoides are 12.06 and 37.60 for upper and lower surfaces, respectively which is a marker character to this plant. Stomatal size is also considered in many cases as distinct features for identification of the plant species. Here, stomatal size is 90.51±3.00 μm × 50.60±2.51 μm on the upper surface and 101.25±3.30 μm × 82.02±5.17 μm in the lower surface of the leaf. The palisade ratio is considered as marker and commonly used in identification of leaf drugs. Here palisade ratio is 8.2 which is also a specific character for this particular species. Trichome features are also very important in proper identification of the plants and considered as one of the valuable taxonomic marker now^{37,38,39,40,41}. Epidermal trichomes of the investigated plant are unicellular, horn shaped and non-glandular type, present on both the surfaces of the leaf epidermis. Size of the adaxial surface trichomes is 72.17±2.18 × 2.17±3.04 µm and it is 69.18±1.77 × 1.96±5.79 μm in case of abaxial surface. In Pharmacognosy, physicochemical characters help in setting standard for a crude drug and are successfully employed in detection of adulterants and improper handling of the crude drug ²⁰. 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 drugs. 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 (10.75%), stem (7.5%) and fruit parts (9%) 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 also. It is considered as an indicator for mineral constituents of the crude drugs or medicinal plants. In this medicinal herb, the total ash content was found greater in leaf (14.29%) than stem (7.30%) and fruit (4.40%) which indicates that leaf part consists of more amount of inorganic minerals like carbonate, oxalate, phosphate including silica and siliceous earthy matters²⁰. Interestingly, a marked difference between values of acid insoluble ash of leaf (2.4%), stem (1.2%) and fruit (1.02%) was observed. It is possibly due to presence of greater amount of siliceous matter and metallic salts in leaf tissues than that of other two parts of this herb⁴². The water soluble ash content is estimated by measuring the amount of ash soluble in water which includes mostly the phosphate salts and some oxalate and carbonate salts. In leaf part, water soluble ash value is 3.2% that is slightly higher than the stem (1.6%) and fruit parts (1.54%). Values for various parameters of ash observed in all the three parts of this plant are different and distinct from one another which can be used as identifying marker in authentication of the crude drugs obtained from the C. scrabaeoides and for quality control of it.

It is now well established fact that phytochemicals present in the medicinal plants play a very important role in curing the diseases and those bioactive phytochemicals provide the lead molecules from which novel drugs are developed. Chemical analysis and biological assay are considered as very important aspects in pharmacognostic evaluation of medicinal plants ^24,43,44. Preliminary phytochemical screening is useful in prediction of the nature of crude drugs and also valuable for detection of phytoconstituents present in it. The important phytochemical groups detected from the leaf, stem and fruit parts of the investigated plant were alkaloids, anthraquinones,phenolics, saponins, tannins, glycosides, etc. Presence of such important phytochemical groups in this medicinal herb clearly indicates its therapeutic properties and also validates to some extant its wide range of ethnomedicinal uses^13-16.

Extractive value is considered as one of the diagnostic features and is used in proper identification of the crude drugs. It also plays an important role to understand the specific solvent where large number and greater quantities of phytochemicals will be extracted out from the crude drugs ^3,20,45. Values of the extractable matters vary according to the polarity of solvent and purity of the crude drug. Here in this study, ethanol was found to be the best extractive solvent among the four solvents used as it extracted out highest yield of the chemical constituents from all the three parts of selected plant species. Stem part showed maximum ethanol extractive value (4%) which indicates that ethanol soluble active constituents in stem (such as phenolics, alkaloids, steroids, plant acids, mucilage, glycosides, inorganic compounds, etc.) are present in greater amount than other two parts. 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 (phytosterols, fixed oils, fats, waxes, etc.) have been leached out from the three parts of the plant. So, it can be concluded that alcoholic solvent is the best option among the solvents taken here for extraction of maximum number of phytochemicals and ethanolic extractive values can be used as marker for identification of the crude samples of all the three parts of this medicinal plant .

The fluorescence analysis of the drug powder is also used as a finger print for proper identification of crude drugs when other physical and chemical parameters of the crude drugs felt inadequate ²⁰. Fluorescence phenomenon exhibited by plant powder is primarily due to its chemical composition. The same material treated with various chemical reagents appears with different colours in different wavelength of light. Here, methanol, ethanol and acetone treated plant powders showed characteristic colour change when illuminated under UV light which is quite distinct from its colour observed under visible light. That marked changes in colours under UV light provide very distinct character which is specific to the crude drugs of this plant.

DIAGNOSTIC CHARACTERS OF THE INVESTIGATED PLANT:

i. Foliar epidermal cells are irregular, sinuous cell wall on the upper surface and irregular, wavy cell wall on the lower surface.

ii. Stomata are of strictly paracytic type; stomatal index- 12.06 (upper surface) and 37.60 (lower surface).

iii. Non-glandular, unicellular trichomes are present on both surfaces of the leaf.

iv. In leaf part: Moisture content-10.75% , total ash content- 14.29%, water soluble ash- 3.2% and acid insoluble ash- 2.4%.

v. In stem part: Moisture content-7.5%, total ash content- 7.30%, water soluble ash- 1.6% and acid insoluble ash- 1.2%.

vi. In fruit part: Moisture content-9%, total ash content- 4.40%, water soluble ash- 1.54% and acid insoluble ash- 1.02%.

vi. UV fluorescence character

Leaf part and fruit part - methanol treated drug powder gives olive green colour in visible light and it fluoresces orange colour under UV light.

Stem part- methanol treated drug powder appears pinkish brown colour in visible light and it shows fluorescent blue colour in UV light.

CONCLUSION:

The diagnostic characters generated from this pharmacognostic study will be useful in proper identification of the crude drugs obtained from different parts of C. scarabaeoides and they will also be helpful in quality assurance of it. The microchemical colour reaction tests conducted on leaves, stem and fruit extracts of C. scarabaeoides revealed the presence of pharmacologically important classes of phytochemicals like, alkaloids, flavonoids, terpenoids, anthraquinones, tannins, glycosides, phenolics and saponins. Presence of such phytochemicals in this medicinal herb clearly indicates its therapeutic properties and also validates to some extant its wide range of ethnomedicinal uses. From the quantitative phytochemicalanlysis, it is found that leaf and stem parts are quite rich in phenolics as well as flavonoids, the phytochemical groups which have a wide range of pharmacological activities. It again proves that leaf and stem parts of this medicinal plant are therapeutically very potent. So, further scientific investigation of these two parts of the Cajanus scarabaeoides has to be carried out to standardize the potent bioactive compounds through various bioassay protocols.

ACKNOWLEDGEMENTS:

We are thankful to the Head, Department of Botany, Visva-Bharati, for providing the necessary laboratory facilities. We are also thankful to the UGC for financial assistance sanctioned in the form DRS-SAP Phase II research programme.

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Received on 23.12.2017 Modified on 11.01.2018

Res. J. Pharmacognosy and Phytochem. 2018; 10(1): 120-131.

DOI: 10.5958/0975-4385.2018.00018.3