pharmacognostic evaluation of leaf and fruit of capsicum ... evaluation... · (resemblance with...

14 Phcog J | May–June 2014 | Vol 6 | Issue 3

O R I G I N A L A R T I C L EP H C O G J

ABSTRACT

Introduction: Capsicum frutescens is a well known spice. Leaves and fruits of the species are used in Ayurveda, Unani and Traditional system of medicines to cure various disorders. Therefore the study was aimed to investigate pharmacognostic parameters of C. frutescens leaf and fruit. Methods: Pharmacognostic studies were carried out in terms of morphological, microscopic characters and physicochemical parameters of C. frutescens samples using standard methods. Results: Smaller fruit size and color of C. frutescens was the distinguishing morphological character observed in the present study. The detailed microscopy of leaf confirmed the presence of rod shaped calcium oxalate crystals, oleoresin cells, pitted parenchyma and fruits with specified oleoresin, sclereid and stone cells with unicellular trichomes on persistent calyx. Physicochemical parameters like ash values, extractive values and nutritive values were determined. Fluorescence analysis of both leaf and fruit powder was determined using organic and inorganic solvents. Preliminary phytochemical screening showed the presence of alkaloids, glycosides, steroids, carbohydrates and proteins. Conclusion: Observed pharmacognostic characters in this study may help in identification and standardization of C. frutescens leaf and fruit.

Keywords: African chili, Powder microscopy, Physico-chemical analysis, Thin layer chromatography.

Pharmacognostic Evaluation of Leaf and Fruit of Capsicum frutescens (Solanaceae)

Shruti V. Hegde*1, Ganesh R. Hegde1, Gangadhar S. Mulgund1 and Vinayak Upadhya2

1Post graduate studies in Botany, Karnatak University, Dharwad- 580 003, Karnataka, India 2Regional Medical Research Centre, Indian Council of Medical Research, Nehru Nagar,

Belgaum – 590 010, Karnataka, India

*Corresponding author.

Shruti Hegde Post graduate studies in Botany, Karnatak University, Dharwad- 580 003, Karnataka, India.

E-mail: [email protected]

DOI: 10.5530/pj.2014.3.3

INTRODUCTION

Genus Capsicum (family Solanaceae) consists of sev-eral wild and cultivated species. Cultivated species viz. C. annum, C. frutescens, C. baccatum, C. chinenses and C. pubes-cens proved their importance as vegetables and spices in our daily diet. Among cultivated species Capsicum frutescence L. commonly called as “African chili” has the importance because of its medicinal properties. It is a perennial shrubby plant, native to tropical America and cultivated universally in all the warmer parts of the globe.[1]

Capsicum frutescence is locally known as “Lavanga Menasu (resemblance with clove bud), Sooji Menasu (pungent

nature), Melmukha Menasu (growing pattern of fruit)” in Kannada language because of small sized fruit nature. The leaves and fruits are used in Ayurveda, Unani and folk medicines to treat various disabilities. Leaves are used to treat headache[2] whereas fruits in native medici-nal practice used to treat typhus, intermittent fever, dropsy, gout, dyspepsia, cholera and stomachache.[3] It is given as local stimulant for the tonsils in tonsillitis which also has a powerful action on the mucous membrane, hoarseness and putrid throat.[4] Antifungal[5] and insecti-cidal[6] activities proved the claims of traditional medicine. Capsaicinoids are the major chemical entities responsible for the pungency in C. frutescens. This compound acts as a local anesthetic and pain reliever. As a rubefacient, the compound increases blood flow and helps to stimulate circulation in rheumatic and arthritic conditions.[4,7] Cap-saicinoid also helps to prevent infection and is excellent remedy for sore throat, laryngitis.[4,7]

Each plant drug possesses unique properties in terms of its botany, chemical constituents and therapeutic potency. So it is important to study pharmacognostic charac-ters of each medicinal plant considering potentiality to

Shruti V. Hegde, et al.: Pharmacognostic Evaluation of Leaf and Fruit of Capsicum frutescens (Solanaceae)

Phcog J | May–June 2014 | Vol 6 | Issue 3 15

differentiate the genuine or novel plant sample. Hence C. frutescence was studied using known pharmacognostic techniques for its standardization.

MATERIALS AND METHODS

Plant material

Fruits and leaves of C. frutescence were collected from areca garden of Siddapur taluka, Uttara Kannada District, Kar-nataka State. The plant was authentically identified using Flora of Presidency of Bombay.[1] Herbarium specimen (Voucher specimen number: KUD/BOT/2012/01) was prepared and deposited in Department of Botany, Kar-natak University, Dharwad, with powder samples (KUD/BOT/2012/09) for future reference study.

The collected fruits and leaves were cleaned and shade dried. Fresh samples were used for anatomical studies and dried parts were powdered, sieved and stored in an air-tight container for further use.

Macroscopic and Microscopic analysis

Key morphological features were observed for easy identification. Macroscopic studies were carried out by using dissecting microscope (AJAY® OPTIK INDI: AJ-2. CM/L-9018771). Histochemial and powder stud-ies were carried out by using reagents and stains like iodine, potassium iodide, ferric chloride, sudan III, ruthenium red, concentrated HCl and phloroglucinol with Con. HCl (1:1).[8,9] Safranin (4%) and toludine blue were used to double stain the transverse sections.[8,10-11] All the reagents of analytical grade were procured from Hi-Media, Mumbai, India. Organoleptic characters like colour, odor, taste were determined for fruit and leaf powder.[8]

Quantitative microscopy

The quantitative examinations such as vein islet number, vein termination number, stomatal index and palisade ratio were studied as per standard methods.[8]

Photo documentation

Photomicrographs of free hand sections and powder microscopy were taken using compound binocular micro-scope at different magnifications (Carl Zeiss Axio Imager M2 model) with inbuilt analogue camera (ProgRess C5- JENOPTIK). Computer images were captured using software ProgRes® CapturePro 2.8- JENOPTIK optical system.

Physico-chemical analysis

Physico-chemical parameters of the powdered drug such as total ash, water-soluble ash, acid-insoluble ash and sul-phated ash were determined. Extractive value and soluble extractive values were determined as per standard proce-dure. The moisture content was detected by loss on dry-ing method.[12] Nutritive contents (ash, moisture, fat, fiber, protein and carbohydrate) of fruit were determined.[13]

Calculation of % carbohydrate and nutritive value

Percentage of carbohydrate was calculated by the follow-ing formula:

% carbohydrates = 100 - (Percentage of ash + percentage Moisture + percentage fat + percentage protein)

Nutritive value was finally determined by:

Nutritive value = 4 × percentage of protein + 9 × per-centage of fat + 4 × percentage carbohydrate

Fluorescence analysis

The powdered sample materials of leaf and fruit powder were analyzed under visible light, short ultra-violet light (254nm) and long ultra-violet light (365nm) after treating with various organic, inorganic solvents and reagents.[14]

Preparation of extracts and preliminary phyto-chemical analysis

The powdered material was serially extracted by cold extraction method15 using petroleum ether, chloroform, ethanol, methanol and water. These extracts were sub-jected for preliminary phytochemical screening.[8,16]

Thin layer chromatography

Thin layer chromatography for C. frutescence was carried out with aluminium plate (5 × 9cm) pre-coated with silica gel 60/UV 254, plate no.25425A-P00 (S. D. fine). The plates were developed using methanolic extract and Rf was calculated. Different combinations of solvent system were tried for best separation of constituents.[17]

Data analysis

Cell size is represented as RDS (radius), DST (distance) and PRM (prism) in leaf and fruit sections. Standard deviation is calculated as mean of three replicates for leaf constants and physico-chemical parameters using SPSS version 16.0, statistical package. Data is represented in tables.



RESULTS

Morphological description

Capsicum frutescens (Figure 1a and b) is 120–135cm high perennial plant with angular branches. Leaves simple, light green, broadly ovate to ovate-lanceolate usually wrinkled, more or less pubescent and vary in size (~10cm x ~ 6cm), pedicels slender, usually 2 or more together, 1–2 inches long. Calyx persistent, embracing the base of the fruit. Fruit a berry, orange to red ( ~ 0.5cm × ~5cm) green when young, ovoid, obtuse sometimes oblong and acuminate (Figure 1c). Seeds creamy yellow, ~ 2 mm and lenticular.

Anatomical description

Leaf

Leaf transverse section (Figure 2a) shows single layered, thick walled epidermal cells (RDS- 1.88-3.73µm) cov-ered by thin cuticle (thickness of DST- 0.69-1.99µm). The epidermal cells have a rectangular shape with slight convex outer margin. Next to epidermis is 4-5 layers of collenchyma (RDS- 2.82-4.20-6.01µm) followed by broad parenchymatous ground tissues. Ground tissue cells are isodimetric/rounded (RDS-3.06- 6.14- 8.832µm) with small intercellular spaces of which some are pitted cells

(DST- 2.19- 2.80µm) and some are filled with starch grains (Figure 2d, RDS- 1.37- 1.59µm), olioresins or oilglobules. The vascular bundles are exarch, conjoint, collateral, closed type enclosed by sclerenchymatous sheath scat-tered within ground tissue; phloem (RDS- 0.89- 1.02µm) consists of companion cells and sieve tubes; xylem (RDS- 2.06- 3.03µm) consists of vessels, tracheids, fibres and xylem parenchyma.

Powder microscopy shows prismatic crystals (Figure 2e), uniseriate, multicellular, septate trichome (Figure 2f), epider-mal cells, pitted parenchyma (Figure 2b) and cells contain-ing oil and olioresin (Figure 2c). Fibres with plane margin, narrow lumen with tapering end were also observed.

Leaf constant

Venation is reticulate with 6 to 8 pairs of alternate lateral veins. Vein islet number is 19.66 ± 0.33 and vein termi-nation number is 20.33 ± 0.57 (Figure 2g). Stomatas are sorrounded by two to four subsidiary cells falling in to Anomocytic type. The stomatal index is 23.7% (Figure 2h) and palicide ratio is 1:6.

Fruit

Fruits are berries. Unicellular trichomes (Figure 3I) have been observed on persistent calyx. Fruit contains 7–9

Figure 1. C. frutescens a and b: Habit; c: Fruits.



seeds embedded in fleshy-solid mass with a free space between seeds and pericarp. Pericarp consists of exocarp, mesocarp and endocarp (Figure 3c); exocarp is single layered with smooth epidermis (RDS- 13.06- 13.16µm) and dense cellulose [(thickness of DST- 1.099µm) (Figure 3b)]; mesocarp and endocarp consists of hypoder-mal collenchyma [circular (RDS- 18.4- 25.20- 33.01µm)]

and parenchyma cells [polygonal (PRM- 67.24- 97.46- 145µm) (Figure 3d)] with markedly wavy zone of sclereid (DST- 61.32- 62.86 × 24.11- 42.15µm). Seeds are albu-minous with creamy white endosperm and embryo. Tegu-ment consists of long, flat and rectangular epidermal cells (DST-61.32 × 39.19 µm) followed by endosperm made up of polygonal cell (PRM- 46.78- 56.63µm) with numerous

Figure 2. a: Transverse section of leaf b: Pitted parenchyma cell c: Section showing oleoresin cells d: Cell containing starch grain e: Calcium oxalate crystals f: Trichome g: Venation pattern h: Epidermal peel showing anomocytic stomata.MS: Mesophyll cell; TRI- Trichome; EPI- Epidermis; CU- Cuticle; ORC- Olioresin cell; X- Xylem; P- Phloem; CAC- Calcium oxa-late crystals; PC- Parenchyma cell; PPC- Pitted parenchyma cell; SG- Starch grain; VI- Vein islet; VT- Vein termination; S- Stomata



starch grains (RDS- 1.229- 1.561µm) and inner embry-onic cell (Figure 3a,3e, 3f, 3g).

Powder study shows anisocytic type of stomata in pedi-cel, epidermal cells, fibers and stone cells.

Organoleptic characters

Leaf powder has dark green color with slightly pungent odour and slightly bitter in taste. Ripen fruits powder shows orange to red color with characteristic pungent

Figure 3. a: Peel of pedicel showing anisocytic stomata; b: Epicarp showing epidermal parenchyma cells in surface view; c: T. S. of fruit; d: Section of epicarp and mesocarp; e: Markedly wavy cells of mesocarp; f, h - Sclerenchyma of mesocarp; g: section of seed, I: Unicellular trichome. S- Stomata; EPI- Epidermis; PC- Parenchyma cell; CO- Collenchyma; CU- Cuticle, SC- Sclerenchyma cell; TE- Tegma; ENS- En-dosperm; EM- Embryo



Table 1. Histochemical test.

Section treated with Reagents Test for Colour change ResultLeaf Fruit Leaf Fruit

Iodine Starch Blue Blue Present Present

Ferric chloride solution (10%) Tannin --------- --------- Absent Absent

Con. Sulphuric acid Cellulose Green Green Present Present

Con. HCl + phloroglucinol Lignin Magenta Magenta Present Present

Con. HCl Calcium oxalate crystals Effervescence Effervescence Present Present

odour and taste. Powder has strong sternutatory property and causes irritation to skin and mucous membrane.

Histochemical analysis

Leaf and fruit sections were treated with different reagents to know various cell components. The results are presented in Table 1.

Physicochemical parameters

Physicochemical characters such as ash value, solubility test, nutritive contents and extractive values in petroleum ether, chloroform, ethanol and water were calculated (Table 2). C. frutescens fruit has shown higher ash values than that of leaf (Table 2). Quantitative estimation of extractive values is represented as percent yield. Extrac-tive percent yield for leaves was higher in ethanol (18.0 ± 0.05% w/w) whereas for fruit it was in aqueous extract (20.5 ± 0.03% w/w). Petrolium ether showed lowest per-cent yield for both leaf (4.05 ± 0.07% w/w) and fruit (4.55 ± 0.02% w/w). Solubility values were higher for leaf and fruit in ethanol (19.9 ± 0.05% w/w) and water (24.4 ± 0.01% w/w) respectively.

The results indicate higher moisture, fat and fiber content in leaf than in fruit. However protein content of fruit is 3.5 times higher than that of leaf. But it is notable that carbohydrate content is almost equal in both leaf and fruit. The nutritive value of fruits (298.65 cal/100g) was higher than the leaf sample (274.20 cal/100g).

Fluorescence analysis

Fluorescence analysis of leaf and fruit powder of C. frutes-cence was carried out after treating with several chemicals. Fluorescence were observed at 254 and 365nm compar-ing its change of colour in visible light. The observations are presented in Table 3 showing variation in colour.

Phytochemical analysis

Successive Soxhlet extraction was performed using dif-ferent solvents. The extract showed sticky nature for all

solvents used and the colour varied from dark brown to yellowish brown for stem; however leaf extracts has dark green colour. Respective extracts were subjected to pre-liminary phytochemical screening and the results (Table 4) certified the presence of alkaloids, glycosides, steroids, carbohydrates, proteins and saponins in leaves where as excluding saponin, all other phytochemical constituents were present in fruit.

Chromatographic Study

Thin layer chromatography of C. frutescens (leaf and fruit) methnolic extracts was carried out by using various sol-vent systems where hexane: acetone in the ratio 8:3 and hexane: ethyl acetate in the ratio 5:5 respectively for fruit and leaf proved best for the separation of phytochemi-cals. Results revealed the presence of several constituents in the extracts. Retention factor (Rf) were summarized in Table 5.

DISCUSSION

Pharmacognostical and physicochemical parameter study is reliable, accurate and inexpensive for the purpose of quality control, assessment of purity and identification of crude drug samples.[18] Observed morphology and mac-roscopic characters of C. frutescens leaf and fruit act as consistent source for detecting a genuine sample. Mor-phologically, smaller sized fruit and seed of C. frutescens are useful in differentiating it from C. annum, C. baccatum, C. chinensis [19] and C. assamicum a newly described species from Assam.[20] C. annum and C. minimum showed calcium oxalate crystals in the mesocarp region[8] whereas it was absent in C. frutescens. Microscopic study of C. frutescens leaf confirmed the presence of various types of cells. The leaf is classified into amphistomatic leaves common in Solanaceae, because of presence of anomocytic stomata on both leaf surfaces.[21] Pandey et al. observed similar type of stomata in C. annum leaves.[22] Unicellular trichome on persistent calyx of C. frutescens is a distinguishing char-acter to differentiate it from C. minimum with fruits show-ing glandular trichomes.[23] Total ash, acid insoluble ash,



Table 2. Physicochemical observations of leaves and fruits.

Parameter Leaf Fruit

1. Ash Value (% w/w):

a. Total ash 6.45 ± 0.028 17.0 ± 0.057

b. Acid insoluble ash 0.49 ± 0.023 0.50 ± 0.003

c. Water soluble ash 2.60 ± 0.057 9.53 ± 0.033

d. Sulphated ash 9.03 ± 0.035 26.0 ± 0.033

2. Extractive value (% w/w):

a. Pet. Ether 4.05 ± 0.07 4.55 ± 0.02

b. Chloroform 5.60 ± 0.05 4.78 ± 0.07

c. Ethanol 18.0 ± 0.05 16.3 ± 0.04

d. Water 15.9 ± 0.03 20.5 ± 0.03

3. Solubility Test (% w/w):

a. Ethanol 19.9 ± 0.05 18.8 ± 0.06

b. Water 18.8 ± 0.03 24.4 ± 0.01

4. Nutritive content (%)

a. Ash 6.45 ± 0.028 17.0 ± 0.057

b. Moisture 26.90 ± 0.010 9.50 ± 0.100

c. Fat 1.52 ± 0.057 0.93 ± 0.088

d. Fiber 2.68 ± 0.010 1.70 ± 0.0053

e. Protein 2.68 ± 0.00 9.60 ± 0.100

f. Carbohydrate 62.45 62.97

5. Nutritive value cal per 100 g powder

274.20 298.65

Table 3. Ultra- violet powder analysis of leaf and fruit of C. frutescence at 245 and 365 nm.

Treatment Visible light U. V. light at 254nm U.V. Light 365nmLeaf Fruit Leaf Fruit Leaf Fruit

P + NaOH (Aqueous) Apple green Omaha orange Ceylon green Ceylon green Apple green Ceylon green

P + NaOH (Alcoholic) Dark apple green Omaha orange Ceylon green Ceylon green Apple green Ceylon green

P + 1N HCl Tan Omaha orange Cream Tan Olive Ceylon green

P + 50% H2SO4 Adiron dark green Black Adiron dark green Black Black Black

P + 50% HNO3 Yellow Light yellow Yellow Yellow Yellow Yellow

P + Lead acetate + NaOH Olive green Omaha orange Ceylon green Ceylon green Adiron dark green Olive

P + HNO3 Yellow Yellow Yellow Yellow Yellow Yellow

P + acetic acid Dark brown Omaha orange Adiron dark green Tan Black Tan

P + FeCl3 Maroon Harvest red Adiron dark green Ceylon green Apple green Apple green

P + HNO3 + NH3 Yellow Yellow Highway yellow Yellow Yellow Yellow

P + H2SO4 Dark maroon Black Purple black Black Adiron dark green Black

P + Pet. Ether Lime green Omaha orange Ceylon green Ceylon green Ceylon light green Light olive

P + Methanol Tom sahnted green Omaha orange Ceylon green Ceylon green Ceylon green Light olive

P + Water Lime green Omaha orange Ceylon green Ceylon green Ceylon light green Light olive

P + Benzene Forest green Omaha orange Apple green Ceylon green Ceylon light green Light olive

P + Glycerin Omaha orange Omaha orange Tan Ceylon green Ceylon light green Light oliveP: Powder

water soluble ash and sulphated ash parameters indicate the amount of inorganic and silica components in the sample studied. Higher ash values in fruit represents high amount of carbonates, phosphates, silicates and silica than leaves of C. frutescens. Higher amount of extractive values in water and ethanol for fruit and leaf indicate the con-centration of secondary metabolite present in it. Mois-ture content of leaves shows considerable amount of moisture in crude drug than that of fruit. Fluorescence is the phenomenon exhibited by various chemical con-stituents present in the plant material. The ultraviolet light produces fluorescence in many natural products, which do not visibly fluoresce in day light.[24] Even some of the non fluorescent natural products may often be converted into fluorescent derivatives by applying different reagents. This nature is helpful to know the florescence character of drug. The florescence result showed colour differen-tiation in ultra violet range and visible range exhibiting presence of various chemical constituents. Preliminary phytochemical screening is helpful in knowing the chemi-cal nature of the drug. The present study tests for various phytochemical components and nutritive values revealed the presence of alkaloids, glycosides, steroids, carbohy-drates and proteins in fruit along with saponins in leaves. TLC studies showed number of banding pattern suggest-ing presence of various chemical components. The phy-tochemical parameters reported in the present study can be ascertained to various biological activities in C. frutescens [5,6]

and in turn its usage in traditional medicine.



Table 4. Qualitative analysis of Phyto-chemical constituents.

Chemical constituents Pet ether Chloroform Ethanol Methanol Aqueous Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit

Saponins + – + – + – + – + –

Tannins – – – – – – – – – –

Alkaloids + + + + + + + + + +

Terpenoids – – – – – – – – –

Triterpenoids – – – – – – – – – –

Glycosides + + + + + + + + + +

Steroids + + + + + + + + + +

Flavonoids – – – – – – – – – –

Carbohydrates + + + + + + + + + +

Protein + + + + + + + + + ++: Present; - Absent

Table 5. TLC for Methanolic extract of leaves and fruits.

Sl. No. Extracts Number of bands Rf Value1. Leaf 11 1.0, 1.02, 1.27, 1.60, 1.72,

1.91, 2.3, 2.37, 2.65, 3.0, 4.6

2. Fruit 03 1.12, 2.40, 2.5

CONCLUSION

The parameters studied can be utilized in identification of C. frutescens in crude drug form and can be used as a potential source for useful therapeutics. The resulted data will be beneficial for quantitative and qualitative standard-ization of genuine drug in herbal preparations.

ACKNOWLEDGEMENT

First author is grateful to UGC- RFSMS, New Delhi, India for their financial assistance. Authors thank P. G. Department of Studies in Botany, Karnatak University, Dharwad for the facilities provided. Authors extend their sincere thanks to USIC, Karnatak University, Dharwad for Instrumental support.

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