Pharmacognostical and Phytochemical Screening of Leaves of Raphanus sativus Linn
Thamizharasi Suresh1*, Shankari Vellaisamy2, Parvatha Raja Kumaran V S3, Chetan Ashok4
1Associate Professor, Department of Pharmacognosy, K.K. College of Pharmacy, Chennai, Tamil Nadu.
2Associate Professor, Department of Pharmacology, K.K. College of Pharmacy, Chennai, Tamil Nadu.
3Saveetha College of Pharmacy, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu.
4Department of Pharmacology, Sri Ramachandra Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu.
*Corresponding Author E-mail: mstarasi@gmail.com
ABSTRACT:
Raphanus sativus (commonly called as Radish) belonging to family Brassicaceae which is a multipurpose plant with high attributes and potential. The study is aimed to evaluate the Pharmacognostic and to reveal the phytochemical profile of Raphanus sativus which serve as authentication and quality control for the crude drug. Microscopical studies and physiochemical parameters like ash value, extractive value, loss on drying and determination of crude fiber content were observed. Preliminary phytochemical screening was performed to identify the various phytoconstituents present in the crude drug. Macroscopical and phytochemical characters such as Fluorescence analysis and Thin Layer Chromatography were studied for the presence of secondary metabolites and High Performance Thin Layer Chromatography fingerprints were studied. Microscopic analysis and phytochemical evaluation provides sufficient data for characterize the drug. Preliminary Phytochemical investigation of Raphanus sativus were performed in various extraction solvents such as Ethanol, Chloroform and methanol and revealed the presence of alkaloids, carbohydrates, flavonoids, phytosterols, Steroids, Tannins and fixed oils. The study concluded that pharmacognostical evaluation data provided will be helpful in characterization of the crude drug and also leads in the development of the pharmaceuticals and research.
KEYWORDS: Phytochemical, Raphanus sativus, HPLC, Chromatography, Extractive value, Thin Layer Chromatography.
INTRODUCTION:
India is the world's largest producer and consumer of herbal medications. In India, there are many different systems of traditional medicine that are used, with plants serving as the primary source of medication for treating and healing illnesses. The treatment of many ailments involves the use of herbal medications. Herbal resources are untapped natural treasures for both conventional and contemporary medical systems.
Because herbal medications are thought to be secure and minimally or completely harmful, the usage of plant-derived treatments has expanded.
Herbalism is a type of traditional medicine or folk medicine that relies on using plants and plant extracts to treat ailments. Botanical medicine, medical herbalism, herbal medicine, herbology, and phytotherapy are other names for herbalism. Healing herbs are important, especially in the present era, as pharmaceuticals, which are believed to have mystical and nearly supernatural abilities, constitute the foundation of medicine. Herbs are founded on the notion that plants have a wide range of therapeutic applications and that natural means safe and synthetic means harmful and hazardous. Herbal medicines might cause issues if they are prescribed with insufficient usage instructions or if the substances are contaminated, adulterated, or labeled wrongly.
Minerals, shells, certain animal parts, as well as fungi and bee products, are occasionally added to the list of things that can be used in herbal therapy1-10. Currently, herbs and spices are recommended as treatments for conditions such as cancer, diabetes, ulcers, tuberculosis, helminthiasis, and analgesia11,12.
Radish, scientifically known as Raphanus sativus L., is a plant that belongs to the genus Raphanus and is a member of the Rapa/Oleacera lineage, according to phylogenetic analysis of the Brassicaceae family. Radish is categorised as a root because it includes specialised structures (hypocotyls) that resemble real roots and the capacity to store starch and other materials. Radishes can have a variety of surface colours, including white in Asia, red in Europe, purple green, and even black. Along with having few calories, radish is a good source of calcium, magnesium, copper, manganese, potassium, vitamin B6, vitamin C, and folate. Radish extracts have been used to treat a number of organ issues13.
Due to its great nutritional content, the tap root of radishes has been consumed all across the world in pickles, salads, and curries. In addition to the roots, the importance of leaves and sprouts for nutrition and medicine has also been highlighted. Since ancient times, radishes' extracts have been used in folk medicine to treat ulcers, hepatic inflammation, urinary infections, constipation, stomach issues, and constipation. Radishes also possess antibacterial, anticancer, antioxidant, and anxiety-reducing qualities, according to numerous investigations12. Researchers and the pharmaceutical sector are now interested in the potential use of radish as a source of bioactive chemicals with clinical and health implications in diseases like hypertension, cardio-metabolic disorders, and as an antibacterial and antioxidant agent14.
MATERIALS AND METHODS:
Collection and Authentication of Plant Specimen:
The leaves of Raphanus sativus were collected from T. Nagar, Chennai. The identification of the leaves was done at Plant Anatomy Research Centre, Tambaram by Prof. Dr. P Jayaraman. An authentic herbarium specimen was deposited in the institution (Sample Reg. No. PARC/2010/598).
Microscopical Studies:
Fixing:
The pre-washed fresh leaves were fixed in FAA (fonnalin-5ml+ acetic acid-5ml + 76% ethyl alcohol-90ml). After 24 hours of fixing, the specimens were dehydrated with graded series of tertiary butyl alcohol as per the schedule given by Sass, 194013. Identification of the specimens was carried by gradual addition of paraffin wax (m.p58-60°C) until tertiary butyl alcohol solution attained super saturation. The specimens were casted into paraffin wax16.
Sectioning:
The paraffin embedded specimens were sectioned with the help of rotary microtome. The thickness of the section was 10-12µm. The sections were de-waxed by customary procedure and stained with toluidine blue as per O’Brien’s method17,18. Since the toluidine blue is a polychromatic stain, the staining results were good and some cytochemical reactions were obtained. The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein’s bodies etc, where ever necessary sections were also stained with safranin and fast green and potassium iodide for starch. Powdered materials of different parts were cleared with NaOH and mounted in Glycerine medium after staining. Different cell components such as stomata, venation pattern, trichome distribution, epidermal peeling were observed, measured and pictured19.
Histochemical studies:
This technique is mostly used for qualitative evaluation for organized crude drugs in entire forms. Microscopic evaluation also covers the study of the constituents by application of chemical method to small quantities of drugs in histological section of the drug20-23.
Physiochemical standards:
Ash value:
The residue remaining after incinerating is the ash content of the drug which represented as inorganic salt naturally occurring in drug or adhering to it or deliberately added to it as a form of adulteration. It is the parameter for the identity and purity of crude drug. Total ash value, acid-insoluble ash value and water-soluble ash value were determined24.
Extractive value:
The extracts obtained by exhausting crude drug are the indication of approximate measure of chemical constituent. This was carried out by determining the water-soluble extractive value and alcohol-soluble extractive value of the powdered drug25.
Loss on drying:
About 1.5mg of the powdered drug was transferred to glass stopper weighing bottle which is previously dried for 30mins in the drier. Then the sample was gently shaken side wise for even distribution and dried in an oven at 100°C to 105°C by removing the stopper. It was cooled in a desiccator and again weighed. The loss on drying was calculated with reference to the amount of air-dried powder taken24.
Determination of crude fiber content:
2g of the powdered drug was taken into a beaker containing 50ml of 10% v/v nitric acid and boiled the whole mixture for about 30sec, later it was strained through fine cotton cloth on a Buchner funnel and washed the residue with boiling water until it is acid free. Rinse the residue back to the flask and added 50ml of boiling 2.5% sodium hydroxide solution and repeated boiling under reflux for 30sec. After filtering, the residue was washed with boiling water until neutral. Dried at 110°C to constant weight and incinerate to constant weight. The weight of the crude drug will be represented by the difference between the weight of the residue and weight of the incinerated residue18,19,22,26-29. It will be expressed as a percentage of the original weight of the material. The crude fiber was calculated on a percent-dry basis as described below.
Dry residue (gm) - Ignited residue (gm)
% Crude fiber (dry basis) = –––––––––––––––––––––––––––––– ×100
Sample (gm)
Phytochemical Studies:
The leaves were dried under the shade and powdered. Extraction was done by chloroform, ethyl acetate and methanol using Soxhlet apparatus.
Preliminary phytochemical screening:
The herbal extract was subjected to preliminary phytochemical screening for the qualitative analysis of active phytochemical constituents like carbohydrates, glycosides, fixed oils and fats, proteins and free amino acids, saponins, Phenolic compounds and tannins, phytosterols, alkaloids, flavonoids, mucilage and gums, waxes, starch22,26,18-33.
Fluorescence Analysis:
The powdered drug was treated with different solvents like 1N Sodium hydroxide aqueous, 1N sodium hydroxide (alcoholic), 1N hydrochloric acid, 50% nitric acid, 50% sulphuric acid, picric acid, acetic acid, nitric acid with ammonia. These solutions were finally subjected to fluorescence analysis in visible and UV light 254nm34-37.
Thin Layer Chromatography:
The TLC plates were prepared by silica gel-G or Silica gel-GF. The slurry of silica gel was evenly distributed over the TLC plate. The samples were spotted on the TLC plate and were placed vertically in tank containing mobile phase. The opening of the tank was covered to allow complete saturation of the inner environment. The solvent rises by capillary action, resolving the same mixture in to discrete spots. The movements of the active compounds were expressed by the retention factor (Rf). The spots were detected with the help of UV light at 254 nm (lower wave length) and 366 nm (higher wave length). The Rf values of individual spots were determined38.
High Performance Thin Layer Chromatography:
Samples were loaded as 8 mm band length in the 10 x 10 Silica gel 60F TLC plate using CAMAG LINOMAT 5 instrument with a 100µl Hamilton syringe and run-in developing chambers saturated with the respective mobile phase upto a solvent front of 80mm. The plates were visualized in a photo documentation chamber CAMAG visualizer: 150503. Images were captured (at 254nm and white light after derivatization) and the plates scanned to display the Peak Table and Peak densitogram39-44.
RESULTS:
Microscopic Studies:
Anatomy of the leaf:
The leaf is distinctly dorsi-ventral with prominent veins. The lamina is about 120µm thick. The epidermal layers are narrow and thin walled.
Lateral veins:
The lateral veins are fairly prominent having short conical adaxial part and wide short abaxial semicircular path. The lateral veins Is about 500µm thick, as in figure 1.
Figure 1. Transverse section of Lamina
Midrib:
Midrib is about 2.6mm wide (horizontal plan) and 1.8mm (vertical plan). It is semicircular in abaxial and more or less flat adaxial part. The ground tissue is parenchymatous. The cells are large lobed and thin walled. The vascular system is 600µm wide and 250µm thick.
Figure 2. Transverse section of Midrib
In figure 2, the lateral stands are smaller and circular. It has several solitary diffusely distributed xylem elements that are circular and thick walled. The sclerenchyma elements occur in the form of an arc, abutting the phloem, the lateral strands are 250-350µm in diameter, Figure 3.
Figure 3. T.S. of Midrib – Lateral Vascular Bundles
Transverse section of petiole:
The petiole is circular with 2 slender adaxial wings. It possesses a thin epidermal layer, parenchymatous ground tissue and a multi-stranded vascular system. The epidermis is thin and continuous comprising squarish cells and prominent cuticle. The vascular system includes about 10 vascular bundles that are organized in deep bowl-shaped outline of the 10 bundles, figure 3. The xylem elements are circular thick walled in parallel rows or in diffuse masses. Phloem occurs in thin arc on the outer part of the xylem strands as shown in Figure 4 and 5.
A
B
Figure 4. Transverse section of petiole – Entire
A
B
Figure 5. Transverse section of petiole-median vascular bundles
Epidermal tissue and stomata:
The epidermal cells are wide and possess thin wavy anti-clinal walls; the cells appear amoeboid in outline. The stomata are surrounded by three subsidiary cells, one of them being smallest as described in the Figure 6. The stomata are fairly large and elliptical in shape as in Figure 7.
Figure 6. Para-dermal section of the leaf
Figure 7. Anisocytic stomata enlarged
Venation pattern:
The veins are thin slender, they are wavy and undulate. The vein islets are fairly wide and irregular in shape. The terminations may be simple and straight, branched once or twice giving rise to dendroid outline as shown in figure 8.
Figure 8. Venation pattern
Figure 9. Myrosine glands
Myrosine glands:
The myrosine producing glands are wide spread in all members of Cruciferae which is pictured in figure 9. Figure 10 show powder microscopy of vessel elements, fibres and parenchyma.
Figure 10. Powder microscopy of vessel elements, fibres and parenchyma
Phytochemical studies:
Histochemical analysis of different extracts of Raphanus sativus were observed and represented in Table 1. Qualitative microscopy of R. sativus is represented in table 2. The values for physio-chemical parameters were accessed and presented in Table 3.
Table 1. Histo-chemical analysis of leaf of R. sativus
S.No |
Reagent |
Observation |
Inference |
1 |
T.S. of leaf + Dragendorff’s reagent |
Reddish brown colour |
Presence of alkaloids |
2 |
T.S. of leaf + Picric acid |
Yellow colour |
Presence of alkaloids |
3 |
T.S. of leaf + iodine solution |
Blue colour |
Presence of starch |
4 |
T.S. of leaf + Ruthenium red |
No red colour |
Absence of mucilage |
Table 2. Quantitative microscopy of leaf of R. sativus
S.No |
Parameters |
Values (%w/w) |
1 |
Stomatal number |
15 |
2 |
Stomatal index number |
50 |
3 |
Vein islet number |
16 |
4 |
Vein termination number |
20 |
5 |
Palisade ratio |
30 |
Table 3. Physico-chemical analysis of leaf powder of R. sativus
S.No |
Parameters |
Values (% w/w) |
I |
Ash Values |
|
|
Total ash |
16.87±0.08 |
|
Acid-insoluble ash |
2.69±0.04 |
|
Water soluble ash |
5.78±0.03 |
II |
Extractive value |
|
|
Water soluble extractive |
9.5±0.09 |
|
Alcohol soluble extractive |
8.8±0.01 |
III |
Loss on drying |
11.5±0.09 |
IV |
Crude fiber content |
40.45±0.06 |
The fluorescence analysis was performed for both visible light and for UV light. The data are tabulated in the Table 4. The qualitative analysis of R. sativus for the presence of various phytoconstituents is determined and record in Table 5. Prior to performing HPTLC, Thin layer chromatography was performed and results are tabulated in the Table 6.
Table 4. Fluorescence analysis of leaf powder R. sativus
S. No |
Particulars |
Visible light |
UV light at 254nm |
1 |
Powder |
Green |
Green |
2 |
Powder + 1N aqueous Sod. Hydroxide |
Light green |
Green |
3 |
Powder + 1N alcoholic Sod. Hydroxide |
Pale green |
Light green |
4 |
Powder + 1N Hydrochloric acid |
Green |
Green |
5 |
Powder + 50%Sulphuric acid |
Dark brown |
Black |
6 |
Powder + 50% Nitric acid |
Brown |
Brown |
7 |
Powder + Ammonia |
Light green |
Green |
8 |
Powder + Iodine |
Reddish brown |
Black |
9 |
Powder + 5% Ferric chloride |
Light brown |
Brown |
10 |
Powder + Acetic acid |
Light green |
Green |
11 |
Powder + Picric acid |
Yellow |
Yellow |
Table 5. Preliminary Phytochemical studies of leaves extracts of R. sativus
S. No |
Test |
Extract |
||
Chloroform |
Ethyl acetate |
Ethanol |
||
1 |
Alkaloids |
+ |
+ |
+ |
2 |
Carbohydrates |
+ |
+ |
- |
3 |
Flavonoids |
+ |
+ |
+ |
4 |
Phytosterols |
+ |
+ |
+ |
5 |
Glycosides |
- |
- |
- |
6 |
Proteins |
- |
- |
- |
7 |
Fixed oils |
+ |
+ |
+ |
8 |
Saponins |
- |
- |
- |
9 |
Mucilage and gums |
- |
- |
- |
10 |
Tannins |
- |
- |
- |
(+) Present, (-) Absent
Table 6. Thin layer chromatography of leaves of R. sativus
S. No |
Solvent system |
Rf values |
Rf values of standard |
||
Chloro-form |
Ethyl acetate |
Ethanol |
|||
1 |
(Toluene: Ethyl acetate: Methanol: Acetic acid) (8:1:0.5:0.3) |
0.66 0.75 0.81 |
0.53 |
0.26 |
- |
2 |
(Toluene: Ethyl acetate: Methanol) (6:3.5:0.5) |
0.46 |
0.1 |
0.43 |
0.45 |
3 |
(Ethyl acetate: Methanol: water) (10:1.5:1) |
0.37 |
0.18 |
0.51 |
- |
High Performance Thin Layer Chromatography:
The HPTLC plates representing the finger printing of various leaves extracts of R. sativus and standard Quercetin at 254 run and 366 run as shown in Figure 11.
Solvent system was selected as Toluene; Ethyl acetate; and Methanol in the ratio of 6:3.5:0.5. Test extracts contained different extracts on Raphanus sativus in chloroform, ethyl acetate and ethanol respectively. Quercetin was selected as standard. The finger printing for three plant extract along with standard drug is provided in Figure 12-15 respectively.
At -254nm At.-366nm
Figure 11. HPTLC plates
Figure 12. HPTLC Finger printing of chloroform extract of leaves of R. sativus
Figure 13. HPTLC Finger printing of Ethyl acetate extract of leaves of R. sativus
Figure 14. HPTLC Finger printing of Ethanol extract of leaves of R. sativus
Figure 15. HPTLC Finger printing of Standard drug
DISCUSSION:
Transverse section of leaves of R. sativus revealed thin wavy anticlinal walls of epidermal layers. The stomata are elliptical in shape and anisocytic (cruciferous) type. Myrosine glands are frequently seen on the surface of the lamina. The vascular system has five large discrete vascular strands with thick-walled xylem elements and thin-walled phloem elements. The transverse section of petiole possesses a thin epidermal layer, parenchymatous ground tissue and multi-stranded vascular system. The vascular system includes about 10 vascular bundles.
Microscopical analysis of powdered drug shows the presence of xylem fibres, vessel elements, and wide fibres. The physicochemical properties were helpful in standardization of powdered plant material. The qualitative chemical tests are useful in detection of active constituents present and adulteration in the drug. The Phytochemical analysis revealed the presence of alkaloids, flavonoids, steroids and tannins. Tannins known for their use as treatment of cancer thus serve as a potential source of bioactive compound in the treatment of cancer. Flavonoids, alkaloids and tannins are considered to possess high antioxidant activities, which are used in the treatment of many diseases, including cancer.
In HPTLC, the Rf value of standard was found to be 0.59. Chloroform extract of leaves of R. sativus showed 9 peaks, ethyl acetate extract showed peaks, ethanol extract showed 9 peaks. The Rf values were 0.57, 0.56, 0.58 of all extracts corresponds with the standard Rf value.
CONCLUSION:
The present study deals with the determination of pharmacognostical, phytochemical of R. sativus. The pharmacognostical parameters which are identified and evaluated from this research work will be helpful to identify the drug in its crude form. The results from physiochemical screening, fluorescence analysis and phytochemical screening, TLC, HPTLC of leaves of R. sativus, will be helpful in developing pharmacopoeial standards.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGEMENT:
The authors would like to express gratitude to the Management of K.K. College of Pharmacy, Chennai for providing all the facilities for the successful completion of the research.
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Received on 31.03.2023 Modified on 14.06.2023
Accepted on 27.09.2023 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Res. 2024; 14(1):25-32.
DOI: 10.52711/2231-5691.2024.00004