Suresh Kumar1, 2*, Anjoo Kamboj3, Anil Kumar Sharma4
1Lord Shiva College of Pharmacy Sirsa, Haryana-125055 India
2Research Scholar, Department of Pharmacy, IK Gujral Punjab Technical University, Jalandhar, Punjab, India-144001
3Chandigarh College of Pharmacy, Landran, Mohali, Punjab-140110 India
4Former Director and Principal in CT Institute of Pharmaceutical Sciences, Jalandhar, Punjab-144020, India
*Corresponding author
Suresh Kumar
Lord Shiva College of Pharmacy, Sirsa, Haryana-125055, India
Abstract
Objective: Present studies deal the quality control parameters of a locally occurring Indian medicinal plant, Fumaria parviflora Lam. which is used as folk medicine in Haryana (India). Materials and methods: In this study the organoleptic, morphological, microscopical characteristics, and physicochemical evaluation (ash value, extractive value, foreign matters and moisture content), fluorescence analysis of the root, stem, leaf and fruit of Fumaria parviflora were investigated. Ethanolic extracts were prepared by extracting the ground powders of root, stem, leaf and fruit. Preliminary phytochemicals analysis of ethanolic extracts of different parts of Fumaria parviflora was carried out for qualitative analysis. Results and conclusion: Preliminary phytochemicals analysis of ethanolic extracts of root, stem, leaf and fruit revealed the presence of carbohydrates, alkaloids, tannins and flavonoids. The pharmacognostic investigation of root, stem, leaf and fruit of Fumaria parviflora would be useful for maintaining the standards for the quality, purity and sample identification.
Keywords: Fumaria parviflora, microscopy, pharmacognostic, flavonoids
Introduction
Fumaria parviflora Lam. (Fumariaceae) commonly known as “fine leaf fumitory, earth smoke”, Indian fumitory, and wax doll in English (Rizvi et al., 2017). It is a loved medicinal herb in Indian system of medicine. It is well known annual weed growing throughout in India from Indus Ganga plain to down Nilgiris in South (Karuna modi et al., 2016). The major chemical constituents reported include alkaloids like Protopine, parfumine, d-bicuculline, hydrastine, N-methlhydrastine, N-methylhydrasteine, microcarpine, sanguinarine, adlumiceine, coptisine, fumaritine, sinactine, N-methylstylopine and sterols of plant are β-sitosterol, stigmasterol,and campesterol etc (Fafal et al., 2007; Paltinean et al., 2013). Entire herb has been widely used in Ayurvedic medicine system as bitter; cooling, expectorant, constipating, increases “vata” removes biliousness, fever, burning of the body, tired feeling, wandering of the mind, intoxication, urinary discharge, vomiting, thirst, enriches the blood, good in leprosy (API, 2004).
The plant shows many biological properties such as hepatoprotective (Khan et al., 2017), antipruritic, antifeedant, Antiprotozoal, antiparasitic, anthelmintic, antidiabetic, antieczema, antioxidant, Antinociceptive, antimicrobial, prokinetic, laxative and spasmodic effect (Kumar et al., 2017).
As plant was not explored in depth but it have lot of ethanopharmacological importance. Therefore, we carried out the present studies which deal with extremely important information on micro morphological characteristics of this medicinal plant which would help in identification and authentification as well as provide basic pharmacognostic parameters. The pharmacognostic and phytochemicals analysis is key steps to develops the herbal pharmacopoeia standards and helpful in identification and quality control of the medicinal plants.
Materials and methods
Plant materials
The fruits bearing plants of Fumaria parviflora Lam. were collected from district Sirsa, Haryana (India) in month of February-March 2015. The collected plant was authenticated from Raw Material Herbarium and Museum, Delhi (RHMD), CSIR-NISCAIR, New Delhi. A slide specimen/ sample have been deposited in the Raw Material Herbarium and Museum, Delhi (RHMD), CSIR-NISCAIR, Ref. No. NISCAIR/RHMD/Consult/2015/2811/04.
Chemicals
All chemicals were analytical grade used in this study purchased from SRL, CDH, SD fine and HIMEDIA. Formalin, absolute alcohol, safranin, haematoxyllin, fast green, glacial acetic acid, clove oil, canada balsam, chloral hydrate, phloroglucinol, H2SO4, NaOH, NH3, lead acetate, FeCl3, potassium hydroxide, ethyl acetate and chloroform etc.
Macroscopic and microscopic examination
Morphological characters were done by using simple microscope. The color, size, shape, odour and taste of roots, stems, leaves and fruits were determined. Microscopic characters were done by preparing thin section of root, stem, leaf and fruit of Fumaria parviflora Lam. (Tekin et al., 2017).
Powdered microscopy
Air dried different parts of plant (leaves, stems, fruits and roots) were finely powdered (# 60) and observed under microscope. Small quantity of different plant parts powder were placed separately on slides and each slide was placed 2-3 drops of chloral hydrate solution and each slide was covered with cover slip then observed under microscope. Different cell contents i.e. epidermis, cork, collenchyma, schlerenchyma, parenchyma, reticulate vessels and stomatal cells were observed and snapshot was done by using digital camera (Sonibare et al., 2014).
Physiochemical examination
Physicochemical parameters of different plant parts of powdered as well as extract of crude drug such as foreign matter, total ash, water soluble ash, acid insoluble ash, alcohol and water soluble extractive values were determined. The moisture content of different plant parts of powdered and extracts were determined by using loss on drying method (Dhingra et al., 2014).
Fluorescence examination
Fluorescence examination of the different parts of plant powder was done by using standard method. The examination was done by treating the plant powder with different solvents including both acidic/basic and organic/inorganic. After treatment they were examined under visible light, short ultra-violet light and long ultra- violet light.
Fluorescence examination is an imperative mechanism for the screening of those constituents which have the assets of showing different colors under UV light. Some components are not fluorescent themselves but when they are reacted with solvents are converted into fluorescent derivatives. This phenomenon may be due to an exacting fluorescent substance or fluorescent derivative formed after treatment with reagents. Still many natural drugs are assessed qualitatively by using this parameter. Powdered roots, stems, leaves and fruits materials were observed under visible light, short ultra-violet light and long ultra-violet light simultaneously after treatment with different organic and inorganic reagents like KOH, NaOH, H2SO4, HCl, FeCl3, iodine solution and HNO3 (Akbar et al., 2014).
Microchemistry examinations of roots, stems, leaves and fruits powder
Microchemistry evaluations of different parts of plant with different chemical reagents were observed. Screening showed the presence of different phytoconstituents along with colour changes under ordinary day light by standard procedure.
Preparation of extract and preliminary phytochemicals examination
The different parts of the plant were air dried in shadow followed by grinding. Extraction of each part was performed with ethanol as solvent on soxhlet apparatus. The each extract was evaporated in rotary evaporator apparatus and air dried at room temperature for 2-3 days. These extracts were stored in refrigerator for further study. The preliminary phytochemicals examination of different plant parts extract of Fumaria parviflora was done by using the standard procedure.
Results
Macroscopic examination of roots, stems, leaves and fruits
The organoleptic and morphological study of Fumaria parviflora leaves as well as powder showed green colour. Leaves are compound, pinnatifid, 5 to 7 cm long, apex acute; petiole is very thin, 2.5 to 4.0 cm long and bitter in taste. The root was branched, cylindrical, about 8-10 cm long, 3 mm thick, cream coloured, and bitter in taste. The stem was pentagonal, pale green, smooth, hollow, about 2-4 mm thick, bitter and slightly acrid in taste. Fruits Capsule, are single seeded, 2 mm long and obovate, subtruncate, obscurely, apiculate, rugose and bitter in taste (Figure 1).
Figure 1. Fumaria parviflora
Microscopic examination
Roots microscopy
Transverse section of root shows a single layered epidermis. The cortex (ct) consisting of thin walled, rectangular, parenchymatous cells, outer 1 or 2 layers irregular and brown in colour. Endodermis is not distinct, secondary phloem very narrow and central part shows a wide zone of xylem and consists of common elements. Xylem vessels (xyv) mostly single having reticulate and spiral thickening, medullary rays (mr) are less developed and mostly uniseriate (Figure 2).
Figure 2. T.S. of Fumaria parviflora roots
Stems microscopy
Transverse section of Stem shows a single layered thin walled epidermis (e) of rectangular cells, covered with thin cuticle (cl), cortex (ct) narrow brown pitted parenchymatous (bpp). Pentagonal outline, of stem having well-known collenchymatous hypodermis (hyp); vascular bundles (vb) collateral, 5 or 6 arranged in a ring; each vascular bundle capped with pericyclic fibres (per). It has centrally hollow wide pith (pi) (Figure 3).
Figure 3. T.S. of Fumaria parviflora stems
Fruits microscopy
Cross section of fruit wall we have observed 3 layers. Outer layer of fruit showed epidermis (e) with thin cuticle cells and some hypodermal cells. Mesocarp (mc) with schlerenchymatous cells. The endocarp (ec) one to many layers of endosperm cells developed under the apical region and folded on inner side of the fruit (Figure 4).
Figure 4. T.S. of Fumaria parviflora fruits
Powder microscopy
The powder characteristics of the root, stem, leaf and fruit were study under microscope and shown in the (Figure 5 a-d).
Figure 5 (a-d). Powder microscopy of Fumaria parviflora (a) roots, (b) stems, (c) leaves, (d) fruits
Physicochemical examination
Present study deals the various physicochemical parameters such as foreign matter, loss on drying, total ash, water soluble ash, acid insoluble ash values, and extractive values were determined in duplicate as depicted in table 1.
Table 1. Physicochemical examination of roots, stems, leaves and fruits of Fumaria parviflora Lam.
S.N. |
Parameters |
Mean ± SD (%W/W) |
||||
Roots |
Stems |
Leaves |
Fruits |
|||
Ash value |
||||||
1 |
Total ash |
16.23±0.21 |
15.23±0.21 |
15.50±0.41 |
8.30±0.22 |
|
2 |
Water soluble ash |
8.5±0.41 |
8.3±0.08 |
9.4±0.29 |
6.16±0.24 |
|
3 |
Acid insoluble ash |
2.33±0.24 |
1.32±0.23 |
5.30±0.22 |
1.17±0.24 |
|
Extractive value |
||||||
1 |
Water soluble |
12.28±0.21 |
26.19±0.05 |
29.73±0.25 |
18.03±0.21 |
|
2 |
Alcohol soluble |
10.19±0.05 |
12.03±0.02 |
14.56±0.40 |
20.83±0.12 |
|
Moisture content |
||||||
1 |
Moisture content |
0.59±0.01 |
0.45±0.02 |
0.82±0.02 |
0.16±0.01 |
|
Foreign matter |
||||||
1 |
Foreign matter |
1.03±0.12 |
0.90±0.08 |
1.33±0.09 |
0.8±0.08 |
Fluorescence investigation
The fluorescence characteristics of the powder with different chemical reagents are depicted in table 2,3,4,5.
Table 2. Fluorescence examination of powdered roots
S.N. |
Reagents |
Colour observed in visible light |
Colour observed under UV light |
|
Short (254nm) |
Long (365nm) |
|||
1 |
Powder |
Light brown |
Light brown |
Light brown |
2 |
NaOH (1N) |
Brown |
Yellowish green |
Pale yellow |
3 |
KOH (1N) |
Brown |
Green |
Yellow |
4 |
Conc. H2SO4 |
Pale yellow |
Yellow |
Yellow |
5 |
Conc. HNO3 |
Reddish brown |
Green |
Green |
6 |
Conc. HCl |
Brown |
Yellow |
Yellow |
7 |
50% H2SO4 |
Reddish brown |
Yellow |
Yellow |
8 |
Iodine solution |
Dull Yellow |
Black |
Green |
9 |
5% FeCl3 |
Brownish black |
Black |
Black |
10 |
Picric acid |
Dull yellow |
Green |
Brown |
Table 3. Fluorescence examination of powdered stems
S.N. |
Reagents |
Colour observed in visible light |
Colour observed under UV light |
|
Short (254nm) |
Long (365nm) |
|||
1 |
Powder |
Yellowish green |
Green |
Light green |
2 |
NaOH (1N) |
Sand |
Lemon |
Creamy |
3 |
KOH (1N) |
Sand |
Lemon |
Yellow |
4 |
Conc. H2SO4 |
Reddish brown |
Green |
Yellow |
5 |
Conc. HNO3 |
Reddish |
Yellow |
Pale yellow |
6 |
Conc. HCl |
Pale yellow |
Creamy |
Creamy |
7 |
50% H2SO4 |
Reddish |
Lemon |
Yellow |
8 |
Iodine solution |
Brown |
Black |
Black |
9 |
5% FeCl3 |
Black |
Black |
Black |
10 |
Picric acid |
Yellow |
Walnut brown |
Walnut brown |
Table 4. Fluorescence examination of powdered leaves
S.N. |
Reagents |
Colour observed in visible light |
Colour observed under UV light |
|
Short (254nm) |
Long (365nm) |
|||
1 |
Powder |
Green |
Green |
Dull green |
2 |
NaOH (1N) |
Pale yellow |
Light green |
Light green |
3 |
KOH (1N) |
Pale yellow |
Light green |
Light green |
4 |
Conc. H2SO4 |
Yellowish green |
Green |
Green |
5 |
Conc. HNO3 |
Reddish brown |
Green |
Green |
6 |
Conc. HCl |
Light brown |
Purple |
Purple |
7 |
50% H2SO4 |
Yellow |
Green |
Green |
8 |
Iodine solution |
Blood red |
Blood red |
Blood red |
9 |
5% FeCl3 |
Bluish black |
Black |
Black |
10 |
Picric acid |
Yellowish green |
Green |
Green |
Table 5. Fluorescence examination of powdered fruits
S.N. |
Reagents |
Colour observed in visible light |
Colour observed under UV light |
|
Short (254nm) |
Long (365nm) |
|||
1 |
Powder |
Dark brown |
Black |
Black |
2 |
NaOH (1N) |
Pale yellow |
Parrot colour |
Green |
3 |
KOH (1N) |
Reddish brown |
Light green |
Light green |
4 |
Conc. H2SO4 |
Reddish |
Purple |
Purple |
5 |
Conc. HNO3 |
Light brown |
Green |
Green |
6 |
Conc. HCl |
Dark brown |
Green |
Green |
7 |
50% H2SO4 |
Red |
Reddish brown |
Reddish brown |
8 |
Iodine solution |
Red |
Parrot colour |
Green |
9 |
5% FeCl3 |
Black |
Dark Green |
Green |
10 |
Picric acid |
Pale yellow |
Green |
Green |
Table 6. Microchemistry investigation of powdered roots, stems, leaves and fruits
S.N. |
Reagents |
Roots |
Stems |
Leaves |
Fruits |
||||
Colour/ppt |
Constituents |
Colour/ppt |
Constituents |
Colour/ppt |
Constituents |
Colour/ppt |
Constituents |
||
1 |
Iodine solution |
Pale yellow |
Cellulose (+) |
Pale yellow colour |
Cellulose (+) |
Pale yellow |
Cellulose (+) |
Pale yellow |
Cellulose (-) |
2 |
Iodine solution+66% H2SO4 |
Bright blue |
Cellulose (+) |
Bright blue colour |
Cellulose (+) |
Bright blue |
Cellulose (+) |
Bright blue |
Cellulose (-) |
3 |
Phloroglucinol + HCl |
Red |
Lignin (+) |
Red colour |
Lignin (+) |
Red |
Lignin (+) |
Red |
Lignin (+) |
4 |
Water |
No change |
Saponin(-) |
No change |
Saponin(-) |
No change |
Saponin(-) |
No change |
Saponin(-) |
5 |
Molisch’S reagent |
Violet colour |
Carbohydrate(+) |
Violet colour |
Carbohydrate(+) |
Violet colour |
Carbohydrate(+) |
Violet colour |
Carbohydrate(+) |
6 |
Aqueous FeCl3 |
Black |
Tannin(+) |
Black |
Tannin(+) |
Black |
Tannin(+) |
Black colour |
Tannin(+) |
7 |
Mg-HCl |
No change |
Flavonoid(-) |
Orange colour |
Flavonoid(+) |
Red to purple colour |
Flavonoid(+) |
Red colour |
Flavonoid(+) |
8 |
Picric acid |
Yellow ppt |
Alkaloid(+) |
Yellow ppt |
Alkaloid(+) |
Yellow ppt |
Alkaloid(+) |
Yellow ppt |
Alkaloid(+) |
Microchemistry investigation of powdered roots, stems, leaves, and fruits
The different parts of the plant powder was treated with different chemical reagent showed the presence of carbohydrate, lignin, tannin, alkaloid, saponin and flavonoids were shown in table 6.
Extractive values and preliminary phytochemicals examination
The extractive values of ethanolic extract of root, stem, leaf and fruit of Fumaria parviflora were calculated (table 7). Preliminary phytochemicals examination of ethanolic extracts of different parts of Fumaria parviflora revealed the presence of carbohydrates, proteins, steroids, alkaloids, glycosides, flavonoids, and tannins (table 8).
Table 7. Extractive values of different parts of Fumaria parviflora extract
Extracts |
Yield (%W/W) |
Colour of extract |
Consistency |
Root ethanolic extract |
6.25 |
Light brown |
Solid |
Stem ethanolic extract |
9.75 |
Yellowish green |
Viscous |
Leaf ethanolic extract |
17 |
Blackish brown |
Gummy |
Fruit ethanolic extract |
14.65 |
Dark brown |
Viscous |
Table 8. Preliminary phytochemicals examination of ethanolic extracts of different parts of Fumaria parviflora
Phytochemical constituents |
Chemical tests |
Roots ethanolic extract |
Stems ethanolic extract |
Leaves ethanolic extract |
Fruits ethanolic extract |
Alkaloids |
Dragendorff’s reagent |
+ |
+ |
+ |
+ |
Mayer’s reagent |
+ |
+ |
+ |
+ |
|
Wagner’s reagent |
+ |
+ |
+ |
+ |
|
Hager’s reagent |
+ |
+ |
+ |
+ |
|
Cabohydrates |
Molisch’s reagent |
+ |
+ |
+ |
+ |
Proteins |
Biuret reagent |
- |
- |
- |
- |
Lipids |
Paper Staining test |
- |
- |
- |
+ |
Saponins |
Foam test |
- |
- |
- |
- |
Glycosides |
Borntrager’s test |
- |
- |
- |
- |
Keller- Killiani test |
- |
- |
- |
- |
|
Tannins |
Ferric chloride test |
+ |
+ |
+ |
+ |
Lead acetate test |
+ |
+ |
+ |
+ |
|
Flavonoids |
Shinoda test |
+ |
+ |
+ |
+ |
66% H2SO4 |
+ |
+ |
+ |
+ |
|
Steroids |
Salkowski test |
+ |
- |
- |
+ |
Conc. H2SO4 |
+ |
- |
- |
+ |
Discussion
As quality control of the allopathic medicine is essential, likewise the pharmacognostic standardization of herbal drug is also necessary for the quality control because substitute or adulterated plant drugs are mainly present in the market. This type of investigation will help to make sure the identity, quality, purity and safety of herbal drug for the medicinal use. The different parameters studied are organoleptic characters, macroscopic analysis, microscopic analysis and fluorescence analysis. Macroscopic and microscopic analysis is one of the easiest and cheapest methods to correctly identify the authentic herbal drug and the surety of raw material. Morphological and microscopical studies of root stem, leaf and fruit will be helpful in the identification of these parts of Fumaria parviflora Lam. Physicochemical analysis of root, stem, leaf and fruit are helpful to establish quality standards of the plant. Therefore, various parameters used for identification of different plant parts are important for drug evaluation. The results of all types of analysis are helpful in establishing quality control standards and purity assurance of drugs. Phytochemicals analysis is also the important part of herbal drug quality parameters. These simple, economical but consistent principles can be useful even for an inexpert person whenever using the drug as folk medicine. This investigation will also be helpful for producer for assessing the quality and purity of raw material. Concisely, the different parameters were described here can be considered as distinguishing to identify and authenticate this herbal drug.
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