Development of Pharmacognostic Standardization Parameters for the Quality control of Aerial parts of Senecio laetus in accordance to WHO Guidelines

 

Nazia Banday*, Z. A. Bhat

Pharmacognosy and Phytochemistry Division, Department of Pharmaceutical Sciences,

University of Kashmir, Hazratbal, Srinagar, J&K, India, 190006.

*Corresponding Author E-mail: nazmuzaffar@live.in

 

ABSTRACT:

The current study was considered with the aim to set up the standardization parameters for Senecio laetus for its identification and to determine its purity and quality. Innumerable parameters for the standardization of plants like macroscopy, microscopy and physicochemical parameters, fluorescence studies and preliminary phytochemical analysis were accomplished. Macroscopical and microscopical studies were carried according to the day-to-day laboratory norms. The guidelines of World Health Organization were considered for phytochemical screening and florescence analysis of the plant. Microscopy of leaf and stem helped to reveal the anatomical structures, which are to be reported for the first time for this plant species. Analysis of the powders of leaf, stem and flower revealed the presence of epidermal cells with stomata, trichomes, lignified vessels, calcium oxalate crystals, starch granules, xylem vessels, parenchyma cells, cork cells and pollen grains. Phytochemical analysis ascertained the presence of carbohydrates, phenols, glycosides, saponins, flavonoids, tannins, etc.  Physicochemical parameters like ash values, extractive values, loss on drying, foreign matter analysis, foaming index, determination of pH and fluorescence were studied.  All these parameters are valuable in differentiating the powdered drug. The conclusions from these experiments will help in providing the values of various parameters for the standardization and identification of S. laetus.

 

KEYWORDS: Senecio laetus, Pharmacognostic standardization, Phytochemical analysis, WHO guidelines, physicochemical parameters, Fluorescence analysis, Microscopy.

 

 


INTRODUCTION: 

It has been observed that in developing nations, in particular, the herbal medicines play a crucial role in health care system. Nevertheless, the difficulty for the developed nations to accept the herbal medicines is due to the dearth of severe quality control procedures and their documentation. Therefore, the standardization of the crude drug material and its documentation is important for its acceptance globally.

 

Pharmacognostic standardization in accordance to WHO guidelines, physicochemical investigation and preliminary phytochemical investigations are acknowledged as a valid proof and endorsement of the genuine plant material. Standardization of herbal drugs is an approach of endorsing certain conditions, qualitative and quantitative parameters that brings quality assurance, effectiveness, safety as well as reproducibility. In herbal medicines a proper identification and quality assurance of the plant material is vital for maintaining reproducibility which in turn improves its efficacy and safety1.

 

Senecio is one of the largest genera2 of flowering plants comprising of about 1250 species and is distributed worldwide with remarkable morphological variations3. It belongs to the genus of daisy family (Asteraceae). Senecio has been used in traditional medicine in more than hundreds of products owing to their anti- inflammatory, antipyretic and detoxification effects.

 

Senecio laetus (Fig. 1) is a prevalent therapeutic herb of the Himalaya region which has been conventionally employed as a medicine for the sore throat and the swelling of mouth4.

 

Fig 1: Senecio laetus

 

MATERIALS AND METHODS:

Senecio laetus (whole aerial part i.e., stem, leaves and flowers) was collected from Lar, Ganderbal, Kashmir, at an altitude of 1650m in July, 2020. The plant was identified and authenticated by Prof. Akhtar. H. Malik, Curator, CBT, University of Kashmir under voucher no. 2823–(KASH) and was deposited in the University of Kashmir’s herbarium for future reference.

 

Macroscopic and microscopic analysis:

The detailed macroscopic evaluation is necessary to differentiate between the related species having similar appearance. It also helps in distinction of the anatomy in adulterants. Stem and leaf were cut into thin transverse sections, then transferred to slides separately and observed under microscope. Some sections were boiled with chloralhydrate, stained using few drops of 0.1% w/v phloroglucinol and few drops of conc. hydrochloric acid. The sections were then transferred to the slide and perceived under microscope.

 

Physicochemical parameters:

Numerous physicochemical parameters viz., ash values (total ash value, acid insoluble ash value, water soluble ash value and sulphated ash value), loss on drying, foreign matter, foaming index, determination of pH, moisture content and extractive values were evaluated in accordance to the “WHO Quality Control methods for herbal materials”.

 

Determination of pH in 1% and 10% solution:

Accurately weighed 1g and 10g of the crushed drug are dissolved in two separate beakers in 100ml of distilled water. The solution is filtered and the pH of the filtrate is measured with the help of the pH meter.

 

Fluorescence analysis:

The powdered drug of the plant is studied for its fluorescence character in visible light as well as UV light (254nm and 365nm) after the treatment with various reagents5,6.

 

Extract preparation:

The aerial parts of the plant were subjected to cold maceration method of extraction with ethanol and water (8:2) as solvent. The plant extract was filtered and then completely dried using rotary evaporator at 40ºC.

 

Qualitative phytochemical screening:

Several fractions of S. laetus were evaluated for qualitative phytochemical analysis using standard procedures. Presence of carbohydrates; proteins; amino acids, alkaloids, steroids; flavonoids; tannins; saponins; glycosides; phenolics and diterpenes was evaluated.7-13.

 

RESULTS:

Senecio laetus is a tall, perennial herb, 2- 4ft tall. It is glabrous below but hairy above. It is grooved and highly branched. The leaves are alternately arranged, 5- 20cm long, dark green in colour. The leaves are irregularly lobed and toothed. The terminal lobe of the leaf is the largest while the basal lobes are stem clasping. The flower heads are yellow coloured 10-15mm across, in large spreading terminal corymbs. There are 8-10 involucre bracts, which are oblong and acute, often tipped dark brown to black. The ray florets are oblong, 10-12 in number. Disc florets are numerous. Achenes are pale-brown, cylindrical, 2.5mm long, strongly ribbed, puberulous or glabrous. The pappus is about 4 mm long and white in colour.

 

The T.S. of Senecio laetus stem reveals the presence of epidermis, collenchyma, xylem, phloem and pith. [Fig 2, 3]. The T.S. of Senecio laetus leaf reveals the presence of upper epidermis, palisade cells, xylem, phloem, spongy parenchyma, collenchyma, trichome and lower epidermis. [Fig 4,5].

 

 

 Fig 2: T.S. of Senecio laetus stem (without stain)

 

Fig 3: T.S. of Senecio laetus stem(stained)

 

 

Fig 4: T.S. of Senecio laetus leaf (without stain)

 

 

Fig 5: T.S. of Senecio laetus leaf (stained)

 

The powder microscopy of leaf reveals presence of epidermal cells with anomocytic stomata [Fig. 6a], covering trichomes [Fig 6b], calcium oxalate crystals [Fig. 6d], group of [Fig. 6c] and starch granules [Fig. 6e].

 

 

Fig 6a: Epidermis in surface view

 

Fig 6b: Covering trichome

 

 

Fig 6c: Group of vessels (spiral)

 

 

Fig 6d: Calcium oxalate crystals (prismatic)

 

 

Fig 6e: Starch granules and part of trichome

Fig 6: Powder microscopy of Senecio laetus leaf

 

The powder microscopy of stem reveals presence of parenchyma cells [Fig 7a], xylem vessels [Fig 7b], cork cells [Fig 7c], calcium oxalate crystals [Fig 7d], and group of fibres [Fig 7e].

 

 

Fig 7a: Parenchyma cell

 

 

Fig 7b: Xylem vessel (spiral)

 

 

Fig 7c: Cork cells

 

 

Fig 7d: Calcium oxalate crystals (prismatic)

 

 

Fig 7e: Group of fibres (lignified)

Fig 7: Powder microscopy of Senecio laetus stem

The powder microscopy of flower reveals presence of abundant pollen grains [Fig 8a], and calcium oxalate crystals [Fig 8b]. These parameters are beneficial for the preparation of the monograph of a particular plant.

 

Fig 8a: Calcium oxalate crystals and pollen grains

 

Fig 8b: Abundant pollen grains

 

Fig 8: Powder microscopy of Senecio laetus flower

 

The total ash, acid insoluble ash, water soluble ash and sulphated ash values were found to be 7.8±0.05, 1.25± 0.03,2.8±0.02 and 19.5±0.21 respectively. The value for loss on drying was observed to be 16.53% and foreign matter present was found to be 0.63%.  The foaming index was found to be over 1000. The pH values of 1 % and 10% solution were noted to be 1.65 and 2.95 respectively.

 

However, the water and alcohol soluble extractive values were found to be 33.6% and 14.4% (Table 1).

 

Table 1: Physicochemical parameters of Senecio laetus powder

Parameter

Values

Total Ash value

7.8±0.05 % w/w

Acid insoluble ash value

1.25±0.03 % w/w

Water soluble ash value

2.8±0.02 % w/w

Sulphated ash value

19.5±0.21 % w/w

Water soluble extractive value

33.6 % w/w

Alcohol soluble extractive value

14.4 % w/w

Loss on drying

16.53 % w/w

Foreign matter content

0.63 % w/w

Foaming index

Over 1000

pH in 1% solution

1.65

pH in 10% solution

2.95

 

Fluorescence characteristics of powdered aerial parts of S. laetus was observed in visible, short and long UV light as shown in Table 2.


 

Table 2: Fluorescence analysis of Senecio laetus powder

Solvent used

Visible Light

UV light

At 254nm

At 365nm

Powder

Yellowish green

Green

Blackish green

Distilled water

Light green

Dark green

Black

Acetone

Olive green

Dark green

Black

Petroleum ether

Dark green

Very dark green

Black

Chloroform

Brown

Very dark green

Black

Ethyl acetate

Brown

Very dark green

Black

Methanol

Green

Dark green

Black

Glacial acetic acid

Brownish green

Very dark green

Black

Ammonia

Brownish green

Dark green

Black

Nitric acid (conc.)

Brownish green

Very dark green

Black

Nitric acid (dil.)

Brownish green

Very dark green

Black

Hydrochloric acid (conc.)

Olive green

Dark green

Black

Hydrochloric acid (dil.)

Olive green

Dark green

Black

Sulphuric acid (conc.)

Dark green

Very dark green

Black

Sulphuric acid (dil.)

Olive green

Dark green

Black

Picric acid

Yellowish green

Lime green

Black

Sodium hydroxide (5%)

Green

Dark green

Black

Ferric chloride (5%)

Dull green

Dark green

Black

 

Preliminary phytochemical analysis revealed the presence of alkaloids, carbohydrates, saponins, flavonoids, tannins, phenols, glycosides, etc. as shown in Table 3.

 

Table 3: Phytochemical analysis of various extracts of Senecio laetus

S. No.

Phytochemical Test

Plant extracts

Hexane

Dichloromethane

Ethyl acetate

Butanol

Residual

1.

Alkaloids

Hager’s Test

+

+

+

-

-

Mayer’s Test

+

+

+

-

-

Wagner’s  Test

+

+

+

-

-

Dragendroff’s Test

+

+

+

-

-

2.

Carbohydrates

Molish Test

-

-

+

+

+

Fehling test

-

-

+

+

+

3.

Proteins

Biuret Test

-

-

-

-

-

Millon’s Test

-

-

+

+

+

4.

Amino Acids

Ninhydrin Test

-

-

+

+

+

5.

Saponins

Foam Test

-

-

-

+

+

Emulsion test

-

-

-

+

+

6.

Tannins

Gelatin test

-

-

-

+

+

7.

Glycosides

Keller-Killiani Test

+

+

-

-

-

Borntranger Test

+

+

-

-

-

8.

Steroids

Salkowski Test

+

+

+

+

-

9.

Flavonoids

Shinoda Test

-

+

+

+

+

Alkaline reagent test

-

+

+

+

+

Lead acetate test

-

+

+

+

+

10.

Gum and mucilage

-

-

-

-

-

11.

Phenolics

Ferric chloride Test

+

+

+

+

+

12.

Fixed oils and fat

+

+

-

-

-

13.

Terpenoids

+

+

-

-

-

14.

Diterpenes

Copper acetate Test

+

+

+

+

+

 

DISCUSSION:

The macroscopic study of the aerial parts of the Senecio laetus reveal that the plant is a perennial herb. The T.S. of Senecio laetus stem reveals the presence of epidermis, collenchyma, xylem, phloem and pith. The epidermis, outermost layer of the stem is wavy, uniseriate consisting of small cells with occasional multicellular covering trichome. The vascular bundles are arranged in a ring and are wedge shaped. They are collateral i.e.; xylem is on the inner surface and phloem on the outer surface. The vascular bundles are placed more towards the epidermis than towards the centre. The xylem stains pink on treatment with phloroglucinol and hydrochloric acid. Pith is the innermost part of the stem formed by a group of loosely arranged parenchyma cells.

 

The T.S. of Senecio laetus leaf reveals the presence of upper epidermis, palisade cells, xylem, phloem, spongy parenchyma, collenchyma, trichome and lower epidermis. The epidermis, both upper and lower, is uniseriate consisting of small rectangular cells. Both the upper and lower epidermis possess covering trichomes, which are 2-8 celled. Trichomes are more in number on the lower epidermis than on the upper epidermis. They are uniseriate, multicellular, and gradually taper to a point. Below the lower epidermis, collenchyma cells are present. The vascular bundles are embedded within the spongy parenchyma and are wedge shaped. The xylem stains pink on treatment with phloroglucinol and hydrochloric acid.

 

The powder microscopy of Senecio laetus leaf reveals presence of epidermal cells with anomocytic stomata, covering trichomes, calcium oxalate crystals, group of vessels and starch granules. The powder microscopy of Senecio laetus stem reveals presence of parenchyma cells, xylem vessels, cork cells, calcium oxalate crystals and group of fibres. The powder microscopy of Senecio laetus flower reveals presence of abundant pollen grains and calcium oxalate crystals.

 

The total ash, acid insoluble ash, water soluble ash and sulphated ash values help in establishing the purity of the crude drug by indicating the amount of impurities present like silicates, carbonates and oxalates. Acid- insoluble ash indicates mostly the presence of silicates while the water-soluble ash value helps to determine quantity of inorganic compounds present. Fluorescence characteristics helps in distinguishing presence of any adulterants in the crude drug.  Preliminary phytochemical analysis revealed the presence of alkaloids, carbohydrates, saponins, flavonoids, tannins, phenols, glycosides, etc.

 

In India most of the medicine systems exploit the crude drugs of plant origin typically. So, it becomes a necessity to put forth identification parameters and quality control measures for a particular plant. Therefore, pharmacognostic standardization of the plant is an utmost parameter.

 

Since, for this plant species there has been no documented reports for the pharmacognostical work, the present study had been taken up with a notion to set up the values of various parameters of standardization, which in turn would be supportive for identification of the plant, its quality and authentication.

 

ACKNOWLEDGEMENTS:

The support provided by the Department of Pharmaceutical Sciences, University of Kashmir is appreciatively acknowledged.

 

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Received on 05.04.2023           Modified on 11.08.2023

Accepted on 18.10.2023          © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):37-42.

DOI: 10.52711/0974-360X.2024.00007