INTRODUCTION:

Herbal medicine is regaining popularity, and people are becoming more interested in green medicine because it is regarded to be safe. Plants and plant extracts were traditionally utilised to treat a variety of ailments and disorders. Medicinal plants have been used as a source of medicine in practically all societies since the dawn of humanity. They are commonly used to treat acute and chronic ailments and are considered the backbone of traditional medicine. According to the World Health Organization, traditional medicines are used by around 80% of the world's population.

In order to strengthen investigation and assessment of the safety and efficacy of herbal products, it approved their use in national policies and drug regulatory procedures. According to the research, 74% of the 119 plant-derived drugs identified by the WHO study were discovered as a result of chemical studies to extract the active chemicals responsible for the traditional use of the original plant. Plants are increasingly being used for healing purposes because they are thought to be healthy and free of adverse effects. Following exposure to a potentially dangerous substance, an unfavourable effect is characterised as an aberrant, undesired, or detrimental change. The most serious side consequence is death, while less serious side effects include changes in food consumption, body and organ weights, apparent pathological abnormalities, or simply changes in enzyme levels 10.As a result, all "natural" compounds used in therapeutics must undergo the same efficacy and safety testing as new synthetic medications¹. Many in vivo investigations on aqueous extracts suggest that herbal medicines are safe, however most in vitro tests on isolated single cells, primarily with extracts other than aqueous extracts, show the opposite, prolonging the dispute on herbal medicine safety. Toxicity studies on herbal medicine are required to reflect their traditional use in order to allow for reasoned arguments about their safety for beneficial application. While research into the safety of numerous herbal medicines in humans continues, careful and responsible use is essential². Poisoning from medicinal plants has been documented, and it is usually due to misidentification of the plants in the form in which they are marketed, or improper preparation and administration by untrained personnel, therefore it is best to be provided by trained personnel³. India has a long tradition of growing ornamental flowers that have recently been exploited both in domestic and international market. However, the floriculture industry has been exploring new species as a result of clients' never-ending hunt for new flowering plants. The bleeding heart vine (Clerodendrum thomsoniae), a powerful decorative plant belonging to the Lamiaceae family, has only recently been utilised by the floriculture industry⁴. The floriculture sector is primarily supported by two major components: first, the commerce of flowering inflorescences and leaf types, and second, nursery stock such as tubers, seeds, bulbs, cutting-grown plants, and tissue culture raised plantlets. Clerodendrum thomsoniae is a well-known cultivated ornamental plant with attractive flowers that has been broadly utilized as pot plants in horticulture due to its gorgeous flowers. Because of its scarcity and low seed production, the species is relatively new to the floriculture business. C. thomsoniae is a tropical West African vine-like shrub with wandering, twining branches. Clerodendrum thomsoniae is also known as Bleeding-heart vine or Bag-flower⁵. This plant has been used in traditional medicine as well as being important in floriculture and horticulture. Other Clerodendrum species, such as C. indicum, C. viscosum, C. serratum, C. colebrookianum and others, are medicinal and utilised in folk medicine⁶. Several medicinally significant phytocompounds are found in the leaves and flowers of C. thomsoniae, are flavonoids, tannins, steroids, alkaloids, etc^7. These phytocompounds protect the human body from oxidative stress and treat a variety of disorders such as bruises, wounds, skin rashes, and sores, among others. Reactive oxygen and nitrogen species (ROS and RNS) cause oxidative stress, which is a crucial factor in the development of various brain disorders, including neurodegenerative diseases, depression⁸and antibacterial activities⁹, antioxidant properties¹⁰.

MATERIALS AND METHODS:

1. Pharmacognostic study:

1.1. Collection, identification and drying of plant material:

The leaves of Clerodendrum thomsoniae were collected from SMBT Campus Igatpuri, authenticated by Assistant director Mr.S.T. Jadhao. The herbarium of the plant specimen has been deposited at parks and garden Aurangabad, the voucher specimen No.CT-1. It was air dried in shade avoiding exposure to direct sunlight and then pulverized in grinder. The powder of leaves was used for standardization and extraction¹¹.

1.2 Macroscopic evaluation:

Organoleptic evaluation refers to evaluation of the plant material by color, odour, taste, size and shape. The organoleptic characters of the samples were determined based on the textual methods¹²¹³.

1.3. Microscopic evaluation:

Free hand section of the materials were taken, suitably stained and subjected to microscopic observations. Photomicrographs were taken using compound microscope by using digital camera¹³.

1.4. Standardization of plant material:

Standardization of leaves powder was done to evaluate the quality and purity of the drug¹⁴. Various Standardization parameters like loss on drying (moisture content), extractive values (water soluble and alcohol soluble) and ash values were calculated as per WHO guidelines^15161718.

1.5. Phytochemical investigation:

1.5.1. Extraction Methodology:

The leaves of Clerodendrum thomsoniae were pulverized in grinder. The pulverizedmaterial (# 60-80) was utilized for extraction by Continuous Hot extraction (Soxhlet apparatus) using Solvent Petroleum ether (60-80°C), Chloroform, Ethanol. The extraction was carried out in several batch¹⁷.

1.6. Characterization of extracts by chemical methods:

A. Test for Alkaloids:

Few mg of each extract was taken separately in 5mL of 1.5% v/v HCl and filtered. These filtrates were then subjected to following tests-

Dragendroff’s test:

The extract was spread on Whatmann filter paper and dried. The test filter paper was made alkaline with ammonia and extracted with chloroform. The chloroform extract was applied on filter paper impregnated with the Dragendroff’s reagent. Development of orange-red colour indicates the presence of alkaloids.

Mayer’s reagent:

The extract was treated with Mayer’s reagent. Formation of cream colour precipitate indicates the presence of alkaloids.

Wagner’s Reagent:

The extract was treated with Wagner’s reagent. Formation of reddish yellow colour precipitate indicates the presence of alkaloids.

Hager’s reagent:

The extract was treated with Hager’s reagent. Formation of yellow colour precipitate indicates the presence of alkaloids.

B. Test for Glycosides:

General test:

Test A: To 5mg of extract, 5mL of dil. H₂SO₄ was added and warmed on waterbath, filtered it. The above solution was neutralised with 5% NaOH solution. 0.5mL Fehling’s solution A and 0.5mL Fehling’s solution B was added and heated on a waterbath for 2 minutes.

Test B: To 5mg of extract, 5mL of water was added and warmed on waterbath, filtered it. The above solution was neutralised with 5% NaOH solution. 0.5mL Fehling’s solution A and 0.5mL Fehling’s solution B was added and heated on a waterbath for 2 minutes.

The red precipitate formed in test A and test B was compared. If test A contains more precipitate than test B, it indicates presence of glycosides. If same precipitate in test A and test B, it indicates absence of glycosides.

C. Test for anthraquinones:

Brontager’s test:

To the 5mg extract, 5mL dil. HCl was added. The soliution was boiled, cooled and 2mL solvent ether was added. Ether layer was separated and strong ammonia solution was added, appearance of pink colour to ammonical layer indicates presence of anthraquinones.

D. Test for Sterols:

Salkowaski test:

Few mg of extract was dissolved in 2mL of chloroform and 2mL of conc.H₂SO₄ was added from the side of test tube. The content of test tube was shake for few min. Development of red colour in the chloroform layer indicates the presence of sterols.

Liebermann test:

Few mg of extract was mixed with acetic anhydride in a test tube and heated gently. Few drops of conc.H₂SO₄were added. Development of blue colour indicates presence of sterols.

E. Test for Triterpenoids:

Libermann-Burchard test:

Few mg of extract was dissolved in chloroform and 0.5 mL acetic anhydride was added to it followed by conc.H₂SO₄ from side of test tube. A development of reddish brown ring at the junction of two liquids indicates presence of triterpenoids.

F. Test for Flavanoids:

Shinoda test:

A small quantity of the extract was dissolved in 5mL of ethanol (95%v/v) and treated with few drops of conc. HCl and 0.5g of magnesium turnings. Development of pink, magenta and crimson colour indicates presence of flavonoid.

G. Test for Tannins:

With FeCl₃:

A small quantity of the extract was dissolved in 5mL of ethanol (95%v/v) treated with FeCl₃solution. Development of blue and brownish green colour indicates presences of hydrolysable and condensed tannins respectively.

H. Tests for Carotenoids:

Carr-price reaction:

Extract was treated with antimony trichloride solution, development of blue colour indicates presence of carotenoids.

I. Test for carbohydrates:

Molisch’s test:

In few mg of extract, 1mL of Molisch’s reagent was added, mixed it. 2mL of Conc, H₂SO₄was added from side of test tube, develoment of violet ring at the junction of two liquids indicates prsence of carbohydrates.

J. Test for Proteins:

Biuret test:

The 5mg of extract was dissolved in 2 mL of alcohol. In alcoholic solution, 2 mL Biuret reagent was added. Development of violet colour indicates presences of protein¹³.

RESULT AND DISCUSSION:

2.1. Pharmacognostic study of Clerodendrum thomsoniae leaves

2.1.1. Macroscopic evaluation of Clerodendrum thomsoniae leaves

Macroscopic identity of herbal materials is based on shape, size, color, odour, taste, characteristics (Figure:1). However, since these characteristics are judged subjectively and substitutes or adulterants may closely resemble the genuine material, it is often necessary to substantiate the findings by microscopy and physicochemical analysis¹³¹⁹.

Fig 1: Leaf of C.thomsoniae

Color: Glossy dark green

Odour: Unpleasant

Taste: Astringent

Size: 12-18cm long and 5-7cm wide

Shape: Elliptic with entire margin

Leaf base- obtuse/ rounded

Apex: Acuminate

2.2. Microscopic evaluation²⁰* of Clerodendrum thomsoniae leaves

T.S. of leaf of Clerodendrum thomsoniae

Figure 2: T.S. of leaf Clerodendrum thomsoniae

Upper epidermis: Single layers of cubical cells covered with cuticle. Many multicellular, glandular covering trichomes are present.

Lower epidermis: Similar to upper epidermis has many different trichomes.

Vascular bundles: xylem and phloem¹³.

Stomatal index

Figure 3: Surface preparation of Epidermis of leaf Clerodendrum thomsoniae for stomatal index

Stomatal index 22

Stomatalnumber

Figure 4: Surface preparation of Epidermis of leaf Clerodendrum thomsoniae for stomatal number Stomatal number –22²¹

Vein islet and vein termination number

Figure5: Vein islet and vein termination

Vein islet number: 12 and vein terminationnumber: 07

2.3. Standardization of Clerodendrum thomsoniae leaves:

Loss on drying is the loss in weight in % w/w resulting from water and volatile matter of any kind that can be driven off under specified conditions. Moisture triggers the enzymatic activity or facilitates growth of microbes which leads to its deterioration^{17, 22}.

Total ash is designed to measure the total amount of material produced after complete incineration of the ground drug at about 450°C to remove all the carbons²³. The ash of any organic material is composed of their non-volatile inorganic components (metallic salts and silica). This value varies within wide limits and is therefore an important parameter for the purpose of evaluation of crude drugs. More direct contamination, such as by sand or earth, is immediately detected by the ash value. Acid insoluble ash is the residue obtained after extracting the total ash with hydrochloric acid¹⁷.

Extractives values represent the amount of active constituents in given amount of medicinal plant material when extracted with solvents. As mentioned in different official books, the determination of water soluble and alcohol soluble extractives is used as means of evaluating crude drugs which are not readily estimated by other means²².

Table 1: Physicochemical parameters of C. thomsoniae leaves

Physicochemical parameters	% w/w
Loss on drying	8
Total ash	7.5
Water extractive value: Hot extraction	30.12
Alcohol extractive value: Hot extraction	23.33
Alcohol extractive value: Cold maceration	21.66
Water extractive value: Cold maceration	36.66

2.4. Phytochemical investigation of Clerodendrum thomsoniae:

The extraction was carried out by continuous hot extraction method using Soxhlet extractor till all constituents removed. The end of completion of extraction was indicated by no color with iodine fumes when spot on TLC plate¹⁷.

Table 2: Extractive values after continuous extraction of C. thomsoniae leaves

Extract	Colour of Extract	% (w/w) of Extract obtained
Petroleum ether	Green	6.65
Chloroform	Green	8.70
Methanol	Brownish green	18.65

2.5. Characterization of Clerodendrum thomsoniae extracts by chemical test:

Table 3: Observation table of preliminary phytochemical test of extracts of Clerodendrum thomsoniae

Sr No.	Test	Extracts
Sr No.	Test	Pet. Ether	Chloroform	Ethanol
1.	Test for Alkaloids	-	+	─
2.	Test for Glycosides	-	+	+
3.	Test for Anthraquinones	─	─	─
4.	Test for Sterols	+	+	─
5.	Test for Triterpenoids	+	+	─
6.	Test for Flavanoids	─	─	+
7.	Test for Tannins	─	─	+
8.	Tests for Carotenoids	+	─	─
9.	Test for Proteins	-	─	+
10	Test for Carbohydrates	-	+	+

Pet-ether extract showed the presence of sterol, carotenoidsandtriterpenoids.

Chloroform extract showed the presence of alkaloids, glycoside, sterols, carbohydrates and triterpenoids.

Ethanol extract showed the presence of glycosides, flavonoids, tannins, and carbohydrates, Proteins.

CONCLUSION:

Ascertaing pharmacognostic and physiochemical standards are an integral part correct identity and quality of a crude drug. Before any drug can be included in the pharmacopeia, these standards must be established. The majority of the information on the identity, purity and quality of the plant material can be obtained from its macroscopy, microscopy and physiochemical parameters. Pharmacognostical and phytochemical parameters of Clerodendrum thomsoniae determined in the present work can serve as major criteria for identity, quality and purity of a crude drug and its extracts.

ACKNOWLEDGEMENT:

The authors are grateful to Principal, S.M.B.T. College of Pharmacy for providing necessary facilities and support.

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Received on 10.08.2022 Modified on 12.12.2022

Res. J. Pharmacognosy and Phytochem. 2023; 15(3):209-214.

DOI: 10.52711/0975-4385.2023.00032