A Study on Physiochemical, Microscopical and Total Alkaloids, Phenolic Contents of Methanolic Extract of Proboscidea louisianica Leaves

Since antiquity, humans have turned to nature in search of remedies for their ailments. The earliest use of medicinal plants was largely instinctive, resembling the natural healing behaviours seen in animals. Over time, through observation, trial, and experience, people began to recognize the therapeutic value of specific plants and their parts. This gradual accumulation of knowledge laid the foundation for traditional medicine systems across different cultures, many of which continue to influence modern pharmacology and healthcare practices today(1) Initially, the use of medicinal plants was guided largely by experience, as little was known about the causes of diseases or the therapeutic properties of plants. Over time, discoveries about why certain plants were effective for specific illnesses allowed their use to move beyond mere empiricism toward a more systematic understanding. Until the 16th century, with the advent of chemistry, plants remained the primary source of both treatment and disease prevention.(2)  Over the centuries, the use of medicinal substances steadily expanded as humans experimented with different plants and remedies. While traditional medicine relied on the direct use of whole plants or their extracts, modern medicine has largely shifted toward synthesized drugs, standardized formulations, and evidence-based therapies. As a result, the direct application of medicinal plants in treatment has been largely displaced, although many contemporary pharmaceuticals still trace their origins to plant-based compounds.(4) For about 75–80% of the global population, particularly in developing nations, herbal medicine remains the mainstay of primary healthcare. This is largely because it is culturally well-accepted, generally safer for the human body, and associated with fewer side effects than conventional medicines.(5) Medicinal plants are a vital source of diverse chemical compounds that possess a wide range of structures and functional properties. These bioactive molecules exhibit numerous beneficial biological activities, including antimicrobial, anticancer, antiviral, antioxidant, and enzyme inhibitory effects. In addition, they are known for their anti-aging, anti-inflammatory, antihypertensive, neuroprotective, and anticoagulant properties. Together, these characteristics highlight the significant therapeutic potential of medicinal plants and their continued relevance in modern healthcare and drug development.(6) Medicinal plants are valued worldwide for their therapeutic significance, serving both as standalone remedies and as complements to conventional medicine. Extensive research documenting their health-promoting properties, combined with centuries of traditional folk medicine experience, has led to a growing interest in the use of natural products. This convergence of scientific evidence and traditional knowledge underscores the continued relevance of medicinal plants in modern healthcare and drug development.(7) The field of phytochemistry explores the chemical constituents of plants and their potential therapeutic applications, highlighting the important role of plant-derived compounds in healthcare. These natural products, ranging from alkaloids and flavonoids to terpenes and phenolics, exhibit diverse biological activities that can address a wide variety of human and livestock ailments. One of the major advantages of plant-based medicines is their generally low incidence of side effects, which makes them safer and more acceptable compared to many synthetic drugs. Additionally, the long-standing traditional use of medicinal plants has reinforced community trust and cultural acceptance, ensuring that these remedies continue to play a vital role in both conventional and complementary healthcare systems worldwide.(8) After harvesting and storing for a period, some medicinal plants produce translucent or white substances on certain organs, commonly referred to as “frost.” This frost forms as specific compounds within the plant gradually migrate to the surface and crystallize. For example, persimmons often show a layer of white frost on their surface after storage, demonstrating this natural process. The presence of such frost can serve as an indicator of bioactive compounds, which may contribute to the plant’s medicinal properties.(9) Nature exemplifies the principle of coexistence, providing a rich source of materials that sustain life. Substances derived from plants, animals, and minerals have long been used to treat human ailments, forming the cornerstone of traditional and modern medicinal practices.(10) Many medicinal plants have been widely investigated for their antioxidant potential in recent years. Increased consumption of foods rich in natural antioxidants is associated with a reduced risk of degenerative disorders, particularly cardiovascular diseases and cancer . Natural antioxidants derived from aromatic, spicy, and medicinal plants have been explored for developing formulations applicable in food, cosmetics, and pharmaceuticals . Plant secondary metabolites are broadly classified into three major groups: terpenoids, phenolic compounds, and alkaloids . Among these, phenolic compounds are the most significant for dietary purposes and have been the focus of extensive research . This group comprises phenolic acids (hydroxybenzoic and hydroxycinnamic acids), polyphenols (such as hydrolyzable and condensed tannins), and flavonoids. These bioactive molecules help protect plants, fruits, and vegetables against oxidative damage and are widely utilized by humans as natural antioxidants. Identifying safe and effective antioxidants from plant sources remains a key area of interest for developing functional foods and nutraceuticals. Phytochemical screening is a common approach employed to identify such antioxidant compounds in plants .(11)

2. MATERIALS AND METHODS

Plant collection

The fresh leaves of selected Proboscidea louisianica plant were collected from the rural area of korangi. kakinada district India. All the plant parts were collected in the rainy season generally in the month of july 2025.

Chemicals:

 Methanol, acetic acid, lead acetate, alkaline reagent ferric chloride, millions regents, Wagner’s reagent, hydrochloride acid, sulfuric acid, gallic acid , sodium bicarbonate, sodium hydroxide, folin-ciocalteu reagent, chloroform, glacial acetic acid, acetic anhydride, hangers’ reagent, phloroglucinol, Sudan red , glycerine, ruthenium red, dil. iodine solution, potassium iodide.

Processing of plant material extract:

Maceration process

Identify the plant Proboscidea louisianica and collect the leaves and wash the plant material to remove dust or impurities. Dry it in shade to prevent loss of active constituents. Grind the dried material into coarse powder Place the powdered plant material in a clean, dry glass container. Add sufficient methanol to completely cover the material (usually 3–5 times the weight of the plant material). Stir the mixture to ensure all particles are soaked. Keep it at room temperature away from direct sunlight. Allow it to macerate for 1 week. Shake or stir the mixture occasionally to enhance extraction. After the extraction period, filter the mixture using filter paper or muslin cloth to remove plant residues. Collect the filtrate, which contains the extracted compounds and steam distillate it.

Microscopical evaluation

Powder microscopy

A small amount of the powdered drug was placed on a slide and observed under the microscope to examine its physical and structural characteristics. The powder was treated with Phloroglucinol, iodine solution, hydrochloride acid, sulfuric acid, glacial acetic acid, Sudan red to improve the visibility of its internal structures. Upon observation, the dry  powder exhibited several diagnostic features including multicellular and lignified trichomes, fibres, calcium oxalate crystals in prismatic and clustered forms, and xylem vessels. These microscopic characteristics provided valuable information about anatomical and structural composition of powdered drug sample(13)

Transverse section of midrib

Fresh leaves of  Proboscidea louisianica were chosen for detailed microscopic studies to understand their internal structure and chemical nature. Thin sections of the leaves were carefully prepared  by using a , free-hand sectioning, which is quicker and useful for general observation. The prepared sections were mounted on glass slides as both temporary and permanent mounts so that they could be examined under the microscope in detail. To study the presence of different substances within the tissues, several special chemical tests were carried out. Hydrochloric acid–phloroglucinol was applied to highlight lignified tissues such as xylem, which turned red in colour. Iodine–iodide solution was used to detect starch grains, giving them a characteristic blue or violet colour. Sudan red   stain was applied to identify fatty or oily substances in the cells, which appeared red. Dragendorff’s reagent was used to confirm the presence of alkaloids, important plant metabolites with medicinal value. Ruthenium red was applied to show mucilage, which stained pink or red. Finally, ferric chloride solution was used to detect phenolic compounds, giving a dark coloration. These observations helped in identifying the structural features of the leaves as well as the presence of important primary and secondary metabolites that play a key role in the plant’s physiology and medicinal properties.(14)

Phytochemical screening

Preliminary phytochemical analysis plays a key role in identifying the nature of chemical constituents present in crude drug samples. In this study, about 50 g of shade-dried leaf powder was subjected to successive extraction using solvent methanol. The extract obtained was weighed, and stored for further analysis. Standard qualitative tests were then carried out on the  extract to determine the presence or absence of major groups of phytoconstituents. These included alkaloids, flavonoids, steroids, tannins, glycosides, amino acids, and other secondary metabolites. The findings provided valuable insights into the chemical profile of the leaves, many of which contribute to their pharmacological and therapeutic significance.(15)

Preparation of plant extracts for qualitative determination of alkaloids and phenolics

About 5 g of each powdered plant sample was mixed with 50 ml of 70% chilled methanol in a beaker and stirred thoroughly. The mixture was then poured into a reagent bottle and left at room temperature overnight. Afterwards, it was centrifuged at 6000 rpm for 15 minutes, and the clear liquid (supernatant) was taken for testing alkaloids and phenolics.

Determination of total alkaloids by titrimetric methods :

The supernatant from the plant sample was used to estimate the total alkaloid content by a titrimetric method. For this, 10 ml of the supernatant was placed in a 100 ml separating funnel, and 10 ml of 0.1 N HCl was added and shaken well for 2–3 minutes. This allowed the alkaloids to dissolve in the acidic layer. In the funnel, the lower layer contained alkaloids in 0.1 N HCl, while the upper layer contained methanol. The acidic portion (HCl layer) was collected in a beaker, and 2–3 drops of methyl red indicator were added, which turned the solution slightly red. This solution was then titrated with 0.1 N NaOH until the color changed from red to pale yellow, indicating the neutralization point. The same procedure was repeated three times for accuracy.

The Total amount of alkaloids was calculated by considering the following equivalent.

1 ml 0.1N HCl ≡ 0.0162 g alkaloid

Determination of total phenolic by Folin-ciocalteu’s method:

The total phenolic content of the plant extract was estimated using the Folin-Ciocalteu colorimetric method, which is based on an oxidation-reduction reaction . A stock solution of gallic acid (1 mg/ml) was prepared in 80% chilled m methanol, diluted ten times, and used as the working standard. Different volumes (0.1, 0.2, 0.3, 0.4, and 0.5 ml) of this standard were taken into separate test tubes. To each tube, 0.5 ml of Folin-Ciocalteu reagent and 1 ml of saturated sodium bicarbonate were added. The final volume was adjusted to 5 ml with distilled water. All tubes were incubated in a boiling water bath for exactly 2 minutes, then cooled to room temperature. The absorbance of each sample was measured at 560 nm using a UV-VIS spectrophotometer (Dynamica HALO DB-20) against a reagent blank. A calibration curve was then plotted (Fig. 1).

For the test samples, 0.4 ml of the plant extract (prepared as described in the materials and methods) was taken in triplicate, and the color reaction was carried out as usual. The phenol content was calculated using a standard procedure. First, a common factor was obtained by dividing the absorbance value by the corresponding concentration for each of the five standard solutions. The average of these five common factors was taken as the final multiplying factor. (16)The total phenol content in the extract was then calculated using the formula

Series of stock solution:

Preparation of standard  solution :

• Weigh 1gm of gallic and dissolve it in the 100 ml of methanoli.e stock A.from stock a prepare 100,250,500,750 1000 concentrations through serial diluations
• Weigh 1 g of gallic acid and dissolve it in 100 mL of methanol to prepare the stock solution with a concentration of 10,000 µg/mL.
• From this stock, take 1 mL of the gallic acid solution and dilute it with 9 mL of methanol to obtain an intermediate solution of 1,000 µg/mL.
• To prepare the first standard solution (100 µg/mL), take 1 mL of the 1,000 µg/mL solution and add 9 mL of methanol.
• To prepare the second standard solution (250 µg/mL), take 2.5 mL of the 1,000 µg/mL solution and add 7.5 mL of methanol.
• To prepare the third standard solution (500 µg/mL), take 5 mL of the 1,000 µg/mL solution and add 5 mL of methanol.
• To prepare the fourth standard solution (750 µg/mL), take 7.5 mL of the 1,000 µg/mL solution and add 2.5 mL of methanol.
• The same procedure is repeated for the preparation of sample solution using the leaf extract.

Physiochemical analysis

The physicochemical parameters of the plant material were analyzed following standard guidelines(17)The tests performed included extractive values, total ash, acid-insoluble ash, water-soluble ash, and loss on drying.

RESULT AND DISCUSSION

Phytochemical screening

• The preliminary phytochemical study revealed that methanol extract of proboscidea lousianica   leaves contains

Transverse Section of Proboscidea louisianica Leaf midrib

The transverse section of the midrib in Proboscidea louisianica shows a distinct dicot structure with certain characteristic features:

Upper epidermis

Epidermis: The outermost layer consists of a single row of compactly arranged epidermal cells, externally covered with a thin cuticle with paracytic stomata The epidermis bears multicellular trichomes, which are abundant and provide protection.

Collenchyma: Just beneath the epidermis, layers of collenchyma cells are present, providing mechanical support to the midrib.

Parenchyma: The ground tissue is made up of compactly packed parenchyma cells, filling the bulk of the midrib region. These cells may store nutrients and assist in maintaining leaf rigidity.

Vascular Bundles: A prominent feature of the midrib is the presence of six  to seven distinct vascular bundles. Each vascular bundle is collateral and surrounded by parenchymatous bundle sheath cells. The xylem is oriented towards the upper (adaxial) side, while the phloem is located towards the lower (abaxial) side.

Lower epidermis

Collenchyma 4-5 layers collnechyma present . Similar to the upper layer, the lower epidermis also shows a single layer of cells, covered with cuticle paracytic stomata  and bearing trichomes

Physiochemical analysis

Loss on drying:

• Weight of the empty crucible =59.23
• Weight of the sample (powder )= 0.50grams
• Weight of the empty crucible+Weight of the powder before drying =59.73g
• Weight of the empty crucible+Weight of the powder after drying=59.68g
• =(59.73-59.68) = 0.05grams
• Percentage of moisture content:
• 0.05gms of crude drug content = 0.05gms
• 100gms of crude drug content = 0.05/0.60x

Swelling index:

Initial swelling in ml =50

After swelling in ml =51.5

After swelling in ml = after swelling – initial swelling/initial swelling ×100

Percentage of swelling index = 51.5 – 50/50 × 100 = 3%

Ash value:

• Weight of the empty crucible(X) =92.43gms
• Weight of the sample+crucible (Y) = 94.43gms
• Weight of the crucible + Ash (Z) = 93.24gms
• Percentage of Ash value = 100/Y × (Z-X)

= 100/92.43 ×[93.24-92.43]

= 1.05 × 0.81

=  0.81%

Total alkaloid content:

1 ml 0.1N HCl ≡ 0.0162 g alkaloid

Volume of NaoH × equivalent factor × N.(NaoH) / weight of HCL × Normality of NaoH

3.63×0.0162×0.09/8.3×0.1×100

1 gram = 0.162

Then 5 grams = 3.1grams

Total phenolic content:

Y = 0.0026x + 0.3464

X = 5.84 GAE/gm

i.e 10 gm = 58.4

Powder microscopy