Phytosomal Formulation of Clerodendrum Infortunatum for Anti- Tubercular Activity and Development of a Therapeutic Suspension

Another earliest recorded forms of healing practices include the use of herbal medicine; the evidence show that it dated even before recorded history. There is sufficient evidence from a variety of sources that links man and his search for drugs in nature to a distant past: from writing, as well as from the first plant remedies[1]. As noted by the WHO, only the conventional medicine is something over 80% of the global population uses as the primary medical remedyIn this context, the use of herbal medicine emerges as a widely accepted component of complementary and alternative medicine systems[2]. Phytotherapy therefore is the science which uses plant based materials in treatment of a certain disease or preventing the occurrence of that particular disease. Herbal medicine is believed to be practiced in many culture to cure various ailments that include cardiovascular diseases, cancer, neurodegenerative diseases, and diabetes mellitus[3]. By and large, plants have served as a very important source of new drugs for a long time and contributed immensely to the medical field. Since about 25% of modern pharmaceuticals are based on plant compounds, their importance cannot be overstated. Plants exhibit a large diversity in secondary metabolites, including alkaloids, flavonoids, and terpenoids, which display multiple kinds of biological activities such as antimicrobial, anti-inflammatory, and anticancer. The age-old ethnobotanical knowledge of indigenous people concerning the medicinal use of plants has led to the discovery of many drugs, including such important drugs as morphine from the opium poppy and quinine from the cinchona tree. Moreover, the growing problem of resistance of pathogens to synthetic drugs underlines the role of novel plant-based compounds in the development of novel therapeutic agents. Since there remains such a wide scope of discovery into plant diversity, the potential for new drugs discovery remains to be great, and thus plants are an exceedingly vital resource for pharmaceutical innovation. Plants represent a crucial source of novel pharmaceuticals, providing a diverse range of bioactive compounds that hold therapeutic promise. Many modern pharmaceuticals, such as paclitaxel for cancer and artemisinin for malaria, have been derived from plant sources, highlighting their significant role in drug discovery[4]. The complexity and versatility of plant biochemistry offers sulky opportunities in the discovery of new disease treatments, including cancers, bacteria, and viruses, and chronic ailments. With new technology, there lies hope that other drugs from plants could be discovered and this means that there is need to conserve plant species and indigenous knowledge on the same[5]. Subsequent studies on these plants may lead to development of other drugs which are way more effective than synthetic drugs and cause little or no side effects[6].

Tuberculosis

Tuberculosis is a viral disease which is very contagious with two thirds of the whole population of the entire world and 40 percent of the Indian population affected by it. However, this concern has arisen due to development of drug resistance of Mycobacterium tuberculosis to both the first and the second line drugs. As a result, MDR and XDR strains of M. tuberculosis are emerging in different parts of the world including India infected it. However, this has become a serious problem as the drug resistance of Mycobacterium tuberculosis has become resistant both against the first line as also the second line drugs. As a result of this, multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of M. tuberculosis have emerged all over the world including India[7]. Anti-TB drugs consist of two groups: First-line or essential therapy which is mainly used in the ordinary TB patient with ordinary Mycobacterium tuberculosis; second-line or reserve anti-TB drugs which are used with MDR-TB. Compared to first-line anti-TB drugs, second line drugs have several fold more adverse effects [8]. There has been increasing concern in the search for natural products against TB based on the efficiencies of medicinal plants in providing Organization of an alterative mode of handling TB in view of the increasing drug resistance. Quite a number of researchers have investigated the effectiveness of different plants in the control of Mycobacterium tuberculosis. For instance, findings on the indicator plant, Andrographis paniculata, demonstrate that the global plant possesses major anti TB element labeled as andrographolide[9]. In the same manner, Curcuma longa (turmeric) has a potential because of its active ingredient, the curcumin that possess a potent antimycobacterial activity[10]. Such an observation indicates that the isolated administration of the medicinal plants may improve the treatment outcomes if combined with the conventional chemotherapy drugs together with the standard TB drugs especially in the areas where healthcare facilities restrain the availability of the conventional anti-TB medications.

Clerodendrum infortunatum

Clerodendrum infortunatum Linn., belongs to the family Verbanaceae, it is a Terrestrial shrub having blackish, square stem its leaves are simple oppositely decussate petiolate exstipulate, coriaceous and hairy, having very obnoxious smell[11]. Clerodendrum infortunatum is a perennial shrub that typically grows to a height of 2-4 feet. It has a slightly woody nature, with blunt, quadrangular stems and branches. The leaves are usually arranged in groups of three or four at a node, although they may occasionally be opposite, and are oblong or elliptical with serrated edges. The plant produces sweetly fragrant, bluish-purple or white flowers that form in terminal, pyramid-shaped panicles. The fruits are four-lobed, purple drupes that are somewhat succulent, each lobe containing a single pyrene. [12].The Clerodendrum of the family Verbenaceae is a very  large family comprising of plants that are distributed mostly in the tropical and sub topic parts of the world with very little penetration into the temperature climate areas. They have estimated the species number five hundred sixty and five hundred eighty. Its origin can be attributed to the year 1753 when Linneus was at last able to set the genus; kind of which is Clerodendrum infortunatum indigenous to India. Later in 1763, Adamson replaced the Latinized form of the family name Clerodendrum to the Greek form of the name Clerodendron. The genus was first described by Linneus for the year 1753 but for the type species that is Clerodendrum infortunatum the descriptions was made from India.The layman’s name, Clerodendrum, then posed to him by Linnaeus, was translated by Adanson in 1763 into the Greek equivalent of Clerodendrondrum infortunatum from India. In 1763, Adanson changed the Latinized form Clerodendrum into Greek form Clerodendron. Linnaeus’s original s-adopted the Latinized form Moldenke in 1942, However, similar to other taxonomists this practice has been continued[13]. In India and several other countries, Clerodendrum infortunatum has a long history of use in the Ayurvedic system for various ailments. In Assam, the roots of this plant are utilized to treat bronchitis and asthma. The leaves, roots, and flowers are often prepared as a paste or ash for medicinal purposes. Additionally, the leaves and flowers are applied to counteract scorpion stings, while the young shoots are used as a remedy for snake bites.Several researchers have established that different fractions of Clerodendrum infortunatum exhibit a range of pharmacological properties. These include antimicrobial, anthelmintic, insecticidal, analgesic, antipyretic, hepatoprotective, wound healing, thrombolytic, antidiabetic, cytotoxic, and anti-inflammatory activities[14].

Herbal Nanotechnology

Herbal remedies and natural products are being used to treat the diseases. Unlike the allopathic system that is in common practice today, these herbal remedies are made of thousands of chemical constituents which all act at once, on the diseases. Phyto therapeutics require an understanding where the components have to be delivered in sustained manner so that patient concordance can be enhanced without having to administer the components repeatedly. This can be realized through the development of NDDSs of the herbal constituents. Besides overcoming the need for repeated administration to overcome non- compliance, NDDSs also enhance the therapeutic value by decreasing toxicity and enhancing entrapment, efficacy, penetration and solubility of the drugs. There are certain benefits of incorporating the herbal extracts in the new formulation systems and these include less absorption that can be solved which is the major drawback seen, and the possibility of attracting major drug companies. Nanotechnology is as area of applied science and technology that focuses on the systematic fabrication of devices and dosage forms in the nanometer range (nanoscale) of 1 to 100 nm. The uses of nanotechnology for therapeutic, diagnostic, sensing, and regulating biological systems have been recently described as nanomedicine. The nanocarriers have been prepared from safe materials such as synthetic biodegradable polymers, lipids and polysaccharides[15]. Herbal nanotechnology is an innovative approach that integrates nanotechnology with plant-based products to enhance their therapeutic potential. By using nanoscale delivery systems such as nanoparticles, liposomes, phytosomes, herbal bioactive compounds can achieve improved bioavailability, stability, and targeted delivery. This technology addresses common challenges faced by traditional herbal formulations, such as poor solubility and rapid degradation. In particular, it enhances the absorption of phytoconstituents, allowing for lower dosages with enhanced efficacy and reduced side effects. Herbal nanotechnology is being increasingly explored in various applications, including in the development of nutraceuticals, cosmetics, and pharmaceuticals, making it a promising field for the efficient use of plant biodiversity[16]. In phyto-formulation research, the preparation of nano dosage forms such as phytosomes, liposomes, cubosomes and nanosponges, and many more have numerous benefits on herbal drugs in terms of solubility, bioavailability, reduction in toxicity, improved pharmacological activity, protection against degradation, increased macrophages distribution in tissues, sustained release and protection from physical-chemical degradations. The portable herbal drug delivery of nano size drug delivery systems could possibly create an added future to the enhancement of activity besides challenging difficulties related to plant medicine in the future. The crucial of NDDS found in the traditional medicine system is the incorporation of nanocarrier to treat more chronic ailments like asthma, diabetes, cancer diseases, microbial diseases, among others. Some specific nanocarriers can transport phytochemicals and endocytose them within the expected cells. Liposomes and phytosomes have enhanced the wound healing properties of the phytochemicals. Yet another advantage of co-delivery of phytochemicals and therapeutic agents is the capability of eliminating the probability of failure or formation of drug resistance. These drug delivery systems are capable of reducing enzymatic degradation in gastrointestinal tract, having bypass effect over the first pass metabolism and reducing toxicity of the off-targeted tissue. More advancements in this particular field may also reveal some concealed opportunities related to liposome and phytosome based delivery of natural products and phytochemicals in therapeutic relevance[17].

Phytosomes

Phytosomes are advanced herbal preparations, which ensure that the bio-actives derived from plants possess high bioavailability in the body. Traditionally used plant extracts have hydrophilic nature, hence possesses low absorption levels in the body. The process of generating phytosome involves complexing phospholipids usually phosphatidylcholine with plant bio actives, thereby producing the lipid-compatible molecular complexes, which helps enhance the solubility and absorption levels of the bioactive compounds across the cell membranes along with their therapeutic efficacy. Phytosomes have been mostly used in the preparations of flavonoids, alkaloids, and other polyphenols. They are most particularly found to be highly useful in chronic treatments wherein improved absorption of the actives of herbal content can lead to better clinical results. Phytosome nanotechnology presents promising therapeutic potential in enhancing drug delivery systems, particularly for bioactive phytochemicals used in topical applications. As lipid-based nanocarriers, phytosomes play a crucial role in influencing the pharmacokinetics and pharmacodynamics of plant-derived polyphenolic compounds. This nanotechnology offers significant potential for advancing the development of topical formulations. Moreover, the penetration of phytochemicals can be enhanced by this nano size delivery system owing to the peculiar physiochemical properties at the biological barriers, and these will enhance its bioavailability[18].

Fig. 1. Diagrammatic representation of a phytosome.

The leaves of Clerodendrum infortunatum were collected from the Thrissur district, Kerala, India, on February 1, 2024. The plant was identified and authenticated by Dr. Ranjusha A. P., Head of the Department of Botany, NSS College, Ottapalam, Kerala. A voucher specimen was deposited at Nehru College of Pharmacy, Thrissur, for future reference.

METHOD

Isolation Of Flavonoidal Bio-Fraction

The flavonoid bio-fraction from the ethanolic extract of Clerodendrum infortunatum leaves was isolated using column chromatography. This technique is employed to separate individual components from a mixture that is dissolved in a fluid, and it falls under the category of chromatography methods.^[19]

Preparation And Evaluation of Phytosome

Evaluation Of Phytosomes

The behavior of phytosomes is influenced by factors like size, shape, stability, and distribution. Therefore, the phytosomes are characterized by the following evaluation parameters:

Scanning Electron Microscopy (SEM)

A scanning electron microscopy study was conducted to assess the surface morphology, size, and shape of the prepared phytosomes. The freeze-dried phytosomes were analyzed using scanning electron microscopy and photographed.

Measurement of particle size (PS)

The particle size of phytosomes was determined using a particle size analyzer.

Measurement of Zeta potential (ZP)

Zeta potential, which refers to the total charge produced by the medium, plays a crucial role in determining the charge of phytosomes within emulsions. Depending on the composition of the phytosomes, the zeta potential can be negative, positive, or neutral. The zeta potential of the optimized phytosome suspension was measured using a zeta sizer.

Drug entrapment efficiency

Entrapment efficiency (EE is assessed using the ultracentrifugation method. In this process, the phytosomes were centrifuged at 12,000 rpm for 45 minutes to separate them from any unentrapped drug. The concentration of the free drug in the supernatant was measured by assessing the absorbance at 462 nm with a UV-Visible spectrometer[22,23].

In Vitro Pharmacological Activity

Determination of minimum inhibitory concentration (Resazurin-based 96-well plate microdilution method)

Test microorganism - Mycobaterium smegmatis (MTCC No :6, Incubation 37°C for 24 hours)

Alamar Blue Susceptibility Assay procedure

• Plates were prepared in a sterile environment, and a sterile 96-well plate was appropriately labeled. A volume of 100 μL of the test substance, either dissolved in 10% (v/v) DMSO or sterile water (usually at a stock concentration of 10 mg/mL), was added to the first row of the plate. For the other wells, 50 μL of normal saline was introduced.

• Serial dilutions were performed using a multichannel pipette, with tips discarded after each use to ensure that each well contained 50 μL of the test substance at progressively lower concentrations.

• Following this, 10 μL of resazurin indicator solution was added to each well, and then 30 μL of a 3.3× strength isosensitized broth was pipetted into each well, achieving a final volume of single-strength nutrient broth.

• Lastly, 10 μL of bacterial suspension was added to each well. To prevent the bacteria from dehydrating, each plate was loosely covered with cling film. Each plate included a set of controls: one column containing three antitubercular drugs (isoniazid, pyrazinamide, and

streptomycin) as a positive control, another column with all solutions except the test compound, and a final column with all solutions excluding the bacterial solution, where 10 μL of nutrient broth was added instead.

• The plates were sealed and incubated at 37°C for 24 hours. One of the control wells was treated with Alamar blue dye and monitored for the development of pink coloration.

• If a pink color was observed in the control well, the test samples and standard drugs were applied to the experimental wells at various concentrations, and the plate was incubated for another 24 hours.

• After incubation, Alamar blue dye was added to all experimental wells, followed by further incubation to allow for pink coloration development. Results were recorded through visual observation and by measuring absorbance at 600 nm.

Experimental Group

• B- Culture Medium +Resazurin reagent (sterility control) (no drug, no culture)
• C- Culture Medium + Microbial culture+ Resazurin reagent (culture and reagent control)
• S- Culture Medium + Microbial culture+Standard drugs + Resazurin reagent
• T- Culture Medium + Test sample + Microbial culture + Resazurin reagent [24,25,26]

Formulation Of Anti-Tubercular Suspension

The formulation for preparing a 100 ml suspension of the sample powder is detailed in Table 13. The fine particles of the drugs, sized at 100 mesh, are thoroughly mixed through triturating. The drug mixture is then blended with water and various additives, including Tween-80, sodium carboxymethyl cellulose (CMC), a sweetener, a flavoring agent, and sodium benzoate, to improve its stability[27,28]

? Organoleptic Properties

O Color: Visually inspect the suspension under natural light.

O Odor: Smell the suspension and note any characteristic or unusual odor.

O Taste: Test a small quantity (with proper consent and safety considerations) to check the taste. This is usually done in trials or on a controlled basis.

O Texture: Rub a small amount of the suspension between your fingers or test it on a glass plate to check for grittiness.

? Sedimentation Volume

Sedimentation volume is a measure used to evaluate the stability of suspensions, indicating the volume occupied by sediment after a certain period. To determine sedimentation volume, a known volume of the suspension is placed in a graduated cylinder and allowed to stand undisturbed for a specific time, usually 24 hours. After this period, the height of the sediment is measured, and the sedimentation volume is calculated by dividing the height of the sediment by the total height of the suspension, expressed as a percentage. This procedure helps assess the stability and dispersion characteristics of the formulation, providing insights into its performance and shelf-life.

? Viscosity

O The sample's viscosity was measured at room temperature using a Brookfield viscometer set to 50 rpm.

? pH Measurement

O The suspension's pH was measured using a pH meter.[29,30]

RESULTS

Preparation Of Phytosomes

The phytosomes was prepared by using rotary evaporation method.

Fig. 2. Prepared Phytosomes

Entrapment efficiency (%)

Scanning Electron Microscopy (SEM)

Fig. 3. SEM image of phytosome

Measurement of Zeta potential (ZP)

Fig. 4. Zeta potential graph of prepared phytosomes

Measurement of particle size (PS)

The particle size of the optimized phytosome was found to be 140.74 nm. This was in accordance with the particle size range of phytosomes. The evaluation of the phytosome formulation revealed promising results, particularly for the optimized phytosome labeled P2, which exhibited a high entrapment efficiency. Scanning Electron Microscopy (SEM) analysis confirmed that the optimized phytosomes possess a spherical shape and a uniform size distribution, essential characteristics for effective drug delivery. Additionally, the zeta potential of the optimized phytosome was measured at -23.6 mV, indicating high stability and minimal tendency to form aggregates. The particle size of the optimized phytosome was found to be 140.74 nm, aligning well with the expected size range for phytosomes.

In-Vitro Pharmacological Activity

Resazurin-based 96-well plate microdilution method

Fig. 6. MABA Assay of Standard drugs and Test samples

Fig. 7. Graph of anti-tuberculosis activity showing absorbance at 600 nm for varying concentrations of Isoniazid (INH), Pyrazinamide (PYZ), Streptomycin (STP), Biofraction (BF), and Phytosome (PHY).

Formulation Of Anti-Tubercular Suspension

Fig. 8. Phytosome loaded anti- tubercular suspensions

Evaluation Of Anti-Tubercular Suspension