ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF HERBAL EXTRACTS

Abstract

The Present invention relates to a novel antimicrobial formulation derived from the stems of Nyctanthes arbor-tristis, a plant traditionally known for its medicinal properties. This invention includes a method for preparing an ethanolic extract from the plant's stems, aiming to harness its antibacterial and antifungal properties for use in pharmaceutical, agricultural, and cosmetic applications. The stems are carefully dried, powdered, and extracted using a Soxhlet apparatus with 95% ethanol to ensure the retention of bioactive compounds. This concentrated ethanolic extract (EENATS) has demonstrated significant antimicrobial activity, particularly against the bacterial strains Serratia marcescens and Enterococcus faecalis, and to a lesser extent against the fungal strain Aspergillus niger. The effectiveness of the extract was assessed through standardized disc diffusion methods, with results indicating measurable zones of inhibition when compared to standard drugs like Ciprofloxacin and Fluconazole.

Specification

Description:FIELD OF INVENTION
The present invention relates to herbal extracts with antibacterial and antifungal properties, specifically those derived from Nyctanthes arbor-tristis, which show significant activity against select bacterial and fungal pathogens. The invention has applications in pharmaceuticals, cosmetics, and agricultural industries where natural antimicrobial agents are desirable.
BACKGROUND OF THE INVENTION
The rise in antimicrobial resistance has become a critical public health issue globally. Bacteria and fungi have developed the ability to resist commonly used synthetic drugs, leading to treatment failures and the spread of infections. Traditional antibiotics and antifungals are increasingly ineffective against resistant strains, posing a challenge in both healthcare and agricultural sectors where pathogen control is crucial. This problem emphasizes the urgent need for new, effective antimicrobial agents, especially those derived from natural sources, which may have fewer side effects and lower risks of resistance.
Natural products, particularly those derived from medicinal plants, have long been used in traditional medicine and are known to contain bioactive compounds with antimicrobial properties. However, despite their known potential, many plant extracts have not been extensively studied or optimized for effective antimicrobial use. Nyctanthes arbor-tristis, commonly known as Night Jasmine or Harsingar, is one such plant with a rich history in traditional medicine for treating infections and other ailments. Preliminary studies suggest that parts of this plant, especially the stem, contain compounds with antibacterial and antifungal properties. Yet, standardized extraction methods and testing for broad-spectrum efficacy against resistant strains are lacking, limiting the practical application of this plant in modern medicine.
To address this gap, the present invention focuses on developing a standardized method for extracting the bioactive components of Nyctanthes arbor-tristis stem using ethanol and evaluating its efficacy against specific bacterial and fungal pathogens. The invention aims to solve the problem of resistance by providing an alternative antimicrobial source that can be integrated into pharmaceutical, cosmetic, and agricultural products. By demonstrating significant zones of inhibition in microbial tests, this extract could provide a natural solution for infection control, reducing reliance on synthetic antibiotics and offering a sustainable approach to managing resistant pathogens.
The following prior art being reported was:
IN201841043475: Synergistic herbal formulation for the management of anti-bacterial and anti-fungal activity of herbal extract formulation in human body with dried, powdered, water extract of selected herbs, wherein the said selected herbs are Aloe barbadensis, Curcuma longa ,tabebuia avellanedae, Glycyrrhiza glabra, Azadirachta indica and Oreganum vulgare. The herbal extract of the present invention is prepared in at least in one of the form such as non-capsulated powdered form, at least in capsulated form or at least in tablet form. The said herbal extract is administered orally along with water. The herbal composition of the present invention has anti-fungal and anti¬bacterial activity. The present invention is differ from this as it relates to herbal extracts with antibacterial and antifungal properties, specifically those derived from Nyctanthes arbor-tristis, which show significant activity against select bacterial and fungal pathogens.
OBJECTS OF THE INVENTION
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative
An object of the present disclosure is to explore the herbal extracts with antibacterial and antifungal properties, specifically those derived from Nyctanthes arbor-tristis, which show significant activity against select bacterial and fungal pathogens
Another object of the present disclosure is the extract provides a plant-based alternative to synthetic antibiotics, reducing potential side effects associated with chemical agents.
Still another object of the present disclosure is it demonstrates efficacy against both bacterial and fungal pathogens, making it versatile for various infection types.
Another object of the present disclosure helps in combat the growing issue of antibiotic resistance by offering an alternative with unique antimicrobial mechanisms.
Still another object of the present disclosure is being a natural extract, it is biodegradable and less likely to harm the environment compared to synthetic antimicrobials.
Still another object of the present disclosure is to Nyctanthes arbor-tristis is readily available in many regions, allowing for sustainable production and reducing reliance on scarce resources.
Yet another object of the present disclosure is suitable for pharmaceutical, cosmetic, and agricultural uses, expanding its utility across multiple industries.
Yet another object of the present disclosure is the method of extraction is straightforward, making it cost-effective and accessible for widespread production.
Yet another object of the present disclosure is as a natural product, it is likely to cause fewer adverse effects, making it a safer option for long-term use.

Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION
The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

The present invention is generally, the stems of Nyctanthes arbor-tristis were sourced from the IFTM University campus and prepared for extraction. The collected stems were carefully air-dried, coarsely powdered, and sieved through a 40-mesh filter to ensure uniform particle size. This powdered material was then subjected to hot percolation using a Soxhlet apparatus with 95% ethanol, conducted at a controlled temperature of 45-50°C for two days. The resulting extract was filtered, concentrated by distillation at 55-60°C, and further dried under reduced pressure to yield a stable ethanolic extract of Nyctanthes arbor-tristis stem (EENATS).

An embodiment of the present invention is to assess its antimicrobial efficacy, solutions of the extract were prepared by dissolving it in DMSO to achieve a concentration of 5 mg/ml. The study utilized nutrient agar and Sabouraud dextrose agar media, which were sterilized and prepared for culturing specific bacterial and fungal strains. The bacterial strains, including Serratia marcescens and Enterococcus faecalis, and fungal strains, Candida albicans and Aspergillus niger, were inoculated on the prepared media. Antimicrobial testing was performed by placing EENATS-saturated discs (ranging from 0 to 5000 µg/disc) on the media plates, with Ciprofloxacin and Fluconazole used as standard reference drugs for antibacterial and antifungal activities, respectively.

An embodiment of the present invention is each plate was incubated under optimal conditions-37 ± 0.5°C for bacterial growth over 24 hours and 48 hours for fungal growth. Zones of inhibition were subsequently measured to evaluate the potential mechanisms of action, including cell wall synthesis inhibition, DNA binding, and membrane penetration. The study was conducted with rigorous controls and in triplicate to ensure accuracy and reliability in assessing the antimicrobial properties of the ethanolic extract of Nyctanthes arbor-tristis stem.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig 1: Zone of Inhibition EENATS and Standard drug on Serraatia marcescens
Fig 2: Zone of Inhibition EENATS and Standard drug on Enterococus faecalis
Fig 3: Zone of Inhibition EENATS and Standard drug on Candida albicans
Fig 4: Zone of Inhibition EENATS and Standard drug on Aspergillus niger

DETAILED DESCRIPTION OF THE INVENTION
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
The present invention relates to a novel antimicrobial formulation derived from the stems of Nyctanthes arbor-tristis, a plant traditionally known for its medicinal properties. This invention includes a method for preparing an ethanolic extract from the plant's stems, its antibacterial and antifungal properties for use in pharmaceutical, agricultural, and cosmetic applications. The stems are carefully dried, powdered, and extracted using a Soxhlet apparatus with 95% ethanol to ensure the retention of bioactive compounds. This concentrated ethanolic extract (EENATS) has demonstrated significant antimicrobial activity, particularly against the bacterial strains Serratia marcescens and Enterococcus faecalis, and to a lesser extent against the fungal strain Aspergillus niger. The effectiveness of the extract was assessed through standardized disc diffusion methods, with results indicating measurable zones of inhibition when compared to standard drugs like Ciprofloxacin and Fluconazole. The mechanism underlying this antimicrobial activity is thought to involve the inhibition of cell wall synthesis, DNA binding, and membrane penetration, making it a promising candidate for natural antimicrobial agents. This invention addresses the growing issue of resistance to synthetic drugs, offering a sustainable, plant-based alternative that could be adapted for a range of antimicrobial applications.

The antimicrobial mechanism of action of the ethanolic extract of Nyctanthes arbor-tristis stem (EENATS) is believed to involve multiple pathways that disrupt the growth and viability of bacterial and fungal cells. One primary mechanism is the inhibition of cell wall synthesis, which weakens the structural integrity of the microorganism, making it more susceptible to environmental stress. Additionally, the bioactive compounds in the extract may irreversibly bind to the DNA of the pathogens, interfering with essential processes such as replication and transcription. This disruption of genetic material prevents normal cellular function and replication. Furthermore, the extract likely penetrates the cell membrane, causing damage to the cell's protective barrier and leading to leakage of cellular contents. Collectively, these mechanisms contribute to the antimicrobial efficacy of the extract against both bacterial and fungal strains.

BIOLOGICAL SOURCE:
Nyctanthes arbor-tristis stems were collected from the IFTM University campus in Moradabad, Uttar Pradesh, India.

EXAMPLE 1: Preparation of Extract
The stems of Nyctanthes arbor-tristis were air-dried, coarsely powdered, and sieved through a 40-mesh filter. This powdered material was then extracted with 95% ethanol using a Soxhlet apparatus through hot percolation at 45-50°C for two days. The resulting extract was filtered and concentrated by distillation at 55-60°C, then dried under reduced pressure to yield a stable, powdered form of the ethanolic extract of Nyctanthes arbor-tristis stem (EENATS).
EXAMPLE 2: Antibacterial Avtivity & Anti- Fungal Activity
In this method, the media were prepared and autoclaved at 121oC, 15 lbs/inch2 pressure for 15 minutes. These media were poured into plates and allowed to solidify. On the surface of the media, the microbial suspension was spread with the help of a sterilized bent shape glass rod. The above said prepared nutrient agar media was taken in a pre-sterilized Petri dish and the microorganisms were grown. The disc of 7 mm was saturated and the concentration of the applied solution on the disc 0 to 5000 µg/disc of extract solution of ethanolic extract of Nyctanthes arbortristis stem and the disc of standard Ciprofloxacin (10 µg/disc) was placed in the centre for antibacterial activity and Fluconazole (500 µg/disc) for antifungal activity in each disc. The plates were kept at room temperature for one hour to allow the diffusion of test compounds and then incubated at 37± 0.50C for an antibacterial activity for 24 hrs and antifungal activity for 48 hrs respectively. The diameters of the zone of inhibition in mm were measured and compared with the standard drugs

Preparation of the Solutions of Extract
The ethanolic extract of the Nyctanthes arbortristis stem, accurately weighed 5 mg of each, were transferred into different10 ml volumetric flasks. These extracts were dissolved into DMSO and the volume in each flask was made up to 10 ml with DMSO. The concentrations of stock solutions were 5 mg/ml prepared. Now from this stock solution 20 ml was taken and sterilized in the empty disc. Thus, the concentration of the applied solution on the disc was 0 to 5000 µg/disc in each Petri dish.
Bacterial and Fungal Strains
In this study total of two bacterial strains, including gram -ve bacteria Serraatia marcescens and gram +ve bacteria Enterococus faecalis and two fungal strains Candida albicans and Aspergillus niger were used for the assessment of antibacterial and antifungal activities.

Preparation of Nutrient Agar Media (2%, pH 6.8±0.2) for Antibacterial Activity
Take about 5 gm beef extract, 5 gm peptone, and 2.5 gm sodium chloride dissolved in 400 ml of distilled water in a 500 ml volumetric flask and warmed it. 10 gm of agar dissolved in 50 ml of warm distilled water. The two solutions were mixed together and the volume in the volumetric flask was made up to 500 ml of warm distilled water. These nutrient agar media were sterilized in an autoclave at121oC, 15 lb /inch2 pressure for 15 minutes (Indian Pharmacopoeia, 1996).

Preparation of Sabouraud Dextrose Agar Media (2%, pH 6.8±0.2) for Antifungal Activity
To prepare the Sabouraud dextrose agar media, take the accurate weight of the 20 gm dextrose monohydrate and 5 gm peptone and dissolved it in 400 ml of distilled water in 500 ml volumetric flask, and warm. Dissolve 7.5 g of agar in 50 ml of warm distilled water. The two solutions were mixed together and make the volume in a volumetric flask up to 500 ml with warm distilled water. This media was sterilized in an autoclave at 121oC, 15 lbs/inch2 pressure for 15 minutes

Preparation of Inoculums
The vial containing lactose dilution (dehydrated powder) of inoculums of bacterial two bacterial strains; gram -ve bacteria Serraatia marcescens and gram +ve bacteria Enterococus faecalis and two fungal strains Candida albicans and Aspergillus niger were prepared individually broken using a sterile scalpel knife in an aseptic condition in a conical flask containing 100 ml of nutrient broth. This flask was incubated for 24 hrs at 370 C in the Biological Oxygen Demand (BOD) incubator. After 24 h, a turbid solution was obtained.

Table 1. Antibacterial activity of bacterial strain Serraatia marcescens of
EENATS (µg/disk) Plate A Plate B Plate C Average
Control (10) 14 14 16 14.6667
0 0 0 0 0
312.5 0 0 0 0
625 0 0 0 0
1250 0 0 0 0
2500 0 0 0 0
5000 7 7 7 7
Table 2. Antibacterial activity of bacterial strain Enterococus faecalis of ethanolic extract Nyctanthes arbor-tristis stem (EENATS)
EENATS (µg/disk) Plate A Plate B Plate C Average
Control (10) 28 29 29 28.6667
0 0 0 0 0
312.5 8 8 8 8
625 8 9 8 8.33333
1250 9 10 9 9.33333
2500 9 9 9 9
5000 10 10 10 10
Table 3. Antifungal activity of fungal strain Candida albicans of ethanolic extract Nyctanthes arbor-tristis stem (EENATS)
EENATS (µg/disk) Plate A Plate B Plate C Average
Control (500) 27 28 28 27.6667
0 0 0 0 0
312.5 0 0 0 0
625 0 0 0 0
1250 0 0 0 0
2500 0 0 0 0
5000 0 0 0 0

Table 4. Antifungal activity of fungal strain Aspergillus niger of ethanolic extract Nyctanthes arbor-tristis stem (EENATS)
EENATS (µg/disk) Plate A Plate B Plate C Average
Control (500) 21 22 21 21.3333
0 0 0 0 0
312.5 6 6 6 6
625 7 7 7 7
1250 7 7 7 7
2500 7 7 7 7
5000 7 7 7 7
Results shows that in the antimicrobial studies using triplicate plates for each bacterial and fungal strain, the ethanolic extract of Nyctanthes arbor-tristis stem demonstrated significant antibacterial activity. Against Serratia marcescens, the zone of inhibition (ZOI) measured 7 mm, while Enterococcus faecalis showed a larger ZOI of 10 mm. These results are compared with the standard drug Ciprofloxacin, which showed ZOIs of 14.67 mm and 28.67 mm for Serratia marcescens and Enterococcus faecalis, respectively (Tables 1 and 2; Figures 2-5). In antifungal activity, the extract showed limited inhibition for Candida albicans (0 mm ZOI) but a 7 mm ZOI against Aspergillus niger, as compared to the standard drug Fluconazole, with ZOIs of 21.33 mm for Candida albicans and 27.67 mm for Aspergillus niger (Tables 3 and 4; Figures 6-9). The potential mechanisms of the antibacterial effect may involve inhibition of cell wall synthesis, irreversible DNA binding, and cell membrane penetration.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
, Claims:We Claim,
1. A method for preparing an ethanolic extract with antimicrobial properties from the stems of Nyctanthes arbor-tristis, comprising the steps of:
• (a) drying and powdering the stems of Nyctanthes arbor-tristis,
• (b) extracting the powdered material with 95% ethanol using a Soxhlet apparatus at a temperature range of 45-50°C for two days,
• (c) filtering and concentrating the extract by distillation at 55-60°C, and
• (d) drying the concentrated extract under reduced pressure to yield a stable ethanolic extract, which is effective against bacterial and fungal pathogens.
2. The method as claimed in claim 1, wherein the ethanolic extract is dissolved in dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 5 mg/ml for antimicrobial testing.
3. The method as claimed in claim 1, wherein the ethanolic extract exhibits antibacterial activity against gram-negative bacteria, specifically Serratia marcescens.
4. The method as claimed in claim 1, wherein the ethanolic extract exhibits antibacterial activity against gram-positive bacteria, specifically Enterococcus faecalis.
5. The method as claimed in claim 1, wherein the ethanolic extract demonstrates antifungal activity against fungal pathogens, specifically Aspergillus niger.
6. The method as claimed in claim 1, wherein the ethanolic extract is applied on an agar disc with a concentration range of 0 to 5000 µg/disc for the purpose of testing antimicrobial activity.
Dated this 6 November 2024

Dr. Amrish Chandra
Agent of the applicant
IN/PA No: 2959

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