1. INTRODUCTION:

Bioactive substances utilized in both conventional and alternative medicine have been mostly derived from medicinal plants. Cyamopsis tetragonoloba (L.) Taub., also referred to as guar or cluster bean, is a leguminous plant that is indigenous to the Indian subcontinent and is widely grown for its seeds, which produce guar gum, a galactomannan polysaccharide that is widely used in a variety of industries, including food, medicine, and cosmetics^.¹

C.tetragonoloba has garnered interest for its therapeutic potential, especially its antibacterial qualities, in addition to its industrial uses. Traditionally, Indian and Ayurvedic medicine has utilized the plant's seeds, pods, and leaves to treat conditions like infections, inflammation, and diarrhea².According to phytochemical screens, the plant has a number of bioactive components with antimicrobial properties, including flavonoids, saponins, tannins, alkaloids, and phenolic compounds^3,4.

The antibacterial activity of C. tetragonoloba extracts against both Gram-positive and Gram-negative bacteria has been the subject of numerous investigations. For example, there have been notable zones of inhibition against pathogens like Staphylococcus aureus, Escherichia coli, in methanolic and aqueous extracts of the seeds and leaves^5,6. The secondary metabolites of the plant are thought to cause these antibacterial actions by either disrupting bacterial cell membranes or inhibiting bacterial enzymes⁷.

Novel and efficient antimicrobial drugs are desperately needed, especially in light of the growing problem of antibiotic resistance. Investigating naturally occurring plant-based antimicrobials, such as C. tetragonoloba, presents a viable substitute for traditional antibiotics. The goal of the current work is to determine the precise components causing the antibacterial activity of different Cyamopsis tetragonoloba extracts and to further explore their antibacterial efficacy.

2. PLANT INFORMATION:

2.1 Cyamopsis tetragonoloba :

Fig. 1 : Cyamopsis tetragonoloba.

Synonyms: Cyamopsis psoraloides (Lam.) DC., Cyamopsis tetragonoloba L. subsp. tetragonoloba L., Cluster bean, Guar.

Taxonomical Description:

Kingdom: Plantae (Plants)

Sub-kingdom: Tracheobionta (Vascular plants)

Division: Magnoliophyta (Flowering plants)

Class: Magnoliopsida (Dicotyledons)

Sub-class: Rosidae

Order: Fabales

Family: Leguminosea

Sub-family: Fabaceae

Genus: Cyamopsis

Species: Cyamopsis tetragonoloba

Native Area and Distribution:

The exact origin of Cyamopsis tetragonoloba L.(Guar), one of the few cultivated species in its genus, remains uncertain. It is believed that guar may have been domesticated in Africa or the Arabian Peninsula before spreading to other regions, including the United States, India, and Pakistan. India is considered the primary center of genetic diversity for the cluster bean. From India, the crop spread to a wide range of countries, including Afghanistan, various parts of Africa (such as Central Africa, Chad, Ethiopia, Sierra Leone, Somalia, South Africa, West Africa, and Zambia), as well as Asia (including China, Indonesia, Indochina, Sri Lanka, Vietnam, and Yemen), Australia, Fiji, and the United States.⁸

Chemistry of Cyamopsis tetragonoloba:

The primary chemical constituent of guar (Cyamopsis tetragonoloba) is guar gum, a type of galactomannan polysaccharide that constitutes up to 85% of the endosperm of guar seeds.

Guar Gum (Galactomannan):

· Composed of a β-D-mannopyranose backbone with α-D-galactopyranose side branches.

· Mannose:Galactose ratio is approximately 2:1.

· Responsible for high water-binding capacity and viscosity.

Proteins:

· Found in the seed germ and cotyledons.

· Guar seeds contain about 5–6% protein, mostly globulins and albumins.

Fibers:

· Both soluble and insoluble dietary fibers are present.

Saponins and Tannins:

· Present in smaller amounts; may contribute to antioxidant and antimicrobial properties.

Minerals:

· Rich in potassium, phosphorus, calcium, and trace elements like iron and zinc.⁹

Therapeutic Uses:

A galactomannan polysaccharide with a variety of therapeutic uses, guar gum is made from the seeds of Cyamopsis tetragonoloba and is non-toxic, biocompatible, and soluble. Its most well-known application is as dietary fiber, which has advantages for medication delivery systems, metabolic regulation, and digestive health.

Cyamopsis tetragonoloba L. (Guar gum) has several important medicinal benefits, including the capacity to control blood sugar levels by postponing stomach emptying and the absorption of carbohydrates, which aids in the treatment of diabetes mellitus. Its high soluble fiber content also lowers cholesterol by binding bile acids in the colon and encouraging their evacuation, which lowers blood cholesterol levels overall. Cyamopsis tetragonoloba L. (Guar gum) also aids in weight management by promoting fullness and decreasing total caloric consumption. Guar gum functions as a prebiotic in gastrointestinal medicine, encouraging the development of advantageous gut flora that enhances nutrition absorption and immunological function. Because of its ability to retain water, which increases stool size and facilitates bowel motions, it is also used to alleviate constipation.¹⁰

3. INTRODUCTION OF BACTERIA’S:

3.1 Escherichia coli: Theodor Escherich, a German pediatrician who lived from 1857 to 1911, identified the germs Escherichia coli from infants' feces in 1885. Gram-negative, non-sporulating, rod-shaped, facultatively anaerobic, and coliform, Escherichia coli is a common bacteria found in food, the environment, and the lower stomach of warm-blooded animals. With in the fields of microbiology and biotechnology, it is the most extensively researched prokaryotic model organism. Long-lived in soil, water, and excrement, it is commonly employed as an indicator organism for water contamination. The bacterium grows quickly in fresh feces under aerobic conditions for two to three days, but then its numbers progressively decline. The bacilli of E. coli are straight, rod-shaped, gram-negative, non-sporing, and non-acid fast. They can be found alone or in pairs. Typically, cells have a rod-like structure, measuring 1-3μm × 0.4-0.7μm (micrometer), measuring approximately 1μm in length, 0.35μm in width, and 0.6-0.7μm in volume. It is motile due to peritrichous flagellar arrangement, and very few strains are non-motile. The optimal growth of E. coli occurs at 37°C (98°F) but some laboratory strains can multiply at temperatures of up to 49°C (120.2°F). It takes as little as 20 min to reproduce in favorable conditions. There are both motile and non-motile imbriated strains.¹¹

Fig. 2: Escherichia coli

3.2 Staphylococcus aureus:

More than 40 species of Gram-positive bacteria make up the genus Staphylococcus; many of these are benign and frequently found on human and animal skin and mucous membranes. However, a variety of infections, ranging from mild skin irritations to serious illnesses, can be caused by some species, including Staphylococcus aureus. Because of their spherical shape and propensity to cluster under a microscope like grapes, these bacteria get their name from the Greek word "staphyle," which means bunch of grapes.

The most harmful species of the genus, Staphylococcus aureus, causes a number of illnesses, including cellulitis, boils, impetigo, and abscesses. More serious illnesses like sepsis, endocarditis, pneumonia, osteomyelitis, and toxic shock syndrome can also result from it. The potential for S. aureus to become resistant to antibiotics is one of the main issues. A major public health problem in both hospital and community settings is the well-known strain of Methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to multiple widely used medications.

Fig. 3: Staphylococcus aureus.

4. Standard drug used for Anti-bacterial activity:

4.1 Gentamicin: An aminoglycoside antibiotic called gentamicin is used to treat a variety of bacterial illnesses. Among other conditions, these could include sepsis, pneumonia, meningitis, endocarditis, inflammation of the pelvis, bone infections, and urinary tract infections. For chlamydia or gonorrhea infections, it is ineffective. It can be used topically, intramuscularly, or intravenously. Topical preparations can be used for external eye infections or burns. Until bacterial cultures identify which particular antibiotics the infection is sensitive to, it is frequently only administered for two days. Blood tests should be used to track the necessary dosage.¹³

Formula : C₂₁H₄₃N₅O₇

Molar mass : 477.603g·mol⁻¹

5. MATERIALS AND METHODS:

5.1 Plant Material Collection: Cyamopsis tetragonoloba (Cluster Beans) seeds Was Collected From The Local Market of Jalgon. The beans were then thoroughly cleaned using tap water and then distilled water. They were then covered with a clear transparent cloth and left to dry in the shade. After that, the beans were crushed with the aid of mortar and Pastle, and the seeds were extracted. After being ground into a coarsed powder, the dried seed components were placed in an airtight container to be used later for solvent extraction.

5.2 Test Organism: Microbial strains of Staphylococcus aureus (Gram Positive); Escherichia coli (Gram Negative) were provided by P.S.G.V.P.M’s Department of Microbiology. They were sub-culture and used throughout the studies.

5.3 Solvents used:

1] Chloroform

2] Petroleum Ether

5.4 Chemicals: (Tween 60): Tween 60 was used to dissolve or disperse the extract more uniformly in the testing medium (e.g., agar plates or broth). This ensures that the antibacterial activity is tested more consistently and represents the true potency of the extract. This chemical (Tween 60) was mainly provided by P.S.G.V.P.M’s Department of Pharmacognosy.

6. Extraction Procedure: Cluster bean (Cyamopsis tetragonoloba) seeds were cleaned, shade-dried, crushed, and 40 grams were used for extraction. First, the crushed seeds were macerated with petroleum ether for 24 hours at room temperature. After collecting the petroleum ether extract, the remaining seed material (34 grams) was then extracted with chloroform under the same conditions. Both extracts, obtained through successive cold maceration, were dried and concentrated by distillation to recover the solvents.

7. Determination of Anti-bacterial Activity:

Procedure: Anti-bacterial activity was assessed by Agar well diffusion method of where in Nuterient agar plates were prepared and was spreaded with 20µl of the available pathogenic cultures. Wells of 8 mm diameter were bored using sterile borer. Wells were loaded with anti-bacterial, Gentamicin as standrad and were incubated at 37°C for 48hours. The zone of inhibition was measured and expressed in mm.

7.1 Preparation of extract: The extract prepared as suspension for evaluation using solvent water and Tween 60. First, Plant seed Petroleum ether extract suspension was prepared with concentration 58 mg /ml and plant seeed chloroform extract suspension was prepared its concentration 48mg ml/ml. To make up the volume distilled water was used as solvent. To enhance the stability of the suspension, Tween 60 was incorporated at a concentration of about 0.5% (v/v). This surfactant helped in evenly dispersing the extract throughout the water. The mixture was stirred thoroughly to ensure complete dissolution and uniformity. After confirming that the extract was well- dispersed, the consistency of the suspension was checked, and additional water or Tween 60 was added as needed to achieve the desired texture. Once the suspension was prepared, it was stored in an appropriate container for further study.

7.2 Preparation of Culture: The culture medium was prepared by dissolving 1.5g of agar-agar and 1.5g of nutrient agar in 100mL of distilled water. The mixture was heated gently with continuous stirring untill all components were completely dissolved and a clear solution was obtained. The prepared medium was then sterilized by autoclaving at 121°C under 15 psi pressure for 15minutes. After sterilization, the medium was allowed to cool to about 45–50°C and was poured aseptically into sterile Petri plates. The plates were left at room temperature to solidify and were later used for bacterial inoculation and incubation.

7.3 Preparation of Species:

Pure cultures of Escherichia coli and Staphylococcus aureus were obtained from P.S.G.V.P.M's department of Microbiology stock cultures. A sterile inoculating loop was used to transfer a small amount of each bacterial culture into separate test tubes containing sterile nutrient broth. The inoculated tubes were then incubated at 37°C for 24hours to allow active bacterial growth. After incubation, the cultures exhibited visible turbidity, confirming successful bacterial proliferation. These actively growing cultures were subsequently used for antibacterial sensitivity testing.

7.4 Antimicrobial Activity: The C. tetragonoloba seed extract prepared above was subjeced to antimicrobial acivity to check its potential agains both gram positive and grams negative sspecies. The do so following protocol was followed.

1. Well Diffusion Method:

Procedure:

· Microbial cultures were evenly spread over agar plates.

· Wells were created in the agar using a sterile punch.

· A fixed volume of the chloroform and petroleum ether extracts was added to each well.

· The plates were incubated at appropriate temperatures for the growth of microorganisms.

· After incubation, the diameter of the clear zone around each well was measured.

Interpretation:

The antimicrobial tests showed that chloroform extracts of cluster bean seeds had significantly stronger activity than petroleum ether extracts, as evidenced by larger zones of inhibition. This suggests that chloroform extracts are a promising source of natural antimicrobial agents.

8. RESULT:

8.1 Plant Extraction: The extraction perfomed on dried extract seeds of C. Tetragonoloba was perfomed and the result of extraction protocol was as follows

Table 1: Result of Petroleum Ether and Chloroform Extract of Cluster Beans Seed.

Parameters	Petroleum Ether Extract	Chloroform Extract
Initial Plant Material Used (seeds)	40 g	34 g (after P.E. extraction)
Solvent Used	Petroleum Ether	Chloroform
Extraction Method	Cold Maceration	Cold Maceration
Duration of Extraction	24 hrs.	24 hrs.
Appearance of Extract	Oily, Pale Yellow	Sticky, Light Brown
Yield of Extract (%)	1.45 %	1.41%
Consistency of Extract	Semi-solid	Semi-solid
Storage Condition	Airtight container, Cool and dry place.	Airtight container, Cool and dry place.

8.2 Zone of Inhibition:

The anti-bacterial Activity of Cyamopsis tetragonoloba (Cluster Beans) were studied against bacterias like Escherichia Coli and Staphylococcus Aureus. The following table shows The results obtained after the antibacterial test:

Bacteria’s	ZOI by Gentamicin	ZOI by Petroleum Ether	ZOI by Chloroform
Escherichia coli	23mm	5mm	30mm
Staphylococcus aureus	13mm	3mm	15mm

Fig.7: Anti-bacterial activity of Petroleum Ether and Chloroform Extract against Escherichia coli.

Fig. 8: Anti-bacterial activity of Petroleum Ether and Chloroform Extract against Staphylococcus aureus.

9. DISCUSSION:

The study evaluated the antibacterial activity of Cyamopsis tetragonoloba (cluster bean) seed extracts against E. coli and Staphylococcus aureus, using gentamicin as a control. Both petroleum ether and chloroform extracts showed activity, but the chloroform extract demonstrated significantly stronger antibacterial effects. It produced a 30 mm zone of inhibition (ZOI) against E. coli and 15 mm against S. aureus, outperforming gentamicin in both cases. In contrast, the petroleum ether extract showed minimal activity, likely due to its non-polar nature, which limits extraction of potent bioactive compounds. These findings suggest that polar and semi-polar compounds in the chloroform extract, such as flavonoids and phenolics, contribute to its potent antibacterial properties.

10. CONCLUSION:

In conclusion, the study confirms that cluster bean seed extracts, especially those obtained with chloroform, have notable antibacterial properties, outperforming even gentamicin in some cases. The minimal activity of the petroleum ether extract suggests that the effective compounds are likely polar. These results support the potential use of cluster bean seeds as a natural antibacterial source and encourage further research to isolate and develop these bioactive compounds for use against antibiotic-resistant bacteria.

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Received on 08.05.2025 Revised on 21.06.2025

Accepted on 27.07.2025 Published on 10.10.2025

Available online from October 18, 2025

Res. J. Pharmacognosy and Phytochem. 2025; 17(4):284-288.

DOI: 10.52711/0975-4385.2025.00045

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