Description:[0001] The present invention relates to a method for preparing an aqueous extract of Psidium guajava (red apple variety) leaves, rich in phytoconstituents suitable for pharmaceutical and nutraceutical applications. Healthy leaves are collected, authenticated, and thoroughly washed with distilled water to remove impurities. The leaves are then shade-dried for 3–5 days to preserve heat-sensitive compounds, coarsely powdered, and accurately weighed. Approximately 30 g of powdered leaf material is refluxed with 300 mL of distilled water in a round-bottom flask for 4 hours with intermittent stirring. The mixture is filtered through muslin cloth or Whatman No. 1 filter paper to separate solid residues, and the filtrate is concentrated and dried on a china dish using a water bath at about 80°C to yield a stable dry extract. The standardized extract retains phytochemicals such as flavonoids, tannins, phenolics, and saponins, making it valuable for antioxidant, antimicrobial, and metabolic health formulations.
BACKGROUND ART
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Plants have long served as sources of therapeutic agents owing to their diverse phytochemical constituents. Traditional medicinal systems have employed guava (Psidium guajava L., Family: Myrtaceae) for centuries in the treatment of diarrhea, diabetes, gastrointestinal disorders, and infectious diseases. Modern research highlights the pharmacological potential of guava leaves due to the presence of flavonoids, tannins, alkaloids, and phenolic compounds.
[0004] Several herbal extracts have been reported for antioxidant and anthelmintic activity. However, existing formulations often suffer from poor aqueous solubility, low stability, and limited bioactivity. Moreover, studies have predominantly focused on common guava varieties, leaving unexplored the Red Apple variety of Psidium guajava, which is particularly rich in anthocyanins and unique phytoconstituents.
[0005] US10293013B2 The present invention provides a method of obtaining Psidium guajava leaf extract standardized to phytochemicals. The extract obtained is highly soluble in water, and contains standardized phytochemicals such as, guijavarin specifically saponins and polyphenols, which may be used in food and beverage products. The method involves the specific method of filtration to obtain highly purified form of phytochemical. The extract obtained is subjected to bio-activity guided fractionation to isolate different compounds to obtain phytochemical enriched fraction followed by purification and isolation of the single phytochemical from the enriched bioactive fraction. The phytochemical is identified as guijaverin, which also exhibits anti-diabetic activity. The Psidium guajava leaf extract is useful in food and beverage industries and is used in different formulations such as chocolates, capsules, and aqua based supplement drinks.
[0006] US8021699B2 The invention provides a method for producing a guava leaf extract powder, including extracting guava leaves with hot water at 90 to 98° C.; concentrating the obtained extract to a Brix level of 20 to 30; and lyophilizing the concentrated extract, and a guava leaf extract powder produced through the method which exhibits limited deterioration in quality after storage in solution for a long period of time.
[0007] Park H. et al., 2024 — GC-MS profiling and biological activity of P. guajava leaf extracts (PMC article). Guava (Psidium guajava) is a plant widely distributed in tropical and subtropical regions. Its leaves contain a large number of physiological molecules such as flavonoid, sesquiterpene, triterpenoid, coumarin, alkaloid, and tannin molecules with antioxidative and anti-inflammatory effects. In this study, the use of concentrated P. guajava leaf extract molecules as a functional natural material was evaluated by confirming the extract’s antioxidative, antibacterial, tyrosinase activity inhibition, and collagenase activity inhibition effects and its trans-2-nonenal removal ability. As a result of the analysis of the antioxidant and antibacterial components of concentrated P. guajava leaf extract molecules through GC-MS, a large amount of aromatic hydrocarbon molecules were detected. When different concentrations of ethanol were used for extraction, the leaf extract concentrated with 70% ethanol showed the most effective active molecules. As a result of measuring DPPH radical scavenging activity, a concentration-dependent antioxidant activity was confirmed. The antioxidant activity tended to increase when the ethanol content used for extraction was increased. Molecules such as 2,4-di-tert-butylphenol, caryophyllene oxide, and γ-muurolene in P. guajava leaf extract concentrate appeared to have antibacterial activities against S. aureus bacteria known to cause atopy. As ethanol content increased, the inhibitory effect on tyrosinase activity was increased. In addition, when ethanol content was 50%, the concentrated leaf extract was able to remove trans-2-nonenal by 52.4%. As a result of determining the concentrated leaf extract’s collagenase inhibition activity, an inhibition rate close to that of ascorbic acid, a positive control, was confirmed. The concentrated guajava leaf extract molecules were confirmed to have whitening and wrinkle-improving functionality. Thus, the P. guajava leaf extract has high potential as a food and natural cosmetic material.
[0008] Gutierrez-Montiel D. et al., 2023 — Review: Psidium guajava as a source of bioactives. Mexico is one of the largest guava producers in the world, so it has access to a huge amount of waste and byproducts obtained after the industrial processing of the fruit. This review discusses the potential recovery of this residue for its application as an antimicrobial agent, considering the phytochemical composition, the bioactivity reported in-vivo and in-vitro, and the toxicology of the plant. Nowadays there is a growing demand for more natural and safer products, so the use of guava extracts is an interesting initiative, especially due to its availability in the country, its wide variety of traditional uses, and its phytochemical profile. This review highlights the importance and potential antimicrobial use of this plant in today's world.
[0009] Accordingly, there exists a need for a stable, water-based extract of Psidium guajava leaves that demonstrates reproducible antioxidant and anthelmintic efficacy, while being suitable for pharmaceutical and nutraceutical applications.
[0010] The invention discloses an aqueous extract of Psidium guajava (Red Apple variety) leaves exhibiting potent antioxidant and anthelmintic activity. The extract is prepared by hot water extraction, yielding 24% w/w of a dark brown, water-soluble material (pH 6.8). Phytochemical analysis confirmed the presence of alkaloids, flavonoids, tannins, phenolics, and terpenoids, while GC-MS profiling identified compounds including 9,12-Octadecadienoyl chloride (Z,Z)-, Glycidyl palmitate, β-Sitosterol acetate, and Avocadenol D.
OBJECTS OF THE INVENTION
[0011] The principal object of the present invention is to overcome the disadvantages of the prior art.
[0012] The primary object of the present invention is to provide an aqueous extract of Psidium guajava (Red Apple variety) leaves with high antioxidant and anthelmintic activity.
[0013] Another objective of the present invention is to develop a reproducible method for the preparation of the aqueous extract.
[0014] Another objective of the present invention is to identify the phytochemical constituents through phytochemical tests and GC-MS analysis.
[0015] Another objective of the present invention is to evaluate antioxidant activity of the extract using the DPPH radical scavenging assay.
[0016] Another objective of the present invention is to investigate the in vitro anthelmintic activity of the extract using Lumbricus terrestris worms, comparing efficacy with standard albendazole.
[0017] Yet another objective of the present invention is to provide a composition suitable for pharmaceutical, nutraceutical, and veterinary applications.
[0018] Another objective of the present invention is to provide a standardized and reproducible method for preparing an aqueous extract of Psidium guajava (red apple variety) leaves while preserving phytoconstituents, including heat-sensitive compounds.
[0019] Another objective of the present invention is to develop a simple, cost-effective, and eco-friendly extraction process using distilled water as a solvent, avoiding harmful organic solvents.
[0020] Another objective of the present invention is to ensure maximum yield of bioactive compounds such as flavonoids, tannins, phenolics, and saponins from Psidium guajava leaves.
[0021] Another objective of the present invention is to obtain a stable dry extract through controlled drying techniques, suitable for storage and further pharmaceutical or nutraceutical formulation.
[0022] Another objective of the present invention is to provide an extraction process adaptable for scale-up in laboratory and industrial applications without compromising quality.
[0023] Another objective of the present invention is to enhance the potential of Psidium guajava extracts in therapeutic domains such as antioxidant, antimicrobial, antidiabetic, and metabolic health formulations.
[0024] Another objective of the present invention is to create a foundation for developing herbal dosage forms such as capsules, tablets, syrups, or functional food products based on standardized Psidium guajava aqueous extract.
SUMMARY
[0025] The invention discloses a novel aqueous extract prepared from dried leaves of Psidium guajava (Red Apple variety). The extract is obtained through hot water extraction, yielding approximately 24% w/w of a dark brown, water-soluble residue with pH 6.8.
[0026] The present invention provides a novel and standardized method for preparing an aqueous extract of Psidium guajava (red apple variety) leaves with retention of bioactive phytoconstituents. The method includes collecting healthy leaves, authenticating the plant material, thoroughly washing the leaves with distilled water, and air-drying them under shade for 3–5 days to preserve heat-sensitive compounds. The dried leaves are coarsely powdered using a mechanical grinder, and an accurately weighed portion (about 30 g) is subjected to aqueous extraction in a round-bottom flask with 300 mL of distilled water. The mixture is heated gently on a heating mantle for approximately 4 hours with intermittent stirring.
[0027] After extraction, the mixture is filtered through muslin cloth or Whatman No. 1 filter paper to remove plant debris, and the resulting filtrate is concentrated and dried in a China dish using a water bath at 80°C. The dry extract thus obtained is rich in phytochemicals such as flavonoids, tannins, phenolic acids, and saponins.
[0028] The invention ensures a cost-effective, reproducible, and eco-friendly extraction process, yielding a stable dry extract suitable for pharmaceutical, nutraceutical, and therapeutic applications, including antioxidant, antimicrobial, and metabolic health formulations.
BRIEF DESCRIPTION OF DRAWINGS
[0029] The accompanying illustrations are incorporated into and form a part of this specification in order to aid in comprehending the current disclosure. The pictures demonstrate exemplary implementations of the current disclosure and, along with the description, assist to clarify its fundamental ideas.
[0030] Fig.1 shows the flow chart of extraction process of Psidium guajava leaves.
[0031] Fig 2 shows the morphological features of Psidium guajava (Red Apple variety) leaf.
[0032] Fig 3 shows the microscopic transverse section of guava leaf.
[0033] Fig 4 shows the powder microscopy features of guava leaf powder.
[0034] Fig 5 shows the graph showing antioxidant (DPPH) scavenging activity.
[0035] Fig 6 shows the comparative chart of anthelmintic activity vs. albendazole.
[0036] It should be noted that the figures are not drawn to scale, and the elements of similar structure and functions are generally represented by like reference numerals for illustrative purposes throughout the figures. It should be noted that the figures do not illustrate every aspect of the described embodiment sand do not limit the scope of the present disclosure.
[0037] Other objects, advantages, and novel features of the invention will become apparent from the following detailed description of the present embodiment when taken in conjunction with the accompanying drawings.
DETAILED DESCRIPTION OF THE INVENTION
[0038] While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and the detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim.
[0039] As used throughout this description, the word "may" is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents acts, materials, devices, articles, and the like are included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.
[0040] In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.
[0041] The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, several materials are identified as suitable for various facets of the implementations.
[0001] The present invention relates to the field of herbal drug formulations, nutraceuticals, and functional foods. More particularly, it relates to an aqueous extract of Hibiscus sabdariffa L. exhibiting potent antioxidant, anthelmintic, and antidiabetic activities, and to the formulation of an herbal jelly incorporating the extract for human consumption.
[0002] In an embodiment of the present invention the method A method for preparing an aqueous extract of Psidium guajava (red apple variety) leaves, comprising the steps of: i) collecting healthy leaves of Psidium guajava from a cultivation farm and authenticating the plant material at a recognized botanical authority; ii) washing the collected leaves thoroughly with distilled water to remove dirt and contaminants; iii) air-drying the leaves under shade for 3–5 days to preserve phytoconstituents, including heat-sensitive compounds; iv) coarsely powdering the dried leaves using a mechanical grinder; v) accurately weighing about 30 grams of the powdered leaf material and transferring it into a round-bottom flask of about 500 mL capacity; vi) adding approximately 300 mL of distilled water into the flask and heating the mixture gently on a heating mantle for about 4 hours with occasional stirring; vii) filtering the mixture through muslin cloth or Whatman No. 1 filter paper to separate solid plant debris from the filtrate; and viii) drying the filtrate on a China dish using a water bath maintained at about 80°C to obtain a dry aqueous extract of Psidium guajava leaves.
[0003] In an embodiment of the present invention, there is provided a plant-based extract derived from the leaves of Psidium guajava (guava) for use in nutraceutical, pharmaceutical, and functional food applications. The invention particularly relates to the preparation, characterization, and evaluation of guava leaf extract for its antidiabetic and anthelmintic activities.
[0004] In an aspect of the invention, the guava leaves are collected, washed, shade-dried, and pulverized to obtain a fine powder. The powdered leaves are subjected to extraction using solvents selected from ethanol, methanol, water, or hydroalcoholic mixtures under controlled conditions. The extraction may be carried out by maceration, Soxhlet extraction, or ultrasound-assisted extraction, followed by filtration and concentration under reduced pressure to yield a crude extract.
[0005] In another embodiment of the present invention, the obtained crude extract is subjected to phytochemical screening to determine the presence of bioactive compounds such as flavonoids, tannins, saponins, terpenoids, alkaloids, and phenolic acids. The extract may further be standardized based on marker compounds such as quercetin and other flavonoid glycosides known to be abundant in guava leaves.
[0006] In an aspect of the invention, the biological evaluation of the guava leaf extract is conducted using a yeast cell glucose uptake assay to determine antidiabetic potential. Yeast cells (Saccharomyces cerevisiae) are incubated with varying concentrations of glucose in the presence and absence of the extract. The glucose uptake by yeast cells is measured spectrophotometrically, wherein an increase in glucose uptake in the presence of the extract is indicative of antidiabetic activity.
[0007] In another embodiment of the present invention, the anthelmintic activity of the guava leaf extract is assessed using Pheretima posthuma (earthworms) as the experimental model. The worms are exposed to varying concentrations of the extract, and the time taken for paralysis and death of the worms is recorded. The activity of the extract is compared with a standard reference drug such as albendazole or piperazine citrate.
[0008] In an aspect of the invention, the extract exhibits a dose-dependent increase in glucose uptake and a significant reduction in the survival time of helminths, thereby demonstrating both antidiabetic and anthelmintic efficacy.
[0009] In another embodiment of the present invention, the guava leaf extract may be formulated into various dosage forms, including but not limited to capsules, tablets, syrups, granules, or functional food products. The formulation may be combined with pharmaceutically acceptable carriers, excipients, or stabilizers, enabling its application in preventive and therapeutic healthcare.
[0010] In an aspect of the invention, the standardized guava leaf extract may also be incorporated into cosmetic formulations for skin health, owing to its antioxidant and antimicrobial properties, in addition to its primary nutraceutical and pharmaceutical applications
[0011] In another embodiment, the invention provides a method of preparing an antidiabetic and anthelmintic composition comprising guava leaf extract, wherein the composition is effective, natural, and economically feasible, thereby addressing the limitations of synthetic drugs which often cause adverse effects and resistance.
[0012] In an embodiment of the present invention, the morphological characteristics of Psidium guajava leaves, particularly of the red apple variety, were documented to ensure proper botanical identification and authentication prior to experimental evaluation. The leaves are simple, with opposite phyllotaxy, and exhibit an elliptical to ovate shape, being broadest at the center and tapering toward both the apex and base. The leaves measure approximately 7–15 centimeters in length and 3–5 centimeters in width. The apex is acute to slightly pointed, while the base is cuneate (wedge-shaped). The leaf margin is entire, displaying no serrations, and the venation is pinnate with prominent, upwardly curved secondary veins. The coloration is reddish to purplish owing to the presence of anthocyanin pigments, particularly characteristic of the red apple guava variety. The texture is coriaceous (leathery), slightly rough to the touch, with the dorsal (upper) surface being glossy and darker in shade, while the ventral (lower) surface is lighter and more textured. The petiole is short or nearly absent.
[0013] In another embodiment of the present invention, the plant material was collected in Thirthahalli, Shimoga district, Karnataka, India. The identity of the specimen was authenticated by the Department of Botany, SRNM College, Shivamogga. Fresh, healthy leaves were carefully selected, washed thoroughly with distilled water to eliminate dirt and surface contaminants, and shade-dried at ambient temperature for a period of 3–5 days to prevent the degradation of thermolabile phytoconstituents.
[0014] In an aspect of the invention, the dried leaves were coarsely powdered using a mechanical grinder to obtain a uniform particle size suitable for subsequent extraction procedures. The powdered material was stored in airtight containers under dry conditions until further use, thereby ensuring the stability and preservation of the bioactive compounds.
Parameter Fresh Leaves Dried Leaves
Moisture content (%) ~70–80% ~5–10%
Color Bright green with red-tinged midrib Pale green to olive-brown
Texture Soft, flexible Brittle, crisp
Odor Characteristic Mild
Thickness (mm) 0.2–0.4 mm (varies with age and water content) 0.1–0.2 mm (due to dehydration)

[0015] In an embodiment of the present invention, microscopic analysis of the Psidium guajava (red apple variety) leaf was carried out to confirm diagnostic anatomical features. A fresh, healthy leaf was thoroughly washed with distilled water to remove surface debris, and thin transverse sections (T.S.) were obtained from the midrib region using a sharp blade.
[0016] In an aspect of the invention, the sections were mounted on glass slides and treated with chloral hydrate solution, followed by gentle heating over a flame to clear the tissues and enhance structural visibility. For specific identification of lignified tissues such as fibers and vessels, the sections were further treated with phloroglucinol in combination with hydrochloric acid, which imparted characteristic coloration to lignified elements.
[0017] In another embodiment of the present invention, a drop of glycerin–water mixture was added to the prepared section on the slide to preserve tissue integrity and prevent drying. A clean coverslip was carefully placed over the section to avoid the entrapment of air bubbles, thereby ensuring accurate microscopic examination.
[0018] In an aspect of the invention, the microscopic observations revealed diagnostic features consistent with Psidium guajava leaf anatomy, which served as confirmatory evidence for the authenticity of the plant material used in subsequent extraction and biological evaluation studies.

[0019] Microscopic Characteristics of Guava Leaf Powder, to perform powder microscopy of Psidium guajava, leaves, healthy and mature leaves are first collected, cleaned, and dried under shade to preserve their phytoconstituents. Once fully dried, the leaves are ground into a fine or moderately coarse powder using a mortar and pestle or mechanical grinder. A small amount of the powder is then placed on a clean glass slide for microscopic examination.
[0020] In an embodiment of the present invention, approximately 30 grams of dried and powdered Psidium guajava (red apple variety) leaves were accurately weighed and transferred into a 500 mL round-bottom flask. To this, 300 mL of distilled water was added, and the mixture was subjected to gentle heating on a heating mantle for about 4 hours with intermittent stirring. The hot mixture was filtered through muslin cloth followed by Whatman No. 1 filter paper to remove coarse plant debris.
[0021] In an aspect of the invention, the filtrate obtained was concentrated by evaporating the solvent on a water bath at approximately 80 °C, thereby yielding a dry extract. The percentage yield of the extract was calculated using the formula:

[0022] The concentrated extract was preserved in airtight containers at 4 °C until further use for phytochemical and biological studies.
[0023] In an embodiment of the present invention, preliminary phytochemical screening of the aqueous guava leaf extract was carried out to detect the presence of primary and secondary metabolites using standard qualitative methods.
[0024] In an embodiment of the present invention, preliminary phytochemical analysis of the aqueous extract of Psidium guajava leaves was performed to identify the major classes of bioactive compounds. The following qualitative tests were conducted:
[0025] In an embodiment of the present invention, the phytochemical screening of the extract was carried out using standard qualitative tests for different classes of bioactive compounds. For carbohydrates, the Molisch test confirmed their presence by the appearance of a violet ring at the interface when the filtrate was treated with alcoholic α-naphthol and concentrated sulfuric acid, while the Benedict’s test indicated the presence of reducing sugars through the formation of an orange-red precipitate upon heating with Benedict’s reagent. For alkaloids, Mayer’s test yielded a cream or pale-yellow precipitate, Hager’s test produced a yellow precipitate, and Wagner’s test gave a reddish-brown precipitate, collectively confirming the presence of alkaloidal constituents. Proteins were identified by a bluish-violet coloration in the Biuret test upon treatment with sodium hydroxide and copper sulfate, and by a blue to violet coloration in the Ninhydrin test after heating with Ninhydrin reagent, confirming amino acids and proteinaceous compounds. Tannins were detected through the Ferric Chloride test, which produced blue-black, violet, or green coloration. The presence of flavonoids was established by multiple tests, including the Sodium Hydroxide test showing yellow coloration, the Lead Acetate test producing a yellow precipitate, the Mineral Acid test yielding an orange coloration, and the Zinc/HCl test giving a red coloration, thereby confirming flavonoid constituents.
[0026] In an aspect of the invention, the positive responses in these tests collectively demonstrated the presence of carbohydrates, alkaloids, proteins, tannins, and flavonoids in the aqueous guava leaf extract, thereby supporting its potential pharmacological activity.
[0027] In an embodiment of the present invention, the aqueous and methanolic extracts of Psidium guajava (red apple variety) leaves were subjected to Gas Chromatography and Mass Spectroscopy (GC–MS) analysis to determine the phytoconstituents present in the extract. The technique enabled separation, detection, and characterization of volatile and semi-volatile compounds based on their retention times and mass spectral fragmentation patterns. The chromatographic peaks obtained were analyzed by comparing the mass spectra with standard spectral libraries, thereby confirming the presence of diverse classes of bioactive compounds such as flavonoids, terpenoids, phenolics, sterols, and alkaloids.
[0028] In an aspect of the invention, the antioxidant potential of Psidium guajava leaf extracts was evaluated using the in vitro free radical scavenging method with DPPH (1,1-diphenyl-2-picryl hydrazyl).
[0029] In an embodiment of the present invention, the reagents employed for the assay included a DPPH working solution prepared to yield an absorbance of 0.9 at 516 nm by dissolving 4.3 µg of DPPH in 3 mL of methanol, a standard solution consisting of ascorbic acid at a concentration of 50 mg per 100 mL of methanol serving as the reference antioxidant, and test solutions comprising aqueous and methanolic extracts of Psidium guajava leaves prepared at a concentration of 1 mg/mL in their respective solvents.
[0030] In an embodiment, 75 µL of the DPPH solution was mixed with different concentrations of ascorbic acid, and the volume was adjusted to 3 mL with methanol. DPPH diluted in methanol served as the blank. The mixtures were incubated for 15 minutes at room temperature, and the decrease in absorbance was recorded at 516 nm. Linear regression was applied between concentration and percentage inhibition to calculate the effective concentration (EC₅₀), defined as the concentration required to reduce the initial absorbance by 50%.
[0031] Test solutions of the guava leaf extracts were evaluated. Both aqueous and methanolic extracts demonstrated free radical scavenging activity, with EC₅₀ values indicating significant antioxidant potential comparable to the standard ascorbic acid.
[0032] In another aspect of the invention, the anthelmintic activity of Psidium guajava leaf extracts was evaluated using adult earthworms (Lumbricus terrestris), chosen due to their anatomical and physiological similarity to human intestinal roundworms.
[0033] In an embodiment of the present invention, the experimental design comprised the evaluation of aqueous extracts of Psidium guajava leaves, which were freshly prepared prior to the assay. Earthworms of approximately equal size were selected as the test organisms, and were divided into groups for exposure to 20 mL of test solutions at varying concentrations of 25 mg/mL and 50 mg/mL. For comparison, albendazole at a concentration of 20 mg/mL was employed as the reference standard, while normal saline was used as the negative control.

[0034] The time of paralysis was recorded when no movement was observed except upon vigorous shaking. The time of death was determined when worms did not respond to any stimulation.
[0035] In an embodiment, the guava leaf extract demonstrated concentration-dependent anthelmintic activity. The extract at 50 mg/mL induced paralysis and death of worms significantly faster than the lower concentration (25 mg/mL), though slightly less potent than the reference albendazole.
[0036] Results of Phytochemical screening of Psidium guajava (red apple variety)
[0037] Report of gas chromatography and mass spectroscopy
[0038] The GC–MS analysis of Psidium guajava leaf extract revealed the presence of various bioactive compounds, including [9,12-Octadecadienoyl chloride (Z,Z)- , Glycidylpalmitate, β-Sitosterol acetate, Avocadenol D], which are known to possess pharmacological activities such as antioxidant, antimicrobial, anti-inflammatory, and antidiabetic effects.
[0039] In an embodiment of the present invention, the in vitro anthelmintic activity of the Psidium guajava leaf extract was evaluated against adult earthworms (Lumbricus terrestris), selected due to their anatomical and physiological resemblance to human intestinal roundworms. The assay was conducted using albendazole (20 mg/mL) as the standard reference drug and normal saline as the control.
[0040] The test extract was prepared freshly and administered in concentrations of 25 mg/mL and 50 mg/mL. Groups of earthworms of nearly equal size were released into Petri dishes containing 20 mL of the respective test and control solutions. The onset of paralysis was determined as the time point at which the worms ceased movement except upon vigorous stimulation, while the time of death was recorded when no response to external stimuli could be observed.
[0041]
[0042] In an aspect of the invention, the results demonstrated that the extract exhibited a clear concentration-dependent anthelmintic effect. The higher concentration (50 mg/mL) significantly reduced both paralysis time and death time compared to the lower concentration (25 mg/mL), indicating a strong dose–response relationship. When compared with albendazole, the extract showed moderate to strong activity, thereby validating its potential as a natural anthelmintic agent.
[0043] In another aspect, the observed activity is attributed to the presence of phytoconstituents such as tannins, alkaloids, and flavonoids in the extract. These compounds are known to impair worm neuromuscular activity, disrupt energy metabolism, and alter structural proteins, thereby inducing paralysis and subsequent mortality.

[0044] Further, the operations need not be performed in the disclosed order, although in some examples, an order may be preferred. Also, not all functions need to be performed to achieve the desired advantages of the disclosed system and method, and therefore not all functions are required.
[0045] Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the embodiments shown along with the accompanying drawings but is to be providing the broadest scope consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the scope of the present invention and appended claims.
, Claims:1. A method for preparing an aqueous extract of Psidium guajava (red apple variety) leaves, comprising the steps of:
i) collecting healthy leaves of Psidium guajava from a cultivation farm and authenticating the plant material at a recognized botanical authority;
ii) washing the collected leaves thoroughly with distilled water to remove dirt and contaminants;
iii) air-drying the leaves under shade for 3–5 days to preserve phytoconstituents, including heat-sensitive compounds;
iv) coarsely powdering the dried leaves using a mechanical grinder;
v) accurately weighing about 30 grams of the powdered leaf material and transferring it into a round-bottom flask of about 500 mL capacity;
vi) adding approximately 300 mL of distilled water into the flask and heating the mixture gently on a heating mantle for about 4 hours with occasional stirring;
vii) filtering the mixture through muslin cloth or Whatman No. 1 filter paper to separate solid plant debris from the filtrate; and
viii) drying the filtrate on a China dish using a water bath maintained at about 80°C to obtain a dry aqueous extract of Psidium guajava leaves.
2. The method as claimed in claim 1, wherein the filtrate is concentrated prior to drying using rotary evaporation for efficient solvent removal.
3. The method as claimed in claim 1, wherein the air-drying process is carried out at ambient temperature away from direct sunlight to prevent degradation of phytoconstituents.
4. The method as claimed in claim 1, wherein the final dried extract is stored in an airtight container to prevent moisture absorption and phytoconstituent degradation.
5. The method as claimed in claim 1, wherein the extract is further subjected to phytochemical screening to identify flavonoids, tannins, saponins, and phenolic compounds present in Psidium guajava leaves.