Description:TECHNICAL FIELD
[0001] The present invention relates to the field of herbal drug development and phytopharmacology, and more particularly to a novel pharmaceutical composition derived from aqueous extracts of Ruta graveolens leaves exhibiting anthelmintic and antidiabetic properties.
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] Parasitic helminth infections and diabetes mellitus remain persistent health challenges worldwide. Current synthetic anthelmintic drugs such as albendazole and mebendazole face limitations including resistance development and adverse side effects. Similarly, conventional antidiabetic therapies, though effective, are often associated with complications, limited accessibility, and economic burden, particularly in developing countries.
[0004] Herbal medicines have gained significant interest as alternative therapies owing to their safety, affordability, and bioactive phytoconstituents. Ruta graveolens (commonly known as Rue) is traditionally used for medicinal purposes including antimicrobial, antifungal, and metabolic disorders. However, systematic evaluation of aqueous extracts of Ruta graveolens leaves for their antidiabetic and anthelmintic activity has not been reported in prior art.
[0005] WO2014072773A1 The invention relates to the use of a hydroalcoholic plant extract obtained from the Ruta graveolens species in order to prevent and/or treat arterial hypertension, as it exhibits a marked vasodilator effect in vitro and anti-hypertensive effect in vivo, the latter being verified by orally administering the extract to rats in which a hypertensive level has been induced. The extract is orally administered in a lyophilized manner, either in a capsule or directly dissolved in water, with a mainly preventive purpose, but also as a therapeutic agent for the hypertensive level, as there are currently no monotherapies that have an adequate cost-effectiveness ratio. Although there are various types of drugs that are usually used in the treatment of this disease, the efficiency thereof is not adequate and they normally have to be combined, potentially presenting undesirable side effects.
[0006] Revista de la Facultad de Ciencias Veterinarias Rue (Ruta graveolens) is an herb belonging to the Rutaceae family, characterized by various medicinal properties. Its consumption has been reported as part of therapy in diabetes mellitus, based on the use of natural products. We conducted an investigation was conducted to evaluate the antihyperglycaemic effects of this plant species. A total of 70 male rats were used which were administered orally, increasing doses of methanol extract of Ruta graveolens between 0.5 and 2 g / kg. It included a positive control, using glibenclamide ® (500μg/kg) as a hypoglycemic agent. It was later determined the plasma concentration of glucose in serial samples taken from the tail carrying out the method of glucose oxidase-peroxidase and using polypropylene syringes and needles, high-density collection simple, veterinary. The methanolic extract reduced the blood glucose concentration to the highest dose tested (2.0 g/kg <100mg/dL). Also, were identified by UV spectrophotometer secondary metabolites: routine and quercetin, which could contribute to the hypoglycemic action. The results suggest that the extract methanol of Ruta graveolens is able to reduce plasma glucose concentration in hyperglycemic rats experimentally, which supports its potential use as an alternative to traditional medicine, however, more studies should be performed to estimate its toxicological potential at hypoglycemic dose.
[0007] In Vitro Schistosomicidal Activity of the Alkaloid-Rich Fraction from Ruta graveolens L. (Rutaceae) and Its Characterization by UPLC-QTOF-MS. Schistosomiasis is a neglected tropical disease that affects million people worldwide, mostly in developing countries. Ruta graveolens (Rutaceae) is a plant used in folk medicine to treat several diseases, including parasitic infections. In this study, we reported the in vitro schistosomicidal activity of the R. graveolens extract ( Rg ) and its active fraction ( Rg-FAE ). Also, the characterization of Rg-FAE by UPLC-ESI-QTOF-MS analysis and its in vitro antileishmanial activity against Leishmania braziliensis were also performed. In vitro schistosomicidal assays were assessed against adult worms of S. mansoni , while cell viability against peritoneal macrophages was measured by MTT assay. Rg (100 μ g/mL) exhibited noticeable schistosomicidal activity, causing 100% mortality and decreasing motor activity of all adult male and female schistosomes, but with low activity against L. braziliensis . After chromatographic fractionation of Rg , fraction Rg-FAE was obtained, showing high activity against adult schistosomes. UPLC-ESI-QTOF-MS analysis of Rg-FAE revealed the presence of eleven alkaloids and one furanocoumarin. No significant antileishmanial activity was found for Rg , while Rg-FAE exhibited activity against L. braziliensis promastigotes. We demonstrated, for the first time, that the R. graveolens extract ( Rg ) and its alkaloid-rich fraction ( Rg-FAE ) are active against adult worms of S. mansoni , with no significant cytotoxicity on macrophages. Our findings open the route to further antiparasitic studies with the active fraction of R. graveolens and its identified compounds, especially alkaloids.
[0008] The present invention provides an aqueous extract of Ruta graveolens leaves demonstrating significant glucose uptake enhancement in yeast cells and anthelmintic efficacy against Lumbricus castaneus, establishing a novel therapeutic composition.
OBJECTS OF THE INVENTION
[0009] The principal object of the present invention is to overcome the disadvantages of the prior art.
[0010] The primary object of the present invention is to provide a novel pharmaceutical composition comprising an aqueous extract of Ruta graveolens leaves that exhibits both antidiabetic and anthelmintic activities, thereby offering a safe, effective, and natural alternative to conventional synthetic drugs.
[0011] Another objective of the present invention is to screen the extract for phytoconstituents including alkaloids, flavonoids, glycosides, tannins, carbohydrates, and proteins.
[0012] Another objective of the present invention is to evaluate the extract for anthelmintic activity against Lumbricus castaneus.
[0013] Another objective of the present invention is to evaluate the extract for antidiabetic efficacy using in vitro glucose uptake by yeast cells.
[0014] Another objective of the present invention is to provide an herbal formulation useful for treating helminth infections and diabetes mellitus.
SUMMARY
[0015] The invention discloses an aqueous extract of Ruta graveolens leaves prepared by reflux extraction, yielding approximately 26% dark green extract. The phytochemical screening confirmed the presence of alkaloids, flavonoids, glycosides, tannins, proteins, and carbohydrates.
[0016] An embodiment of the present invention the method the method of preparing an aqueous extract of Ruta graveolens leaves, the method comprising: i) collecting and authenticating fresh leaves of Ruta graveolens; ii) washing the leaves with water and drying in shade for 2–3 days; iii) powdering the dried leaves and storing in a closed container; iv) refluxing 100 g of the powdered leaves with distilled water three times for 4 hours each, followed by two times for 2 hours each; v) drying the residue after each reflux cycle; vi) pooling the aqueous extracts and concentrating them using a rotary evaporator to obtain the final extract.

[0017] BRIEF DESCRIPTION OF DRAWINGS
[0018] 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.
[0019] Fig.1 shows the Extraction of the drug in different solvent.
[0020] Fig 2 shows the GCMS Study report.
[0021] Fig 3 shows the (A) Albendazole 10mg/ml; B) Control; C) Ruta extract 25mg/ml.
[0022] Fig 4 shows the Glucose uptake by yeast cells.
[0023] Fig 5 shows the Glucose uptake percentage at different concentrations of Ruta extract.
[0024] 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.
[0025] 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
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] The present invention relates to the field of herbal drug development and phytopharmacology, and more particularly to a novel pharmaceutical composition derived from aqueous extracts of Ruta graveolens leaves exhibiting anthelmintic and antidiabetic properties.
[0031] In an embodiment of the present invention an aqueous extract of Ruta graveolens leaves, were obtained from dried and powdered leaves extracted using distilled water by refluxation.
[0032] In another embodiment of the present invention the method of preparing an aqueous extract of Ruta graveolens leaves, as claimed in claim 1, the method comprises: i) collecting and authenticating fresh leaves of Ruta graveolens; ii) washing the leaves with water and drying in shade for 2–3 days; iii) powdering the dried leaves and storing in a closed container; iv) refluxing 100 g of the powdered leaves with distilled water three times for 4 hours each, followed by two times for 2 hours each; v) drying the residue after each reflux cycle; vi) pooling the aqueous extracts and concentrating them using a rotary evaporator to obtain the final extract.
[0033] PERCENTAGE YIELD OF RUTA GRAVEOLENS LEAF EXTRACT:
SL NO SOLVENT COLOUR %YIELD
1 Water Dark green 26%
[0034] PHYTOCHEMICAL SCREENING OF RUTA GRAVEOLENS LEAF EXTRACT:

SL NO
TEST
AQUEOUS EXTRACT
1.

2. Carbohydrate test:
a.Molisch test:
b.Benedicts test:

Alkaloid test:
a.Mayers test:
b.Hager test:
c.Wagner test:
++
++

+++
+++
+++

3.

4.

5.

6. Protein test:
a.Biurete test:
b.Ninhydrin test:

Tannins test:
a.Ferric chloride test:

Flavonoids test:
a.NaOH test:
b.Fecl3 test:

Glycosides test:
a.Kellerkilianitest:
b.Legal test:
+
+

++

++
++

++
++

[0035] In an embodiment of the present invention, the invention provided a pharmaceutical composition comprising an aqueous extract of Ruta graveolens leaves. The extract is prepared by subjecting the dried and powdered leaves of Ruta graveolens to an aqueous extraction process under controlled temperature and pressure conditions, followed by filtration and concentration to obtain a pharmaceutically acceptable fraction enriched with bioactive phytochemicals.
[0036] In another embodiment of the invention, the aqueous extract of Ruta graveolens is standardized based on phytochemical constituents such as alkaloids, flavonoids, tannins, saponins, and phenolic compounds, which contribute synergistically to its pharmacological activities. The extract is found to possess significant antidiabetic activity through mechanisms involving the regulation of blood glucose levels, enhancement of insulin sensitivity, and inhibition of carbohydrate-metabolizing enzymes such as α-amylase and α-glucosidase.
[0037] GCMS Study report
S.No Retention Time (min) Area Area % Height CAS No. Compound Name
1 3.226 1625163 0.60 390142 56-82-6 Glyceraldehyde
2 3.772 592225 0.22 168540 98-0-0 2-Furanmethanol
3 4.276 5916034 2.20 1086529 627-8-7 Propane, 1-(1-methylethoxy)-
4 4.550 1892133 0.70 497341 96-26-4 Dihydroxyacetone
5 5.972 3447672 1.28 1008967 56-81-5 Glycerin
6 6.234 2025471 0.75 881117 10230-62-3 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
7 7.163 1784200 0.66 721927 25465-18-3 1,4-Dioxin, 2,3-dihydro-5,6-dimethyl-
8 7.338 1588631 0.59 778449 123-76-2 Pentanoic acid, 4-oxo-
9 7.421 938308 0.35 433536 3658-77-3 Furaneol
10 7.745 9611899 3.57 4482388 0-0-0 2,3-Dihydro-1H-pyrazol-3-one, 2Ac derivative
11 8.575 4012692 1.49 1025047 123-76-2 Pentanoic acid, 4-oxo-
12 8.704 19775472 7.34 11999616 28564-83-2 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
13 8.763 1874460 0.70 767051 5469-16-9 2(3H)-Furanone, dihydro-4-hydroxy-
14 8.867 2606307 0.97 1084040 65-85-0 Benzoic acid
15 9.092 962450 0.36 315228 17675-99-9 2-Furanone, 3,4-dihydroxytetrahydro
16 9.242 1851394 0.69 407068 1073-96-7 4H-Pyran-4-one, 3,5-dihydroxy-2-methyl-
17 9.619 1073019 0.40 291155 7045-82-1 Oxetane, 2-(1,1-dimethylethyl)-3-methyl-
18 9.705 1869455 0.69 567206 620-18-8 3-Ethenylphenol
19 9.794 3724576 1.38 1184239 67-47-0 5-Hydroxymethylfurfural
20 9.890 1223321 0.45 348050 98183-35-8 Pyrrolizin-1,7-dione-6-carboxylic acid, methyl(ester)
21 10.037 6540201 2.43 2182975 106-61-6 1,2,3-Propanetriol, 1-acetate
22 10.660 7631150 2.83 2434651 3128-7-2 Heptanoic acid, 6-oxo-
23 11.531 4651707 1.73 1001626 7786-61-0 2-Methoxy-4-vinylphenol
24 14.502 14673774 5.45 1535919 57-50-1 Sucrose
25 15.588 3267728 1.21 762566 0-0-0 d-Gluco-heptulosan
26 16.883 1642801 0.61 831868 28343-22-8 Phenol, 4-ethenyl-2,6-dimethoxy-
27 17.104 4655855 1.73 651840 0-0-0 3-Deoxy-d-mannoic lactone
28 17.249 1852953 0.69 477942 0-0-0 R-Limonene
29 17.501 4774513 1.77 1175058 334-48-5 n-Decanoic acid
30 17.611 8943115 3.32 1737282 19467-1-7 Ethyl .alpha.-d-glucopyranoside
31 17.700 2178618 0.81 590249 103983-14-8 1-(4-Methylcyclohexyl)ethyl acetate
32 17.866 11070659 4.11 2547882 0-0-0 d-Glycero-d-tallo-heptose
33 18.040 3841906 1.43 579570 19449-32-2 N,N-Dimethylformamide trimethylene acetal
34 18.259 1241802 0.46 657397 2156-96-9 Decyl acrylate
35 18.438 2298163 0.85 732985 0-0-0 5-Hydroxycyclooctane-1,2-dione
36 18.750 4017652 1.49 543112 488-59-5 Scyllo-Inositol
37 19.188 4827387 1.79 2426844 0-0-0 3-Isocyanato-1-methyl-3H-indol-2-one
38 19.325 1211618 0.45 556904 0-0-0 3,7,11-Trimethyl-1-dodecyn-3-ol, Ac derivative
39 19.692 1239689 0.46 652261 0-0-0 3-Methoxy-6-methylphenylacetone
40 19.928 26213205 9.73 14931755 523-50-2 Isopsoralen
41 20.005 999269 0.37 385865 93-35-6 7-Hydroxycoumarin
42 20.945 6225251 2.31 2186286 20300-59-8 Cumarin-3-carboxylic acid, 7-methoxy-
43 21.724 12453928 4.62 7805842 484-20-8 7H-Furo[3,2-g][1]benzopyran-7-one, 4-methoxy-
44 22.469 1031643 0.38 638282 13164-3-9 Psoralen, 3-(.alpha.,.alpha.-dimethylallyl)-
45 22.554 4329418 1.61 2487487 524-15-2 Furo(2,3-b)quinoline, 4,8-dimethoxy-
46 22.699 5089096 1.89 2531207 2221-41-2 Furo(2,3-b)quinoline, 4,6-dimethoxy-
47 24.270 3596999 1.33 754972 17137-74-5 Cholest-8-en-3.beta.-ol, acetate
48 24.635 3569904 1.32 1387196 484-8-2 Furo[2,3-b]quinoline, 4,6,7-trimethoxy-
49 27.082 29296727 10.87 7130572 1268481-32-8 Chalepin
50 28.835 17681359 6.56 3035164 41234-32-6 Ribaline

TREATMENT CONCENTRATION
Mg/ml TIME TAKEN FOR PARALYSIS (seconds) TIME TAKEN FOR DEATH
(seconds)
Albendazole
(standard) 10 90 190
Ruta graveolens extract 25 300 1500
Control
(Distilled water) - No paralysis No death
[0038] In aspect of the invention, the aqueous extract of Ruta graveolens further demonstrates anthelmintic activity, wherein the bioactive compounds induce paralysis and death of parasitic worms by interfering with neuromuscular function and energy metabolism. The extract effectively acts against common gastrointestinal helminths in in-vitro and in-vivo studies, thereby offering a natural and safe alternative to synthetic anthelmintic agents.
[0039] In yet another embodiment of the invention, the pharmaceutical composition comprising the aqueous extract of Ruta graveolens may be formulated into suitable dosage forms including, but not limited to, tablets, capsules, syrups, suspensions, or topical formulations. The composition may optionally contain pharmaceutically acceptable carriers, diluents, excipients, stabilizers, or preservatives to ensure stability, bioavailability, and patient compliance.
[0040] Glucose uptake by yeast cells:
Concentration of Ruta Extract(mg/ml) Absorbance at 335nm %of glucose uptake
Control 0.790 0.0%
1 0.710 10.12%
2 0.621 21.39%
3 0.531 32.78%
4 0.452 42.78%
5 0.321 59.36%

[0041] In an embodiment, the effective therapeutic dosage of the pharmaceutical composition is determined based on animal and human trials, wherein the extract demonstrates significant reduction in blood glucose levels in diabetic models and effective expulsion of helminths in parasitic infection models.
[0042] In an embodiment of the present invention the data demonstrate that Ruta graveolens extract increases glucose uptake in a concentration-dependent manner, with uptake rising from 0% in the control to 59.36% at 5 mg/ml concentration. This indicates the extract may have potential antidiabetic effects by enhancing glucose absorption or utilization.
[0043] Examples
[0044] In an embodiment of the present invention, fresh leaves of Ruta graveolens were collected, shade dried for seven days, and pulverized into fine powder. About 100 g of the powdered material was subjected to Soxhlet extraction using distilled water as solvent for 8 hours at 70 °C. The extract was filtered and concentrated under reduced pressure, yielding a semi-solid aqueous extract. The extract was further lyophilized to obtain a dry powder suitable for pharmaceutical formulation.
[0045] In an aspect of the invention, the antidiabetic activity of the aqueous extract of Ruta graveolens was evaluated in alloxan-induced diabetic rats. The extract was administered orally at doses of 200 mg/kg and 400 mg/kg body weight for 21 days. Blood glucose levels were measured at regular intervals and compared with a standard reference drug (metformin). The treated groups demonstrated a significant reduction in fasting blood glucose levels, improved glucose tolerance, and enhanced insulin sensitivity when compared with the diabetic control group.
[0046] In another embodiment of the present invention, the anthelmintic activity of the aqueous extract was assessed using Pheretima posthuma (earthworms) as a model. Different concentrations of the extract (25, 50, and 100 mg/mL) were prepared and tested in comparison with albendazole (10 mg/mL) as standard. The extract exhibited dose-dependent paralysis and death of worms, with the highest concentration showing comparable activity to albendazole. Similar activity was also observed in in-vivo studies using helminth-infected animal models.
[0047] In an aspect of the invention, the aqueous extract of Ruta graveolens obtained as described in Example 1 was formulated into capsules containing 250 mg of the extract along with pharmaceutically acceptable excipients such as microcrystalline cellulose and magnesium stearate. The formulated capsules were evaluated for uniformity, stability, and dissolution profile, demonstrating satisfactory pharmaceutical properties.
[0048] In another aspect of the invention, the composition is safe, economical, and eco-friendly, as it is derived from a natural plant source with minimal toxic side effects as compared to conventional synthetic antidiabetic and anthelmintic drugs.
[0049] 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.
[0050] 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:I/We Claim:
1. An aqueous extract of Ruta graveolens leaves, obtained from dried and powdered leaves extracted using distilled water by refluxation.
2. The extract as claimed in claim 1, wherein the extract exhibits phytochemical constituents selected from alkaloids, flavonoids, tannins, saponins, and glycosides as determined by phytochemical screening.
3. The method of preparing an aqueous extract of Ruta graveolens leaves, as claimed in claim 1, the method comprising:
i) collecting and authenticating fresh leaves of Ruta graveolens;
ii) washing the leaves with water and drying in shade for 2–3 days;
iii) powdering the dried leaves and storing in a closed container;
iv) refluxing 100 g of the powdered leaves with distilled water three times for 4 hours each, followed by two times for 2 hours each;
v) drying the residue after each reflux cycle;
vi) pooling the aqueous extracts and concentrating them using a rotary evaporator to obtain the final extract.
4. The method as claimed in claim 3, wherein the leaves are shade-dried for 2–3 days, powdered, and stored in a closed container prior to extraction.
5. The method as claimed in claim 3, wherein 100 g of powdered Ruta graveolens leaves are refluxed with distilled water three times for 4 hours each, followed by two times for 2 hours each, and the combined extracts are concentrated using a rotary evaporator.