Description:TECHNICAL FIELD
[0001] The present invention relates to the field of herbal drug extraction and phytopharmaceutical development, and more particularly to a method for preparing aqueous extracts from different parts of Balanites aegyptiaca, including fruit pulp, leaves, and thorns. The invention further relates to the use of these extracts for exhibiting antioxidant, anthelmintic, and anti-diabetic activities in vitro, making them suitable for incorporation into pharmaceutical, nutraceutical, and herbal 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] Balanites aegyptiaca, commonly known as the desert date, is a plant widely recognized in traditional medicine for its diverse therapeutic properties. Various parts of the plant, including fruit pulp, leaves, and thorns, have been reported to possess pharmacological activities such as antioxidant, anthelmintic, anti-diabetic, antimicrobial, and anti-inflammatory effects. These properties are largely attributed to its rich content of bioactive phytochemicals, including flavonoids, saponins, alkaloids, tannins, and phenolic acids.
[0004] Despite its medicinal potential, there is a need for standardized extraction methods that can yield consistent, high-quality bioactive extracts while being safe, eco-friendly, and commercially viable. Conventional extraction techniques often use organic solvents, which may leave harmful residues, increase costs, and limit the scope for pharmaceutical or nutraceutical applications.
[0005] US20080287662A1 The invention relates to stable preparations of saponin nanovesicles, which can encapsulate active biological or chemical materials such as drugs, pesticides, vitamins, minerals and nutrients, and protect them. The preparations can be used, for example, in agricultural, pesticidal, dermatological and cosmetic compositions. The saponins per se can further be used as adjuvants for irrigation water. The invention further relates to novel saponins extracted from Balanites aegyptiaca.
[0006] US20080271364A1 The invention provides methods for production of biodiesel from Balanites aegyptiaca oil or crushed nuts, and further relates to the biodiesel obtained. The Balanites aegyptiaca biodiesel obtained has a composition of triglycerides of mainly C16:0 and C18:0 saturated and unsaturated fatty acids, with a very high content of linoleic acid and of oleic acid, and it further contains Balanites saponins, acting as surfactants, which reduce the rate of corrosion and improve the performance of the engyne.
[0007] Aqueous extraction offers a safer and more sustainable alternative, but existing processes may not maximize phytochemical yield or maintain the stability of bioactive compounds. The present invention addresses these challenges by providing an optimized aqueous refluxation method for extracting bioactive constituents from Balanites aegyptiaca fruit pulp, leaves, and thorns, producing multifunctional extracts with antioxidant, anthelmintic, and anti-diabetic properties for use in health-promoting formulations.
[0008] OBJECTS OF THE INVENTION
[0009] The principal object of the present invention is to overcome the disadvantages of the prior art.
[0010] The primary objective of the present invention is to develop a simple, eco-friendly, and efficient aqueous extraction method for obtaining bioactive phytochemical-rich extracts from Balanites aegyptiaca fruit pulp, leaves, and thorns.
[0011] An object of the present invention is to authenticate and process plant material to ensure botanical purity and consistency in extract quality.
[0012] Another object of the present invention is to optimize a refluxation-based extraction process using demineralized water as a safe, non-toxic solvent.
[0013] Another object of the present invention is to isolate and concentrate aqueous extracts containing a broad spectrum of phytochemicals, including flavonoids, alkaloids, saponins, tannins, and phenolic compounds.
[0014] Another object of the present invention is to provide multifunctional herbal extracts that can be incorporated into pharmaceutical, nutraceutical, or herbal formulations.
[0015] Another object of the present invention is to offer a scalable and cost-effective extraction process suitable for commercial production.
[0016] SUMMARY
[0017] The present invention provides a novel aqueous extraction method for obtaining bioactive phytochemical-rich extracts from different parts of Balanites aegyptiaca, including fruit pulp, leaves, and thorns. The process involves collection and botanical authentication of the plant material, drying of leaves and thorns under ambient conditions, manual separation of fruit pulp, and powdering of dried plant parts for storage in airtight containers.
[0018] Aqueous extraction is carried out using demineralized water as the solvent through a three-stage refluxation process—first for approximately 4 hours, then 3 hours, and finally 1 hour—with drying of the plant residue between cycles. The extracts from all cycles are pooled, concentrated under reduced pressure using a rotary evaporator, and their yield calculated with respect to the initial dried plant weight.
[0019] The resulting aqueous extracts exhibit antioxidant, anthelmintic, and anti-diabetic activity in vitro, making them suitable for pharmaceutical, nutraceutical, and herbal formulations targeting metabolic, infectious, and oxidative stress-related conditions.
[0020] These and other features will become apparent from the following detailed description of illustrative embodiments thereof, which is to be read in connection with the accompanying drawings. While the invention has been described and shown with reference to the preferred embodiment, it will be apparent that variations might be possible that would fall within the scope of the present invention.
BRIEF DESCRIPTION OF DRAWINGS
[0021] 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.
[0022] Fig.1 shows the aqueous extracts tablet.
[0023] Fig 2 (a) Control and (b) Standard
[0024] Fig 3 (a) Fruit pulp (10mg) and (b) Fruit pulp (25mg).
[0025] Fig 4 (a) Leaf (10 mg) and (b) Leaf (12mg).
[0026] Fig 5 (a) Thorns (10 mg) and (b) Thorns (25 mg).
[0027] 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.
[0028] 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
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] The present invention discloses a method for preparing aqueous extracts from different plant parts of Balanites aegyptiaca for in-vitro evaluation of antioxidant, anthelmintic, and anti-diabetic properties. The invention also relates to the resulting extracts and their applications in pharmaceutical and nutraceutical formulations.
[0034] In an embodiment of the present invention a method for preparing aqueous extracts from plant parts of Balanites aegyptiaca, comprising the steps of i) collecting plant material selected from fruit pulp, leaves, and thorns of authenticated Balanites aegyptiaca; ii) drying the leaves and thorns for approximately four days under ambient conditions, and manually separating the fruit pulp from the fruit; iii) powdering the dried leaves and thorns, and storing the powder in a closed container until use; iv) weighing approximately 25 g of each plant part separately, and subjecting the weighed material to aqueous extraction using demineralized water as the solvent; v) performing extraction by refluxing the plant material in water for about four hours in a first cycle, followed by about three hours in a second cycle, and about one hour in a third cycle, wherein the residue is dried between cycles; vi) pooling the aqueous extracts from all cycles and concentrating them using a rotary evaporator; and vii) calculating the percentage yield of the extract with reference to the weight of the dried plant material.
[0035] In an aspect of the present invention fresh young tender shoots, fruits, leaves, and thorns of Balanites aegyptiaca were collected from a local market. The plant material was botanically authenticated at SRMC College of Applied Sciences, Department of Botany, Shivamogga, and confirmed as Balanites aegyptiaca.
[0036] The fruit pulp was manually separated from the seeds for subsequent extraction. Leaves and thorns were dried under ambient conditions for approximately four days to reduce moisture content. The dried leaves and thorns were powdered using a mechanical grinder and stored in closed, airtight containers to prevent contamination and moisture absorption.
[0037] Approximately 25 g of each plant part fruit pulp, powdered leaves, and powdered thorns was weighed separately. Demineralized water was used as the extraction solvent. Extraction was performed using the refluxation method as reported by Forsyth (1955). First cycle: The plant material was refluxed in demineralized water for about 4 hours. Second cycle: The plant residue was dried and refluxed for about 3 hours. Third cycle: The dried residue was again refluxed for about 1 hour. Extracts from all three cycles were pooled together. The pooled extract was concentrated under reduced pressure using a rotary evaporator until a semi-solid mass was obtained. The percentage yield of each extract was calculated with reference to the weight of the dried plant material.
[0038] Balanites aegyptiaca (L.) Delile: Commonly known as Desert Date or Hingot, it is a thorny tree widely distributed in dry tropical and subtropical regions of Africa, the Middle East, and parts of South Asia. The plant is well known for its medicinal fruit, rich in saponins and other bioactive compounds. Traditionally, various parts of the plant are used in the treatment of liver disorders, diabetes, infections, and as an anthelmintic. Table number: 01
Botanical source Balanites aegyptiaca (L.) Delile
Family Zygophyllaceae
Origin Africa, Middle East, South Asia
Sub family Tribuloideae (under Zygophyllaceae)
Synonym Hingot, Desert Date, Soapberry Tree, Egyptian Balsam
[0039] Taxonomical classification
[0040] Table Number: 02
Kingdom Plantae
Division Magnoliophyta
Class Magnoliopsida
Order Zygophyllales
Subclass Rosidae
Tribe Tribuloideae
Species Balanites aegyptiaca
Genus Balanites
Family Zygophyllaceae
[0041] Balanites aegyptiaca contained saponin, furanocoumarin and flavonoid. The mesocarp of its fruit contains protein, sugars, organic acids, other constituents like 3-rutinoside and 3rhamnogalactoside of isorhamnetin, diosgenin, it also contain a mixture of 22R and 22S epimers of 26-(Oβ-D-glucopyranosyl)-3-β-[4-O-(β-D-glucopyranosyl)-2-O-(α-rhamnopyranosyl)-β-D-lucopyranosyloxy]-22,26dihydroxyfurost-5-ene. Its kernel contained the xylopyranosyl derivative of above saponin present in mesocarp while nine saponins have been reported from kernel cake of Balanites aegyptiaca. The leaves and fruit kernels of Balanites aegyptiaca were found to contain six diosgenin glucosides including di-, tri-, and tetraglucosides. Its bark contains furanocoumarin bergapten and dihydrofuranocumarin D- marmesinIt also contains beta-sitosterol, bergapten, marmesin,and beta-sitosterol glucoside balanitin-1-2, and -3;balanitin-1 for example possesses a yamogenin aglycone with a branched glucose and rhamnose side chain.
[0042] The young tender shoots of Balanites aegyptiaca were collected from local market. Authentication of plant was done at SRMC, College of applied sciences, Botany Department, Shivamogga. Authenticated as Balanites aegyptiaca.
[0043] The Fruit pulp, Leaf, Thorns of Balanites aegyptiaca were collected separately, leaves and thorns were dried for 4 days. Fruit pulp is manually separated from the fruit for further subsequent evalution.
[0044] The dried leaves and thorns were powdered and stored in a closed container for further studies. Extraction of the drug in aqueous solvents. To isolate different extracts, the dried powder of Balanites aegyptiaca was extracted by refluxation method as reported by Forsyth (1955). Extraction with water was done.
[0045] Water (De mineralized water)
[0046] Example:
[0047] 25 gms of Balanites aegyptiaca fruit pulp ,dried leaves and thorns was refluxed with all the aqueous solvent for 4 hrs, followed by another 3hrs and 1 hr respectively, residue was dried each time before extraction with next solvent. The extracts were pooled together & concentrated by rotary evaporator. All the extracts were screened for the phytochemical analysis. The % yield was calculated with reference to dried powder drug & the results are tabulated in table no 09.
[0048] EVALUATION OF Balanites aegyptiaca EXTRACT
[0049] Phytochemical screening:
[0050] The following extracts were screened for their phytochemical activity.
[0051] Aqueous extract:
[0052] Carbohydrate Test
[0053] Molisch Test:
[0054] Filtrate was treated with 2 drops of alcoholic alpha-naphthol solution in a test tube and 0.2 ml of con. Sulphuric acid was added carefully along the sides of the test tube. Formation of violet ring at the junction indicates the presence of carbohydrates.
[0055] Benedicts test:
[0056] Filtrate was treated with benedict’s reagent and boiled on water bath. Formation of orange red precipitate indicates the presence of reducing sugars.
[0057] Mayers Test:
[0058] Few ml of extract was treated with Mayers reagent. Formation of cream pale-yellow precipitate indicates the presence of Alkaloids.
[0059] Hagers Test:
[0060] Few ml of extract was treated with Hager’s test reagent. Formation of yellow ppt indicates the presence of alkaloids.
[0061] Wagner Test:
[0062] Few ml of extract was treated with Wagner’s test reagent. Formation of reddish brown precipitate indicates the presence of alkaloids.
[0063] Dragendorff’s Test:
[0064] Few ml of extract was treated with Dragendorff’s reagent. Formation of reddish brown precipitate indicates the presence of alkaloids.
[0065] Protein Test:
[0066] a.Biurete Test:
[0067] Add 2ml of sodium hydroxide and 5 to 6 drops of copper sulfate solution to it. Shake the test tube gently to mix the ingredients thoroughly and allow the mixture to stand for 4 to 5 minutes if there is appearance of bluish –violet color, it indicates the presence of protein
[0068] b. Ninhydrin Test:
[0069] A 1 percent solution of amino acid is prepared in distilled water and a few drops of Ninhydrin solution is added to this solution. The test tube is kept in a hot water bath for approximately 5 minutes. After a few minutes a blue or violet colour indicates the presence of amino acids, amine and protein groups.
[0070] c. Millon’s Test:
[0071] Dissolve the given extract in 1ml of distilled water and add 5 to 6 drops of Millon’s reagent. If there is appearance of red precipitate, it indicates the presence of protein.
[0072] Phenolic compounds and Tannins Test:
[0073] a. Test with Ferric chloride:
[0074] To the solution of sample added ferric chloride, a blue, black, violet or green ppt or color confirms the presence of tannins.
[0075] Lead acetate Test:
[0076] Add a solution of 10% lead acetate to the extract, yellow color confirms presence of tannins/ phenols compound.
[0077] Alkaline reagent Test:
[0078] Treat the extract solution with Sodium hydroxide solution, yellow color to colorless confirms the presence of tannins/ phenols compound.
[0079] Gelatin Test:
[0080] Add an aqueous solution of gelatin and sodium chloride to the extract solution, presence of precipitate confirms the presence of tannins/ phenols compound.
[0081] Flavonoids Test:
[0082] a. NaOH test:
[0083] To the extract add 10 percent NaOH. Formation of yellow color indicates presence of Flavonoids
[0084] b. Ferric chloride test:
[0085] Extract was treated with ferric chloride solution. Formation of green to black color indicates presence of Flavonoids.
[0086] Glycosides Test:
[0087] a. Borntrager’s Test:
[0088] Boil the plant extract with 5ml of 10% Dil. Sulfuric acid for one or two min and filter. Add an organic solvent such as benzene, ether or chloroform to the filtrate and shake. Separate the organic layers, then add 5ml of 10% ammonium sol to organic layer. Formation of pink or red color, confirms the presence of glycosides.
[0089] Steroids/Phytosterols test:
[0090] Liebermann – Burchard’s Test:
[0091] Dissolve the given extract solution in acetic anhydride, heat to boiling, then cool the mixture. Add 1ml of Con. sulfuric acid along the sides of the test tube. Formation of reddish color ring shows the presence of Steroids’s.
[0092] b. Salkowski Test:
[0093] Shake the given extract with chloroform. To the chloroform layer add sulphuric acid along the sides of test tube. Red or violet ring confirms the presence of steroids.
[0094] Saponin Test:
[0095] Dilute about 1 ml of alcoholic and aqueous extracts separately with distilled water to a final volume of 20 ml. Shake each solution in a graduated cylinder for 15 minutes. Froth formed confirms the presence of saponin.
[0096] Resin Test
[0097] Dissolve the plant material in acetone and then pour the solution into distilled water. Precipitate formation shows the presence of resins
[0098] Evaluation of Anti-oxidant activity:
[0099] In –vitro free radical scavenging activity of the extracts and isolated compounds was determined using a method based on the reduction of a methanolic solution of the coloured DPPH (1-1-diphenyl 2-picryl hydrazyl). The activity was expressed as effective concentration at 50% reduction (EC50) or the concentration of the test solution requires giving a 50% decrease in absorbance compared to that of blank solution.
[00100] Reagents:
[00101] 1. DPPH solution: A working solution DPPH having an absorbance of .9 at 516nm was used. This was prepared by taking 75µl of stock solution containing 4.3µg of DPPH in 3ml of methanol.
[00102] Blank solution: Solution was prepared without sample and used as a Blank. Test solution: The aqueous extracts were tested. The 1mg per ml test solution of the extracts was prepared by dissolving them in respective solvent.
[00103] To 75µl of DPPH solution in methanol, different concentrations of Balanites aegyptiaca samples were added and the volumes were made up to 3ml with methanol. DPPH diluted to 3ml was taken as blank. Decrease in absorbance in the presence of Balanites aegyptiaca leaf, thorns, fruit pulp was noted down after 15min.Linear regression was applied for concentration and % inhibition was calculated.
[00104] Evaluation of Anthelmintic activity:
[00105] The anthelmintic activity was carried out as per method of NeerajChoudhary et al. The assay was performed in in vitro using Indian adult earthworms (Lumbricusterrestris)that process similarity with human intestinal parasites for preliminary evaluation of Anthelmintic activity.
[00106] Test samples of was prepared at the concentrations ,10, 25 mg/ml of fruit pulp, leaf, thorns in distilled water and 15 earthworms (Lumbricusterrestris) in each group of approximately equal size (same type) were placed in each petridish containing 25ml of above test solution of Balanites aegyptiaca extract. Albendazole (400mg/100ml) was used as reference standard and double distilled water was used as control. All the test solution and standard drug solution were prepared freshly before starting the experiment. The time taken for the paralysis and death of the earthworms were noted.
[00107] Determination of Glucose Adsorption Capacity: -
[00108] Glucose uptake by yeast cells:
[00109] 250mg of the extract was weighed and dissolved in small amount of distilled water, sonicated when necessary and diluted up to 50ml to get 5mg/ml (5000μg/ml), concentration solution. Further series of dilutions were made by withdrawing 2ml, 4ml, 6ml and 8ml from 5mg/ml concentration of solution and diluted up to 10ml with distilled water to get 1, 2, 3 and 4mg/ml concentration respectively. Procedure 1ml of each concentration of the extracts (1 to 5mg/ml) was mixed with 1ml of glucose solution and incubated at 37°c for 10min. After 10min of incubation 100μg/ml of yeast suspension was added and again incubated for 1hour at 37°c temperature. After 1 hour of incubation the solutions were subjected to centrifugation for 5minutes at 2500xg rpm. Supernatant was collected for glucose content. The absorbance was recorded at 520 nm in UV spectroscopy. Simultaneously a control was also performed without adding sample.
[00110] Formula: Increase in the percentage of glucose uptake by yeast cells was calculated by the given formula:
Increase in Glucose Uptake (%) = A control - 𝑨 𝒔𝒂𝒎𝒑𝒍𝒆 × 100/ A control
Where,
A control = Absorbance of control
A sample = Absorbance of sample
[00111] Principle: In the presence of atmospheric oxygen, glucose present in the specimen is oxidized by the enzyme glucose oxidase (GOD) to gluconic acid and hydrogen peroxide (H2O2). Thus formed H2O2 oxidatively couples with 4-aminoantipyrine and phenol in presence of peroxidase (POD) to form red-colored quinoneimine dye, which is measured calorimetrically at 514 nm. The intensity of the colour is directly proportional to the concentration of glucose present in the specimen.

Preparation of stock solution
Glucose solution- about 18.016gm of glucose was weighed and dissolved in small amount of distilled water then diluted up to 100ml to make 1000mM (stock solution).
Preparation of series of dilutions
From the stock solution dilutions were made to prepare 5mM (0.005M), 10mM (0.01M), 20mM (0.02M), 50mM (0.05M) and 100mM (0.1M) solution i.e., 0.5ml, 1ml, 2ml, 5ml, and 10ml of the stock solution was taken and diluted up to 100ml with distilled water to get 5, 10, 20, 50 and 100mM glucose solution respectively. From each above series of dilutions 10μl of the solution was taken into the ephendroff’s tubes separately to which 1ml of working reagent was added (prepared from GOD-POD kit) and observed for original glucose concentration i.e., G1 using colorimeter at 514 nm.
Preparation of 1% Extract solution: 250mg of the extract was weighed and added into small amount of distilled water and sonicated, diluted to 25ml to make it 1% of the solution.
Procedure:
To 25ml of the each 5, 10, 20, 50 and 100mM of glucose 1ml of 1 percent solution of plant was added and mixed properly. The mixture was then incubated on a shaker water bath which was maintained at 37°c for 6hrs for thorough mixing of the solution. After 6hrs of the incubation the mixture was collected separately in ephendroff’s tubes and centrifuged at 4000rpm for 20minutes. The supernatant was analysed using GOD-POD kit in colorimeter at 514 nm for the determination of glucose bound using the given formula. Formula: The amount of glucose bound was determined by using given formula and the results were expressed in the terms of mM/L.
PERCENTAGE YIELD OF Balanites aegyptiaca FRUIT PULP:
SI NO SOLVENT COLOUR ⁒YIELD
01 Water Dark brown 72

PERCENTAGE YIELD OF Balanites aegyptiaca LEAF:
SI NO SOLVENT COLOUR ⁒YIELD
01 Water Dark green 48
PERCENTAGE YIELD OF Balanites aegyptiaca THORNS:
SI NO SOLVENT COLOUR ⁒YIELD
01 Water Light brown 12
PHYTOCHEMICAL SCREENING OF Balanites aegyptiaca EXTRACT:
The Balanites aegyptiaca fruit pulp, leaves, thorns extract was subjected to qualitative chemical analysis for identification of various plant constituents like carbohydrates, alkaloids, protein, Phenolic compounds and tannins, flavonoids, glycosides, Steroids/Phytosterols, and Saponins. Observed phytochemical screening reports of Balanites aegyptiaca.
SI NO TEST Aqueous extract of fruit pulp Aqueous extract of leaf Aqueous extract of thorns
1

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

Alkaloid test
a.Mayers test:
b.Hager test:
c.Wagner test:
d.Dragendorff’s test:
+
+
+
+
+
+
+
+
+
+
+
+
3 Protein test
a.Biurete test:
b.Ninhydrin test:
c.Millon’s test:

-
-
-
-
+
-
-
+
-
4 Phenolic compounds and tannins test:
a.Ferric chloride test:
b.Lead acetate test:
c.Alkaline reagent test:
d.Gelatin test:

-
+
+
-

+
+
+
-

-
+
+
-
5 Flavonoids test
a.NaOH test:
b.Ferric chloride test:
+
+
+
+
+
+
6 Glycosides test
a.Borntrager’s test:
+

-

-

7 Steroids/Phytosterols test:
a.Liebermann-Burchard’s test:
b.Salkowski test:

+

+

+

+

-

+
8 Saponins test + + +
9 Resins test +

PEAK#
R.Time Area Area% Name
1 3.289 13973621 1.79 Butanoic acid, 3-methyl-
2 3.488 7638825 0.98 Butanoic acid, 2-methyl-
3 3.937 7234083 0.92 Methylvinylacetic acid
4 4.281 3038808 0.39 Propanoic acid, 2-oxo-, methyl ester
5 4.531 3195750 0.41 Dihydroxyacetone
6 5.574 5864753 0.75 2,5-Furandione, dihydro-3-methylene-
7 5.958 13714831 1.75 Glycerin
8 6.235 5943408 0.76 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-on
9 6.410 1394230 0.18 Cyclopentanol
10 7.159 2102650 0.27 1,4-Dioxin, 2,3-dihydro-5,6-dimethyl-
11 7.744 10501688 1.34 2,3-Dihydro-1H-pyrazol-3-one, 2Ac deivative
12 8.540 2560077 0.33 Glyceraldehyde
13 8.573 4265551 0.55 Pentanoic acid, 4-oxo-
14 8.705 14753426 1.89 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6
15 8.761 1506193 0.19 2(3H)-Furanone, dihydro-4-hydroxy-
16 9.091 2745528 0.35 2-Furanone, 3,4-dihydroxytetrahydro
17 9.242 1422197 0.18 4H-Pyran-4-one, 3,5-dihydroxy-2-methyl-
18 9.795 3873396 0.49 5-Hydroxymethylfurfural
19 10.033 7702041 0.98 1,2,3-Propanetriol, 1-acetate
20 10.656 10181084 1.30 Heptanoic acid, 6-oxo-
21 11.537 1740325 0.22 2-Methoxy-4-vinylphenol
22 14.517 23618049 3.02 1,3-Propanediol, 2-(hydroxymethyl)-2-nitro-
23 15.590 1527128 0.20 D-Allose
24 17.090 1531073 0.20 3-Deoxy-d-mannonic acid
25 18.265 271138883 34.65 3-O-Methyl-d-glucose
26 18.348 67665791 8.65 2-Methoxy-3-methyl-butyric acid, methyl ester
27 18.423 236199850 30.18 4-O-Methylmannose
28 18.867 15160151 1.94 4-O-Methylmannose
29 19.085 1970016 0.25 2-Amino-3-hydroxypyridine
30 19.247 3773293 0.48 Adenosine 3',5'-cyclic monophosphate
31 19.433 7831921 1.00 Myo-Inositol
32 21.619 1415359 0.18 9-Octadecenoic acid (Z)-, methyl ester
33 22.857 2231309 0.29 Tetracosane
34 23.592 2821154 0.36 Tetracosane
35 24.245 1367698 0.17 Cholest-5-en-3-ol (3.beta.)-, tetradecanoate
36 24.458 5632193 0.72 Tetracosane
37 25.500 3222359 0.41 Tetracosane
38 26.307 4056663 0.52 Nonacosan-10-ol
39 26.770 3932358 0.50 Tetracosane
40 28.327
2111303
0.27
Hexacosane 78

782559016 100.00
In vitro anti radical scavenging activity by using DPPH model. The extracts and isolated compounds were tested for invitro anti radical scavenging activity. Using DPPH reduction model as reported by Vani,et.al. (1997).
SAMPLE MG/ML ⁒OF INHIBITION
Balanites aegyptiaca fruit pulp extract 4 mg 99.4
Balanites aegyptiaca leaf extract 1mg 99.75
Balanites aegyptiaca thorns extract 1mg 96.19

The finding if the Balanites aegyptiaca fruit pulp, leaf, thorn extract anthelmintic activity test showed that it had various level of anthelmintic activity at all of the tested concentrations, the extract demonstrated worm death in addition to worm paralysis
Albendazole which as concentration of 400mg/100ml, the tablet was shown to how the activity at 1171 sec (paralysis time) & 2100 sec (death time).
SI NO GROUP MG/ML Lumbricusterrestris
Time taken for paralysis in second Time taken for death in second
1 Control (distilled water) - - -
2 Standard (albendazole) 400mg/100ml 1171 sec 2100 sec
3 Balanites aegyptiaca fruit pulp 10 mg 616 sec 926 sec
25 mg 394 sec 751 sec
4 Balanites aegyptiaca leaf 10 mg 799 sec 967 sec
25 mg 306 sec 750 sec
5 Balanites aegyptiaca thorns 10 mg 1324 sec 1622 sec
25 mg 603 sec 981 sec

Anthelmentic activity screening results show that aqueous extracts of Balanites aegyptiaca leaf caused worm paralysis for 306 seconds and death for 750 seconds at a dosage of 25 mg , Balanites aegyptiaca fruit pulp caused worm paralysis for 394 seconds and death for 751 seconds at a dosage of 25 mg, Balanites aegyptiaca thorns caused worm paralysis for 603 seconds and death for 981 seconds at a dosage of 25 mg. The results show that water extract of leaf has more Anthelmentic action than Albendazole.

[00112] In vitro anti-diabetic activity was evaluated using the Glucose uptake by Yeast cells, alpha amylase activity, GOD-POD method. The extract and isolated compound were tested for in vitro inhibition activity. At various concentration Balanites aegyptiaca shows potent Anti-Diabetic activity.
Sample Mg/ml % Inhibition
1 Balanites aegyptiaca fruit pulp 1mg 97.72
2 Balanites aegyptiaca leaf 1mg 100
3 Balanites aegyptiaca thorns 1mg 96.19
[00113] 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.
[00114] 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 aqueous extracts from plant parts of Balanites aegyptiaca, comprising the steps of
i) collecting plant material selected from fruit pulp, leaves, and thorns of authenticated Balanites aegyptiaca;
ii) drying the leaves and thorns for approximately four days under ambient conditions, and manually separating the fruit pulp from the fruit;
iii) powdering the dried leaves and thorns, and storing the powder in a closed container until use;
iv) weighing approximately 25 g of each plant part separately, and subjecting the weighed material to aqueous extraction using demineralized water as the solvent;
v) performing extraction by refluxing the plant material in water for about four hours in a first cycle, followed by about three hours in a second cycle, and about one hour in a third cycle, wherein the residue is dried between cycles;
vi) pooling the aqueous extracts from all cycles and concentrating them using a rotary evaporator; and
vii) calculating the percentage yield of the extract with reference to the weight of the dried plant material.
2. The method as claimed in claim 1, wherein the plant material is authenticated botanically prior to extraction.
3. The method as claimed in claim 1, wherein the extraction method is based on a refluxation process as reported by Forsyth (1955).
4. The method as claimed in claim 1, wherein the pooled aqueous extracts are subjected to phytochemical screening to identify bioactive constituents.
5. The method as claimed in claim 1, wherein the antioxidant activity is determined using a radical scavenging assay, the anthelmintic activity is determined using in-vitro parasite immobilization tests, and the anti-diabetic property is assessed using in-vitro enzymatic inhibition assays.
6. The aqueous plant extract as claimed in claim 1, wherein said extract is mostly bioactive phytochemicals extracted from at least one plant part of Balanites aegyptiaca selected from fruit pulp, leaves, and thorns.