PHYTOCHEMICAL PROFILING OF ASTERACANTHA LONGIFOLIA AND MORINGA OLEIFERA: A COMPARATIVE ANALYSIS OF SOXHLET AND MICROWAVE-ASSISTED EXTRACTION METHOD

PHYTOCHEMICAL PROFILING OF ASTERACANTHA LONGIFOLIA AND MORINGA OLEIFERA: A COMPARATIVE ANALYSIS OF SOXHLET AND MICROWAVE-ASSISTED EXTRACTION METHOD

Nazneen Azad and Chumkijini Chhatria *

P.G. Department of Biotechnology and Bioinformatics, Sambalpur University, Jyoti Vihar, Odisha, India.

ABSTRACT: Plants have rich deposits of phytochemical constituents, which are responsible for their medicinal uses. Different extraction methods like Soxhlet and microwave-assisted extractions are used to isolate these phytochemicals from plant materials. However, there is a lack of studies on a comprehensive comparison of phytochemical profiles obtained from different plant materials using Soxhlet and microwave-assisted extraction methods. Therefore, this research is carried out to investigate the presence and quantification of phytochemicals in Asteracantha longifolia and Moringa oleifera using Soxhlet extraction and Microwave-assistedextraction methods. The extraction process is carried out using solvents like ethanol, hydro-alcoholic solution, and distilled water. In both extraction methods, the highest yield is obtained for the stem and leaves of Moringa oleifera with distilled water. Soxhlet extraction with distilled water exhibited the highest extract yield potential i.e., 12.68% for Moringa oleifera (Stem and leaves) and 9.62% for Asteracantha longifolia (Root). In the microwave-assisted extraction method, Moringa oleifera (Stem and leaves) obtained the highest yield of 19.2%, 28.7%, and 33.2% with the solvent’s ethanol, hydro-alcoholic solution, and distilled water, respectively. Asteracantha longifolia (Root) exhibits a low yield of 0.91% with ethanol and 1.06% with distilled water. The phytochemical screening shows the presence of phytochemical compounds. Furthermore, the result shows that the highest total phenol content is obtained using microwave-assisted extraction, and the highest total flavonoid content is obtained using the Soxhlet extraction methods.

Keywords: Asteracantha longifolia, Moringa oleifera, Phytochemical, Soxhlet Extraction, and Microwave-assisted extraction

INTRODUCTION: The world is enriched with varieties of medicinal plants. Their medicinal and pharmacological properties are attributed to bioactive components that exert specific physiological effects on the human body ¹. Asteracantha longifolia and Moringa oleifera are plants with significant medicinal properties and are used for different medicinal purposes.

Asteracantha longifolia is traditionally used for diuretic and hepatoprotective properties, while Moringa oleifera is known for its rich nutritional and antioxidant profile. Both the plants are rich in phytochemicals ^2,3. Phytochemicals are biologically active, naturally occurring chemical compounds found in plants that provide health benefits for humans as medicinal ingredients and nutrients ⁴.

The major phytochemicals include carbohydrates, proteins, amino acids, chlorophylls, alkaloids, saponins, steroids, flavonoids, and tannins. Plants are a rich source of polyphenols and flavonoids, which protect the body against oxidative stress and maintain the balance between oxidants and antioxidants ^{5, 6}.

Various extraction techniques have been used to obtain the phytochemicals from different plant parts. These extraction techniques include the conventional solvent-based (Soxhlet extraction method) and the more robust modern and green extraction techniques (Microwave-assisted extraction method) ^{7, 8}. Soxhlet and microwave-assisted extraction are automated techniques that enhance extraction efficiency. While Soxhlet is a continuous solvent-based process, microwave-assisted extraction provides reduced solvent usage and shorter extraction time. Both extraction methods are considered as commonly used methods for phytochemical extraction from plants ^{9, 10}.

The extraction methods and employed solvents significantly effect on extraction of phytochemicals. The results revealed that the extraction efficiency for the maceration with methanol showed significant residue yield and the content of Betulin. Also, ultrasound-assisted extraction with ethanol demonstrated better selectivity for phytochemicals of Asteracantha longifolia (L.) Nees collected from the western ghat of Maharashtra ¹¹. The yield obtained by the Soxhlet extraction method using ethanol, hexane, and petroleum ether was 42%, 38.9%, and 38%, respectively, in Moringa oleifera seeds of Brazil. Microwave-assisted extraction with hexane and petroleum ether showed oil yields of 35.9% and 35.8%, respectively ¹².

The optimum formulation was obtained with an optimal extraction time of 2.119 minutes, ethanol concentration of 57.618%, and solid-solvent ratio of 1:15 g/mL using microwave-assisted extraction ¹³. Moringa oleifera oil from microwave-assisted and Soxhlet extraction methods had good yields of 34.25% and 28.75%, respectively, and a low moisture content of 0.008% and 0.011%, respectively ¹⁴. Existing studies focused on various conventional and modern methods of phytochemical extraction from plants. Many studies expressed these views, particularly on the extraction of bioactive compounds from plant materials. However, limited studies have directly compared the efficiency of Soxhlet extraction and microwave-assisted extraction in isolating phytochemicals from specific medicinal plants. Therefore, this research is carried out to investigate the presence and quantification of phytochemicals in Asteracantha longifolia and Moringa oleifera using Soxhlet extraction and microwave-assisted methods.

MATERIALS AND METHODS:

Sample Collection: The plant parts, such as the aerial parts and roots of Asteracantha longifolia and the stem and leaves of Moringa oleifera were collected from nearby areas of Sambalpur. Dust particles were removed by washing with tap water followed by distilled water and then the plant materials were air-dried. The dried plant material was ground to a fine powder using a mechanical grinder and kept in airtight polybags.

Soxhlet Extraction: The powder samples were taken for Soxlet extraction. 60g powder of aerial parts and 54g of Asteracantha longifolia root; 54g of Moringa oleifera stem and leaves were used for Soxhlet extraction. Ethanol, distilled water, and a hydro-alcoholic solution (ethanol: distilled water, 70:30) were used as solvent. To guarantee the highest phytochemical output, the extraction procedure was conducted for 48 hours and included continuous cycles of solvent reflux and condensation. After passing through the Whatman filter paper, the extracts were concentrated at 50°C in a rotary evaporator. Extraction of different plant material is shown in Fig. 1.

FIG. 1: SOXHLET EXTRACTION OF PLANT SAMPLES (a) AERIAL PART, (b) ROOT, (c) STEM AND LEAVES

Microwave-assisted Extraction: For microwave-assisted extraction, 25g powder sample of aerial parts and 15g of root of Asteracantha longifolia and 30g powder sample of stem and leaves of Moringa oleifera, were taken and extracted using ethanol, hydro-alcoholic solution, and distilled water. The extraction was performed at 500W microwave power for 5 minutes per cycle, followed by centrifugation at 7000 rpm for 10 minutes. The supernatant was collected, and the solvent was evaporated at room temperature. The dried extracts were scraped from Petri plates for further analysis, as shown in Fig. 2.

FIG. 2: MICROWAVE-ASSISTED EXTRACTION OF PLANT SAMPLES (a) AERIAL PART, (b) ROOT, (c) STEM AND LEAVES

Determination of Percentage Yield: Percentage yield measures the effectiveness of the entire extraction process. % yield was calculated using the formula given below:

Percentage yield = (Weight of extract) / (Weight of powder sample taken) × 100

Qualitative Phytochemical Screening: Qualitative phytochemical screening is a method used to identify the presence or absence of various classes of natural compounds, such as alkaloids, glycosides, flavonoids, diterpenes, phenols, proteins, carbohydrates, saponins, tannins, and sterols. Qualitative phytochemical screening was done by using various standard methods, such as Wagner’s Test, Hager’s Test, Concentrated H₂SO₄ Test, Alkaline Reagent Test, Lead acetate Test, Copper acetate Test, Ferric Chloride Test, Folin Ciocalteu Test, Biuret test, Fehling’s Test, Benedict’s test, Froth Test, Gelatin Test, and Salkowski Test, which is shown in Table 1.

TABLE 1: METHODS TO IDENTIFY PHYTOCHEMICAL CONSTITUENTS

Quantitative Analysis of Phytoconstituents:

Estimation of Total Phenol Content: The total phenolic content was determined using the modified Folin-Ciocalteu technique ¹⁹. A standard calibration curve was prepared using gallic acid (10–50 µg/mL). The plant extracts (10 mg/mL) were reacted with 1 mL of Folin-Ciocalteu reagent (diluted 1:10 v/v) and 1 mL of 7.5% sodium carbonate solution. After incubation at room temperature for 15 minutes, the absorbance was measured at 765 nm using a UV-Vis spectrophotometer (Shimadzu UV-1800). The results were expressed as mg of gallic acid equivalents (GAE) per gram of extract.

Estimation of Total Flavonoid Content: Total flavonoid content was determined using the aluminium chloride colorimetric assay ¹⁹. A standard quercetin curve (5–25 µg/mL) was prepared. 3 mL of plant extract (10 mg/mL) was mixed with 1 mL of 2% AlCl₃ solution and allowed to incubate at room temperature for 15 minutes. The absorbance was measured at 420 nm, and flavonoid concentration was expressed as mg quercetin equivalents (QE) per gram of extract.

RESULTS:

Yield Potential of Extract: Extracts from various parts of the Asteracantha longifolia and Moringa oleifera were obtained using a variety of solvents, thus resulting in significant differences in the yield for each solvent. Table 2 illustrates the extract yield obtained in different solvents using the Soxhlet method and microwave-assisted method.

TABLE 2: YIELD POTENTIAL OF PLANT EXTRACT

Phytochemical Screening using Soxhlet and Microwave-assisted Method: All the extracts were evaluated for the occurrence of the phytochemical constituents. The results of the qualitative phytochemical analysis are given in Table 3, where the ‘ü’ sign indicates the presence of that constituent or the ‘û’ sign indicates the absence.

TABLE 3: PHYTOCHEMICAL SCREENING

FIG. 3: MICROWAVE-ASSISTED EXTRACTION USING (i) ETHANOLIC EXTRACT FOR (a) AERIAL PART, (b) ROOT, (c) STEM AND LEAVES, (ii) HYDRO-ALCOHOLIC EXTRACT FOR (d) AERIAL PART, (e) ROOT, (f) STEM AND LEAVES, (iii) AQUEOUS EXTRACT FOR (g) AERIAL PART, (h) ROOT, (i) STEM AND LEAVES

FIG. 4: SOXHLET EXTRACTION USING (i) ETHANOLIC EXTRACT FOR (a) AERIAL PART, (b) ROOT, (c) STEM AND LEAVES, (ii) HYDRO-ALCOHOLIC EXTRACT FOR (d) AERIAL PART, (e) ROOT, (f) STEM AND LEAVES, (iii) AQUEOUS EXTRACT FOR (g) AERIAL PART, (h) ROOT, (i) STEM AND LEAVES

Estimation of Total Phenolic Content and Total Flavonoid Content: The total phenolic and flavonoid contents of ethanol, hydro-alcoholic solution, and distilled water extracts from the aerial part and root of Asteracantha longifolia and stem and leaves of Moringa oleifera were tested. The result of the total phenolic contents was expressed as mg/100mg of the gallic acid equivalent of dry extract sample using the equation of a calibration curve g= 0.019x+0.021, R² = 0.998  as shown in Fig. 5(A), where x is the gallic acid equivalent and y is the absorbance. Total flavonoid contents were expressed as quercetin equivalent using the equation g= 0.047x+0.016, R² = 0.998   as shown in Fig. 5(B), where x is the quercetin equivalent.

FIG. 5: CALIBRATION CURVES FOR (a) GALLIC ACID, (b) QUERCETIN

The folin-Ciocalteu technique and aluminium chloride colorimetric assay were used to determine the total phenol and flavonoid content. Table 4 shows the total phenol and flavonoid content obtained using the Soxhlet and microwave-assisted extraction methods.

TABLE 4: TOTAL PHENOL AND FLAVONOID CONTENT

The result showed that the highest total phenol content was obtained for ethanolic extract of Moringa oleifera (Stem and leaves) using Soxhlet extraction and Asteracantha longifolia (Aerial part) and Moringa oleifera (Stem and leaves) using microwave-assisted extraction. Also, the highest total flavonoid content was obtained for the ethanolic extract of Moringa oleifera (Stem and leaves) using Soxhlet extraction and Asteracantha longifolia (root) using microwave-assisted extraction. However, the root extract of Asteracantha longifolia using the soxhlet extraction method with solvents, such as ethyl acetate, ethanolic, and aqueous extracts was examined ²⁵. The study found that the total phenol content and total flavonoid content with ethanolic extract were 0.529 mg/100mg and 0.608 mg/100mg, respectively, and with aqueous extract were 0 and 0.251 mg/100mg, respectively.

DISCUSSION: In the present work an attempt has been made to study the presence and quantification of phytochemicals in Asteracantha longifolia and Moringa oleifera using Soxhlet extraction and microwave-assisted methods. Soxhlet extraction with distilled water exhibited the highest extract yield potential i.e., 12.68% for Moringa oleifera (Stem and leaves) and 9.62% for Asteracantha longifolia (Root). In the microwave-assisted extraction method, Moringa oleifera (Stem and leaves) obtained the highest yield of 19.2%, 28.7%, and 33.2% with the solvent’s ethanol, hydro-alcoholic solution, and distilled water, respectively. Asteracantha longifolia (Root) exhibits a low yield of 0.91% with ethanol and 1.06% with distilled water. In both extraction methods, distilled water solvent extract had the maximum yield. Further, the microwave-assisted extraction method obtained the highest yield (33.2%) in stem and leaves of M. oleifera. In contrast to the present study, ²⁰ found that the highest yield of 28.40% was obtained by Moringa oleifera using the Soxhlet extraction method with n-hexane as the solvent. Also, the yield obtained through the microwave-assisted extraction method was 6.90%. In another study, ²¹ examined the aerial parts, leaves, and seeds of Hygrophila auriculata (Asteracantha longifolia) using conventional extraction techniques. They found the plant extract yield of 4.15 % for aerial parts with the ethanolic extract and a high yield of 22.8 % and 35.64 % for seeds with ethanolic and hexane extracts, respectively, using the Soxhlet extraction method.

Another researcher ²⁷ analyzed the leaves of Moringa oleifera using a microwave-assisted extraction method and showed an extraction yield of 14.64 to 17.65% with a hydroethanolic extract, which is less than the present study (28.7%). As the distilled water and ethanol are polar solvent that exhibit much plant extract yield potential in both extraction techniques. The polar solvents have the potentialities to extract polar compounds like flavonoids and polyphenols. Also, solvent-to-solid ratio is responsible for extract yield potential. The present study found that there were no sterols in the stem and leaves of Moringa oleifera with ethanolic, hydroalcoholic, and aqueous extracts. Carbohydrates were only present in the stem and leaves of Asteracantha longifolia with ethanolic extract using the Soxhlet extraction method. Also, Saponins were present in the steam and leaves of Moringa oleifera. Similar to the present study, a separate study ²² found that there was a presence of saponins in the leaf extracts of Moringa oleifera ²³. tested in the methanolic extract from the leaves of Asteracantha longifolia for the availability of phytochemicals and confirmed the presence of terpenoids, alkaloids, flavonoids, phenols, steroids, glycosides, saponins, tannins, carbohydrates, and proteins. The study of ²⁸ revealed that the presence or absence of particular phytochemicals is determined by the polarity of the solvents used for extraction. Also, justified that M. oleifera contain rich phytochemicals, including alkaloids, flavonoids, phenolics, terpenoids, tannins, and saponins, that are known to have pharmacological properties. In the present study, the Folin Ciocalteu Test showed that phenols are present on the root of Asteracantha longifolia with ethanolic, hydroalcoholic, and aqueous extract using both Soxhlet and microwave-assisted extraction methods. Also, the study showed that glycosides are only presented in the roots of Asteracantha longifolia using the microwave-assisted method and stem and leaves of Moringa oleifera using the soxhlet method with the ethanolic extract.

Alkaloids are absent in root extract of Asteracantha longifolia. Compared to the current study, another author ²⁴ stated that there were no glycosides present in the leaves of Hygrophila auriculata (Asteracantha longifolia) with the ethanolic extract. In the present study, the total phenol and flavonoid content with ethanolic extract for the root of Asteracantha longifolia were 0.66 mg/100mg and 0.92 mg/100mg, respectively. Researchers ²⁶ examined the seeds of Moringa oleifera for the total phenol and flavonoid content using the Soxhlet extraction method and found a high value for methanolic extract. Similar to the present study, ²⁷analyzed the leaves of Moringa oleifera using a microwave-assisted extraction method and revealed a total phenol content of 63.36 to 76.40 mg GA/gram. Phenolic compounds act as a potent antioxidant and for instance, medicinal plants are rich reservoirs of phenolic compounds ²⁹.

CONCLUSION: The present study examined the phytochemicals in Asteracantha longifolia and Moringa oleifera using two extraction methods: microwave-assistedand Soxhlet extraction. The study further investigated the yield obtained from various parts of Asteracantha Longifolia and Moringa Oleifera using solvents, such as ethanol, hydro-alcoholic solution, and distilled water. Among them, Moringa oleifera (Stem and leaves) obtained the highest yield of 33.2% with distilled water using the microwave-assisted extraction method and 12.68% using the soxhlet extraction method. The phytochemical screening showed the presence of phytochemical compounds, such as alkaloids, glycosides, flavonoids, diterpenes, phenol, proteins, carbohydrates, saponins, tannins, and sterols. The research showed that microwave-assisted extraction obtained a higher total phenol content of 1.14 mg/100mg with ethanolic extract for the aerial parts of Asteracantha longifolia and stem and leaves of Moringa oleifera. Further, soxhlet extraction obtained a higher total flavonoid content of 1.28 mg/100mg for the stem and leaves of Moringa oleifera with ethanolic extract. Using distilled water for both the microwave-assisted extraction method and the Soxhlet extract method could provide a high extraction yield for plant materials. Also, the microwave-assisted extraction method could be efficient for obtaining higher total phenol content. Furthermore, the Soxhlet extract method could be efficient for obtaining higher total flavonoid content. There is need of further study on antioxidant, anti-inflammatory, antimicrobial and anti-cancer activity of this plant extract.

ACKNOWLEDGEMENT: Authors remain thankful to the Head of the Department, and COE, NPT, P.G. Department of Biotechnology and Bioinformatics, Sambalpur University for necessary laboratory facilities.

CONFLICT OF INTEREST: No conflicts of interest to declare.

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    How to cite this article:

    Azad N and Chhatria C: Phytochemical profiling of Asteracantha longifolia and Moringa oleifera: a comparative analysis of Soxhlet and microwave-assisted extraction method. Int J Pharm Sci & Res 2025; 16(10): 2820-28. doi: 10.13040/IJPSR.0975-8232.16(10).2820-28.

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