PHYSICOCHEMICAL AND PHYTOCHEMICAL ANALYSIS OF METHANOLIC EXTRACT OF LEAVES AND FRUITS OF FLACOURTIA JANGOMAS (LOUR.) RAEUSCH

PHYSICOCHEMICAL AND PHYTOCHEMICAL ANALYSIS OF METHANOLIC EXTRACT OF LEAVES AND FRUITS OF FLACOURTIA JANGOMAS (LOUR.) RAEUSCH

Akshaya Pai and K. Chandrakala Shenoy ^*

Department of Biosciences, Mangalore University, Mangalagangothri, Dakshina Kannada - 574199, Karnataka, India.

ABSTRACT: Plants have been used extensively as medicine for the treatment of various ailments for centuries. The present study was conducted to analyze physicochemical and phytochemical parameters of leaves and fruit of Flacourtia jangomas (Lour.) Raeusch  Physicochemical profile showed the high total ash, water-soluble ash, and acid-soluble ash content in F. jangomas leaf when compared to the fruit. The water extractive value of F. jangomas leaves and fruit was more a better extractive compared to alcohol. Moisture content, swelling index, and foaming index of leaves were found to be 6.22%, 5.466 ml, and >100 units, whereas fruit was 8.973%, 3.433 ml, and <100 units, respectively. Phytochemical constituents of dried leaf and fruit were analyzed using qualitative and quantitative methods. Preliminary phytochemical screening showed the presence of alkaloids, glycosides, tannins, flavonoids, steroids, phenols, quinones, lignin, resins, coumarins, carbohydrates and proteins. Quantitative analysis showed high flavonoid content (868.04 ± 3.185, 80.91 ± 0.796 mg QE/g) followed by tannin (253.10 ± 0.738, 23.96 ± 0.071 mg TAE/g) then phenol (144.35 ± 0.99, 13.226 ± 0.035 mg GAE/g) and alkaloids (4.98 ± 1.161, 7.130 ± 0.317 %) in both leaf and fruit of F. jangomas respectively. GC-MS analysis showed the presence of 19 phytoconstituents in leaf and 7 phytoconstituents in fruit extract.  EDS-SEM analysis showed the presence of N, O, K and C, Ca in leaf and N, O, K, C in fruit. The results provide referential information of the plant, and various phytoconstituents suggest that this plant exhibit pharmaceutical significance.

Physicochemical parameters, Phytochemical screening, GC-MS, EDS-SEM, Flacourtia jangomas

INTRODUCTION: India has one of the oldest, richest, and most diverse traditions associated with the use of medicinal plants ¹. Plants have been used extensively for the treatment of various ailments. According to the World Health Organization (WHO), around 85-90% of the world’s population uses herbal medicines ². Plants used in traditional herbal medicines may not be as useful as asserted or may have therapeutic properties in addition.

Therefore, proper scientific understanding is requiredto explore the medicinal plants for curative purpose ³. Due to the lack of inadequate synthetic drugs WHO has recommended the evaluation of physicochemical and phytochemical parameters of medicinal plants for its efficacy. These parameters help in identification and authentication of the plant material ⁴.

Flacourtia jangomas (Lour.) Raeusch (family Salicaceae) is commonly known as Paniala or Indian plum. Leaves are alternate, spirally arranged, rarely ovate-lanceolate, and pale pink when young. The fruits are dark-red when ripe. Traditionally, various parts of the plant are used for the treatment of numerous ailments. Sasi et al. reported that the leaves are astringent and stomachic. The fruits are used to overcome various disorders such as quench thirst, biliousness, fevers, stomach upset, and diarrhea ⁵. Therefore, the aim of the present work was to ascertain chemical standards like physicochemical and phytochemical testing of the leaves and fruits of Flacourtia jangomas (Lour.) Raeusch.

MATERIALS AND METHODS:

Plant Material: Flacourtia jangomas (Lour.) Raeusch was collected from Bellikoth, Kasargod District of Kerala, India. It was taxonomically identified and authenticated by Dr. T. S. Swapna, Professor, and Head, Department of Botany, University of Kerala, Kariavattom, Thir-uvananthapuram, and the voucher specimen was deposited with Accession number KUBH10169. The voucher specimen of the plant F. jangomas (Lour.) Raeusch was preserved in the Herbarium of Department of Botany, University of Kerala, Kariavattom.

Physicochemical Analysis: The leaves and fruits of F. jangomas were collected, cleaned and shade dried for about 3 weeks and 7 weeks, respectively. Dried leaves and fruits were then homogenized to obtain coarse powder and stored in an airtight container for further use.

The various physicochemical parameters such as moisture content, ash values including total ash, acid insoluble and soluble ash, water-soluble and insoluble ash and sulphated ash, water-soluble extractive value, alcohol soluble extractive value, foaming index, swelling index, and pH of powdered plant material were determined by the methods described in Indian Pharmacopoeia ⁶ and WHO guidelines ⁷.

Fluorescence analysis: The leaf and fruit powder of F. jangomas was subjected to fluorescence analysis, after treating with various chemical reagents like methanol, chloroform, ethanol, diethyl ether, petroleum ether, acetone, benzene, glacial acetic acid, sulphuric acid, hydrochloric acid, nitric acid, 1N sodium hydroxide and distilled water against visible and UV light (312 nm) ⁸.

Phytochemical Screening:

Qualitative Analysis: For preliminary phyto-chemical screening, the powdered leaf and fruit material were extracted with methanol in Soxhlet apparatus, and the solvent extract was concentrated using a rotary vacuum evaporator and stored at 4ºC until further use. The extract thus obtained was subjected to different qualitative analyses to discern the bioactive components. The presence of alkaloids, glycosides, tannins, flavonoids, fat and fixed oils, steroids, phenol, quinones, lignin, resins, saponins, coumarins, chalcones, proteins, and carbohydrates were determined according to the Harborne method described by Joshi et al., ⁹ and Irum et al.,¹⁰.

Quantitative Analysis:

Determination of Total Phenolic Content: The total phenolic content was determined by the method of Jose and Anilkumar ¹¹ with slight modifications. Gallic acid was used as a standard, and the total phenolics were expressed as mg/g gallic acid equivalents (GAE). Briefly, 1 ml of a standard solution of different concentrations (10-100 µg/ml) of gallic acid and 1 mg/ml of plant extract were prepared in methanol. 0.5 ml of sample mixed with 2.5 ml of a 10% Folin-Ciocalteu’s reagent and 2ml of 7.5% sodium carbonate. The tubes were covered and allowed to stand for 30 min at room temperature, and the absorbance was read at 760 nm spectrometrically.

Determination of the Total Flavonoid Content: Total flavonoid content was determined using the Aluminum chloride method of Jose and Anilkumar ¹¹ with slight modifications. In this method quercetin was used as standard, and flavonoid contents were measured as quercetin equivalent (QE). Briefly, 1 ml of standard solution (100-500µg/ml) and 1mg/ml extract were prepared in methanol. 0.5 ml of sample was taken into a test tube containing 4ml of distilled water. 0.3 ml of 5% NaNO₂ added to the test tube. After 5 min, 0.3 ml 10% AlCl₃ and 2 ml of 1M NaOH were added to the mixture and made up the volume to 10 ml with distilled water. The absorbance was noted at 510 nm using a UV-Visible spectrophotometer. Standard was prepared in the same manner.

Determination of the Total Tannin Content: The total tannin content was determined by Folin-Ciocalteu method of Chandran and Indira ¹² with slight modifications. Tannic acid was used as standard, and tannin content was expressed as mg/g of tannic acid equivalents (TAE). Briefly, 1 ml of standard solution (10-150 µg/ml) was prepared in distilled water, and 1mg/ml extract was prepared in methanol. 0.5 ml of sample was taken into a test tube; containing 7.5 ml of distilled water.0.5 ml of Folin-Ciocalteu’s phenol reagent and 1 ml of 35% sodium carbonate solution were added to the test tube and diluted to 10 ml with distilled water. The mixture was shaken well and kept at room temperature for 30 min. Standard was prepared in the same manner as described earlier. Absorbance for test and standard solutions were measured against the blank at 700 nm with an UV/ Visible spectrophotometer.

Determination of Alkaloid Content: Alkaloid content was determined gravimetrically by the method of Roghini and Vijayalakshmi ¹³. One gram of the sample was taken in a beaker. 200 ml of 10% acetic acid in ethanol was added to it and left as such for 4 h. It was then filtered and the extract was concentrated on a water bath to one quarter of the original volume. Concentrated NH₄OH was added drop wise to the extract until the precipitation was complete. The whole solution was left to settle and the precipitate was collected and washed with dilute NH₄OH and then filtered. The residue is the alkaloid, which was dried and weighed.

GC-MS analysis: The phytochemical investigation of methanolic extract of both leaf and fruit of F. jangomas was performed on Shimadzu GC-MS (Model Number: QP2010S) instrument and GC-MS Software - GCMS Solutions was used for analysis. Experimental conditions of the GC-MS system were as follows: Rxi-5Sil MS column, dimension: 30 Mts, ID: 0.25 mm, film thickness: 0.25 μm. A flow rate of mobile phase (carrier gas: He) was set at 1.0 ml/min. In the gas chromatography part, the temperature program (oven temperature) was 80 °C raised to 280 °C at 5 °C / min, and injection volume was 1 μl. Samples dissolved in methanol were run fully at a range of 50-500 m/z and the results were compared by using Wiley Spectral library search program (Wiley 8) and NIST mass spectral database (NIST 11).

EDS Analysis: The semi-quantification elemental analysis of leaf and fruit of F. jangomas was performed using Scanning Electron Microscope- Energy Dispersive Spectrometer (SEM-EDS) equipment. OXFORD INCA software was used for Digital Imaging and Spectral Collection.

Statistical Analysis: Values have been expressed as mean ± standard deviation (n=3). Statistical comparisons of physicochemical and phyto-chemical parameters F. jangomas leaf and fruit were made with Student t-test

RESULTS AND DISCUSSION:

Physicochemical Analysis: Evaluation of physicochemical parameters is important in identifying adulteration or inappropriate handling of drugs ⁴. The physicochemical profile of leaf and fruit of F. jangomas is shown in Table 1.

TABLE 1: PHYSICO-CHEMICAL STANDARDIZATION OF LEAVES AND FRUITS OF F. JANGOMAS

The values represent Mean ± SD of three replicates. F. jangomas fruit vs F. jangomas leaf:  * p<0.01 ** p<0.003 *** p<0.0001 (Student t- test).

The total ash content, water-soluble ash and acid-soluble ash of F. jangomas leaf was found to be more when compared to the fruit.

Ash values are used to ascertain the quality and purity of crude drug. The water-soluble ash is used to evaluate the amount of inorganic matter and acid insoluble ash measures the amount of silica present and it shows contamination with earthy material. Water soluble ash is the water-soluble part of the total ash ¹⁴. Water extractive value of F. jangomas leaves and fruit was more, a better extractive compared to alcohol. Moisture content, swelling index and foaming index were found to be 6.22%, 5.466 ml and >100 whereas fruit was 8.973%, 3.433 ml and <100 units respectively. Moisture content of drug was found to beless, thus prevents the growth of microorganisms during storage ¹⁴.

Fluorescence Analysis: In fluorescence analysis characteristic color changes were observed with various chemical reagents in both leaf and fruit of F. jangomas under visible and UV light (312 nm), it showed different colors of the powder in the presence or absence of chemical constituents Table 2.

When the substances are not fluorescent by themselves, the chemical reagents promote their transformation into fluorescent derivatives ⁴.

TABLE 2: FLUORESCENCE ANALYSIS OF F. JANGOMASLEAF AND FRUIT POWDER

Phytochemical Screening: Methanolic extract of leaf and fruit of F. jangomas were subjected to qualitative chemical tests. The preliminary phytochemical analysis has revealed the presence of various phytochemicals such as alkaloids, glycosides, tannins, flavonoids, steroids, phenols, quinone, lignin, resin, coumarin, carbohydrates and proteins but trace amounts of fats and fixed oils were present and saponins were absent in both leaf and fruit extract Table 3A and 3B. Quantitative analysis of methanolic extract of Flacourtia jangomas leaf and fruit was found to possess phenols (144.35 ± 0.99, 13.226 ± 0.035 mg GAE/g), flavonoids (868.04 ± 3.185, 80.91 ± 0.796 mg QE/g), tannins (253.10 ± 0.738, 23.96 ± 0.071 mg TAE/g); alkaloids (4.98 ± 1.161, 7.130 ± 0.317 %) respectively Table 4. When compared to fruit extract, leaf extract possesses more phenol, flavonoid, tannin, alkaloid content (p<0.0001, Student t-test). Thus, results indicate that the plant holds high profile values and can be used to cure various ailments.

TABLE 3A: QUALITATIVE PHYTOCHEMICAL SCREENING OF METHANOLIC EXTRACT OF F. JANGOMAS LEAVES AND FRUITS

++ present in moderate; +++ present in more quantity; - Absent

TABLE 3B: QUALITATIVE PHYTOCHEMICAL SCREENING OF METHANOLIC EXTRACT OF F. JANGOMAS LEAVES AND FRUITS

++ present in moderate; +++ present in more quantity; - Absent

TABLE 4: QUANTITATIVE PHYTOCHEMICAL ESTIMATION OF METHANOLIC EXTRACT OF F. JANGOMAS LEAVES AND FRUITS

Results are mean of triplicate determinations on the basis of reference standard ± standard deviation. F. jangomas fruit vs F. jangomas leaf:  * p<0.03 **p<0.0001 (Student t-test). GAE*- Gallic acid equivalent; QE*- Quercetin equivalent; TAE*- Tannic acid equivalent.

GC-MS Analysis: The spectrum profile of GC-MS analysis confirmed the presence of nineteen major constituents in the methanolic extract of F. jangomas leaves and seven major constituents in the methanolic extract of F. jangomas fruits as presented in Table 5 and Table 6, respectively.

Fig. 1 and Fig. 2 revealed the retention time and the spectral peaks of the bioactive compounds present in F. jangomas leaf and fruit extract respectively.

FIG. 1: GC-MS CHROMATOGRAM OF METHANOLIC EXTRACT OF F. JANGOMAS LEAF

TABLE 5: PHYTOCONSTITUENTS IDENTIFIED IN THE METHANOL EXTRACT OF F. JANGOMAS LEAF

TABLE 6: PHYTOCONSTITUENTS IDENTIFIED IN THE METHANOL EXTRACT OF F. JANGOMAS FRUIT

FIG. 2: GC-MS CHROMATOGRAM OF METHANOLIC EXTRACT OF F. JANGOMAS FRUIT

EDS Analysis: Results of the elemental composition of F. jangomas leaf and fruit powder using SEM-EDS technique is showed in Table 7 and SEM-EDX spectra are showed in Fig. 3.

F. jangomas leaf sample showed a high concentration of Oxygen and Carbon, a moderate amount of calcium, and trace quantities of nitrogen and potassium. While F. jangomas fruit sample showed a high concentration of Oxygen and Carbon and a moderate amount of nitrogen and potassium.

TABLE 7: THE PERCENTAGE OF TRACE ELEMENTS PRESENT IN THE LEAF AND FRUIT OF F. JANGOMAS

FIG. 3: SEM-EDS SPECTRUM OF F. JANGOMAS LEAF AND FRUIT

CONCLUSION: The present study provides information on physicochemical parameters and phytochemicals present in methanolic extract of leaves and fruit of Flacourtia jangomas (Lour.)

Raeusch can be used for the identification and authentication of plant material. Quantitative and GC-MS analysis reveals that the leaves and the fruit are a rich source of phytoconstituents indicating that the plant material can be further subjected to elucidate the possible mechanism of action in order to explore its medicinal use.

ACKNOWLEDGEMENT: The author’s acknow-ledge the Department of Biosciences, Mangalore University, for providing DST-FIST and UGC-SAP supported laboratory facilities and also acknowledge DST-PURSE laboratory Mangalore University.

The authors wish to acknowledge the Council of Scientific and Industrial Research University Grants Commission (CSIR-UGC), New Delhi, for financial assistance.

CONFLICTS OF INTEREST: Authors have no conflicts of interest.

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    How to cite this article:

    Pai A and Shenoy KC: Physicochemical and phytochemical analysis of methanolic extract of leaves and fruits of Flacourtia jangomas (lour.) raeusch. Int J Pharm Sci & Res 2021; 12(3): 1671-78. doi: 10.13040/IJPSR.0975-8232.12(3).1671-78.

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