Evaluation of Physiochemical and Phytochemical Parameters of Eichhornia crassipes (Mart.) Solms

 

Anuradha Shukla¹, Pankaj Jain², Rashmi Tripathi³ ⃰

¹Department of Biosciences and Biotechnology, Banasthali Vidyapith, Banasthali - 304022 Rajasthan.

²Department of Pharmacy, Banasthali Vidyapith, Banasthali - 304022 Rajasthan.

³Assistant Professor, Banasthali Vidyapith, Banasthali - 304022, Rajasthan.

 

ABSTRACT:

The study aimed to analyze the phytochemical and pharmacological activities of the leaf extracts of E. crassipes. The hydroethanolic extract fractionated with different solvents (Hexane, Chloroform, ethyl acetate, n-Butanol and aqueous) was used for the analysis. The extracts were analyzed qualitatively for the presence of several phytochemical components. The plant powder was put through a series of different chemicals, and fluorescence emitted was analyzed. The fluorescence was analyzed in daylight and in ultraviolet-light (254nm, and 365nm). The fluorescence examination revealed that the plant powder fluoresces in various colours of dark green, brown, yellow in visible light, and varied shades of green, grey, reddish brown, and brown in UV light. The moisture content (25.1±1.38), and total ash value (16.18±1.39)was observed. The hydroethanolic extracting value was determined to be higher than hexane, chloroform, ethyl acetate, n-butanol, and aqueous. The findings of this study reveal that leaves contain a variety of phytoconstituents that can be employed as a natural source of antioxidants and for the creation of plant-based medications.

 

 

 

INTRODUCTION: 

Plants have always been the backbone of the health-care system, dating back to ancient times¹. Because different active ingredients exist in raw herbal medications, the efficacy, and safety of herbal products are dependent on the quality, and appropriate identification of the plant source. As a result, it has become increasingly important to comprehend the therapeutic and potential worth of plants². Medicinal plants continue to make a substantial contribution to current medications by providing lead chemicals for the development of novel drugs^3,4. The water hyacinth (Eichhornia crassipes (Mart.) Solms) is a Pontederiaceae flowering plant that grows in water. The plant is a freshwater hydrophyte that can be found in both subtropical and tropical climates regarded as an unwanted weed. 

 

Numerous studies have demonstrated its benefits as anti-inflammatory, antifungal, antibacterial^5,6,7 and different uses such as production of ethanol, phytoremediation⁸; production of biofuel⁹; in animal feed¹⁰ and its use in pharmaceutical industry¹¹.

 

The purpose of this study was to determine the physical properties of E. crassipes leaves as indices to indicate the quality of herbal medicine.

 

MATERIALS AND METHODS:

Collection of plant sample:

The E. crassipes have been chosen for the medicinal properties and procured in the summer months from Chekhla, Sanand, Ahmedabad district, Gujarat, India. The collected fresh green leaves were washed with water. The leaves were dried, grounded and stored in airtight containers. In the Patanjali Research Centre, Haridwar, the plant was identified and authenticated (Authentification no: 2626).

 

 

Physical estimation of powdered drug:

According to the standard methodology of Indian pharmacopeia (1996), the Physico-chemical parameters of the drug were evaluated.

 

Ash value:

The 2gm of the drug powder was taken in an asphalted crucible and carbonized for 4 h to 450°C. The weight was taken after the sample was cooled. The ash value was calculated using the formula:

                            Wt. of the sample

% Total ash= -----------------------------×100

                       Wt. of the crucible dish

 

Acid-insoluble ash:

25ml of dil HCl was mixed with ash and heated to boiling point for 5min. The insoluble ash was gathered and washed with warm water on an ash less filter paper. It was then ignited for 4h to 450°C in a crucible and the proportion of ash insoluble in acid was calculated.

 

Water-insoluble ash:

25ml of water was boiled with total ash for 5min and was filtered through the ash less filter paper. The undissolved content was collected on the filter paper; hot water was passed to wash the content. It was then ignited for 4h at 450°C in a crucible. The water-soluble ash content was calculated for the dried drug using the formula: 

      (Wt. of total ash-Wt. of water insoluble ash sample)

%Water insoluble ash=----------------------------------×100

                                 Wt. of crucible dish

 

Extractive Value:

The solvents non-polar to polar i.e., Hydroethanolic fractions of hexane, chloroform, ethyl acetate, n-butanol, and aqueous were utilized for extraction. The powdered drug (5gm) was infused with 100ml of each solvent. The drug is added to the solvent and was kept on a rotator shaker in a closed flask for 24h and allowed to stand for 18h and filtered using Whatman filter paper. The 25ml of the filtrate collected was dried and weighed.

 

Determination of moisture content:

2 gm of the drug powder was placed for 2h at 105°C in an oven in a crucible. It was cooled in desiccators at room temperature. The method was repeated until a constant weight is noted and the percentage of drying loss has been calculated using the formula as given:

                                   Loss in wt. of sample

%Loss on drying = --------------------------------×100

                                        Wt. of sample

 

Fluorescence analysis of powder drug:

The Fluorescence analysis of powder drug was done with different chemical reagents and the resultant color was determined under visible and ultraviolet light. Take about 0.5gms of plant powder into clean and dried watch glass. To each watch glass 2ml of different organic solvents.

 

Preparation of extract:

The powder was macerated with the hydroethanolic for 72h. Hydroethanol crude extract was fractionated with the different solvent (Hexane, Chloroform, ethyl acetate, n-Butanol and aqueous) successively to yield fractions in different solvents. The extracts for further use were dried and stored at 4°C.

 

Phytochemical Assessment:

Phytochemical assessment to identify components in the drug was conducted on E. crassipes.

 

Detection of protein:

Biuret test: 4 to 6 drops of NaOH was added to the extract mixed thoroughly. Copper sulfate was added drop by drop mixed resulting in purple or violet color.

 

Detection of amino acid:

Tyrosine test: 3ml of extract was heated with the 3 drops of millions reagent and the dark red colored solution was observed indicating the presence of tyrosine.

 

Determination of carbohydrate:

Fehling’s test: Solution Fehling’s A and B was mixed and heated with extract. This leads to the formation of cuprous oxide as a red precipitate indicating the presence of reduced sugar.

 

Benedict test:  Same volume of Benedict reagent and extract, were heated in water bath at boiling point. The purple, red or green color appears indicating the presence of sugar in the extract.

 

Detection of starch (non-reducing polysaccharides):

Iodine test: To the extract was added iodine solution. Blue colour was observed which disappeared on boiling and again appears when cooled.

 

Detection of saponins:

Foam test: Extract was shaken with distilled water frothing and its persistence on heating indicates the presence of saponins.

 

Detection of fats and oils:

Sudan test: 0.2gm extract was shaken with Sudan red, purple-colored droplets are formed indicating the presence of fats and oils.

 

Qualitative analysis of secondary metabolites:

Detection of alkaloids:

Dragendroff’s test: Few drops of dragendroff’s reagent were added to the extract. The alkaloids are indicated by the appearance of orange brown color.

Detection of flavonoids:

NaOH test: Extract mixed with aq NaOH and HCl the appearance of yellow orange colour confirms the existence of flavonoids.

 

H₂SO₄ test: Treatment with conc. H₂SO₄, resulted in the formation of dark orange color confirms the presence of flavonoids.

 

Detection of tannins:

10% Ferric chloride test: Extract when treated with 10% ferric chloride brownish blue or black color appears indicating the existence of tannins.

 

Detection of quinines:

The extract when mixed with conc. HCl, results into a yellow colour precipitate confirming the presence of quinines.

 

Detection of terpenoids:

Extract mixed with 10ml of ethanol was shaken well and filtered. To the filtrate (5ml) was added chloroform (2ml) and H₂SO₄(3ml), reddish-brown color was formed indicating the presence of terpenoids.

 

Detection of phenols:

Potassium permanganate test: The extract when treated with potassium permanganate resulted in discoloration.

 

Detection of anthraquinone glycosides:

Borntrager’s test:  Dil. H₂SO₄was added to the mixture of extract and filtered after boiling. To the filtrate the same volume of benzene or chloroform was added and mixed. The pink colour formed on adding ammonia after removal of organic solvent indicates the presence of anthraquinone glycosides.

 

Detection of anthocyanin:

NaOH Test: To the extract was added 2M NaOH resulting in the formation of blue green colour.

 

Detection of glycoside:

Coumarin test: The extract when mixed with 1ml of 10% NaOH forms yellow colour indicating the presence of coumarins.

 

RESULTS AND DISCUSSION:

Plants are a great source of a variety of medicinal substances. One of the major benefits of using plants is that they rarely cause the adverse effects¹². For the use of the plant in traditional medicine it becomes important to regulate it for the use as a drug for which the determination of pharmacognostical properties is useful for setting the standards of the drug. Physical constants are important in the detection of adulteration. Phytochemicals present in the plant dissolve in different solvents and so non-polar to polar solvents were selected for the study.

 

Physicochemical analysis:

Moisture content:

The drug was estimated to contain 25.1±1.38 the moisture content in the leaves was found to be significant hence the powder needs to be kept dried to prevent microbial contamination (Table A.1). The moisture content needs to be minimum to prevent microbial contamination¹³. 

 

Ash value:

The purity and quality of the drug are determined by the ash values indicating the carbonate, oxalate and silicate impurities. The purity of the drug depends on the ash value which indicates the presence of inorganic matter was reported by Chaudhari and Girase¹⁴. The water-soluble and acid insoluble ash value indicates the presence of inorganic elements and the presence of silicates. In the study conducted (Table1), the total ash content of leaves in E. crassipes was found to be higher followed by water-soluble and acid insoluble ash value. The acid insoluble ash content was used to illustrate the quality of herbal medicine^15,16. The lower amount of acid-insoluble ash indicates a lesser carbonate, phosphates, silicates, and silica in the plant. The estimation of ash content is important as it indicates pollution that effects pharmacological activities and is indicative of quality of herbal remedies.

 

Table A.1 Physico-chemical parameters in leaf powder of E. crassipes (Data are Mean± SD, where n=3)

Physicochemical parameters	Values (in % w/w)
Moisture content	25.1±1.38
Total ash value	16.18±1.39
Water soluble ash value	11.28± 0.35
Acid insoluble ash value	5.25±1.11

 

Extractive value:

Percentage of the extractive value indicates the nature of the phytochemicals, the value varied with the extract used and the part of the plant^17,18. Extractive value provides the yield percentage of phytoconstituents in the solvent. The hydro-alcohol was found to be most effective followed by aqueous and n-Butanol fractions. The value of extraction yield was high among the polar solvents and low among nonpolar solvents¹⁹. The extracts in polar solvents exhibited higher extraction yield on being collated with other extracts. This indicates the abundance of more polar compounds in the extracts (Table A.2). Polar solvents can effectively extract substances like sugars, proteins, glycosides, organic acids, tannins, salts, and mucus from the given plant material is the reason for the higher extraction yield of the n-butanol and aqueous extracts. Ethyl Acetate extraction product mainly contains medium hydrophobic compounds and polar-chain carbonated polymers²⁰.

 

Table A.2   Represents the extractive value of the hydro-alcohol extract of E. crassipes leaves and its fractions in different solvents from non-polar to polar. (Data are Mean ±SD Where n=3)

S. No.	Extract	Extractive value (%)
1	Hydro-alcohol	48.88±2.7
2	Hexane	3.36±0.18
3	Chloroform	4.48±0.25
4	Ethyl acetate	2.64±0.14
5	n- Butanol	7.36±0.41
6	Aqueous	32.32±1.79

 

Fluorescence analysis of powder drug:

These distinct and reproducible color variations can be attributed to different phytochemicals solvent properties¹⁸. Ultraviolet light induces fluorescence in many natural products that do not fluorescence clearly in daylight such as alkaloids, phenols, tannins, etc. Most of the compounds may also be transformed into fluorescent products using specific chemical reagents, but they are not fluorescent. We can also use fluorescence to test these basic drugs qualitatively because that is the most significant parameter for pharmacognostical evaluation. It can serve as a method for detecting adulterants and replacements, which will help to preserve the consistency, reproducibility, and potency of natural drugs²⁰. This fluorescent analysis technique can be used effectively to assess the pharmacognostical parameters of any synthetic drug in a qualitative method and can also help to detect it (Table A.3)

 

Phytochemical analysis Qualitative phytochemical screening:

Identification of different groups of plant phytochemical components is a significant parameter that indicates the active pharmacological metabolites present in the plant. E. crassipes qualitative study revealed the existence of specific phytochemicals (Table A.4). The existence of various phytochemicals such as carbohydrates, proteins, alkaloids, flavanoids, tannins and terpenoids, phenols, anthocyanin, anthraquinone, and fats and oils were demonstrated in several tests used. These bioactive compounds exhibit biological properties such as antioxidant, anti-carcinogenic, anti-inflammatory, anti-microbial, hepatoprotective^21,22,23. The pharmacological and chemical research of therapeutic plants relies heavily on phytochemical screening²⁴. Phenolic chemicals are also good antioxidants because they are effective hydrogen donors²³.

 

 

 

Table A.3Analysis of fluorescence in E. crassipes leaf powder under UV (short / long), and visible radiation

S. No.	Treatments	UV Short (254nm)	UV Long (366nm)	Visible
1	Powder	Dark green	Olive green	Dark green
2	Powder + DW	Dark green	Light green	Green
3	Powder +picric acid	Black	Black	Yellowish green
4	Powder+ glacial acetic acid	Dark green	Reddish brown	Green
5	Powder  + 1N HCl	Dark green	Dark green	Green
6	Powder + 1N H2SO4	Black	Black	Brown
7	Powder + Conc. HNO3	Black	Grey	Yellow
8	Powder + Ferric chloride	Black	Black	Green
9	Powder + Iodine solution	Black	Black	Olive green
10	Powder + Ammonia solution	Dark green	Dark green	Green
11	Powder + 1N NaOH	Black	Dark green	Olive green
12	Powder + Potassium dichromate	Black	Black	Yellowish green
13	Powder + HNO3+NH3 Solution	Black	Black	Dark green
14	Powder + Ethanol	Dark green	Dark green	Dark green
15	Powder +Methanol	Dark green	Green + fluorescence	Dark green

 

Table A.4 Represents the bioactive constituents in the different fractions of Hydro-alcoholic extract of leaves of E. crassipes

S. No.	Test	Hexane	Chloroform	Ethyl Acetate	n-Butanol	Aqueous
1	Carbohydrate test
	Fehling’s	-	-	-	-	-
	Benedict	+	+	+	+	++
	Molish	-	-	-	+	+
2	Protein
	Millions	-	-	-	-	-
	Ninhydrine	-	-	-	-	-
	Biuret	+	+	+	+	+
3	Alkaloids
	Dragendroff’s	+	+	++	+	+
4	Flavanoid test
	NaOH	-	-	-	-	-
	Shinoda	-	-	-	-	-
	H2SO4	-	-	+	+	+
5	Tannins test
	10% FeCl3	+	+	+	+	+
6	Quinones	-	-	-	-	-
7	Terpenoids	-	-	++	+	+
8	Phenols test
	Dil.KMNO4	++	++	++	++	++
	5 % FeCl3	-	-	-	-	-
	Lead acetate	-	-	++	+	+
9	Anthocynanin test
	NaOH	+	+	+	+	+
10	Glycosides test
	Coumarin	+	+	+	+	+
11	Amino acid test
	Tyrosine	-	-	-	-	-
	Xanthoprotein test	-	+	++	++	+++
12	Anthroquinones test	-	-	+	+++	+
13	Fats and oils test	+	+	+	++	++
	Sudan	+	+	+	+	++
14	Saponines test	-	-	-	-	-
15	Non-reducing polysaccharides
	Iodine test	-	-	-	-	-

 

 

CONCLUSION:

Secondary metabolites found in the leaves of E. crassipes are an important source for the production of plant-based medications. The constituents of the ash also vary with time, and part of the plant, and usually represent the inorganic part of the plant. It helps to evaluate the chemical constituents present. The distinctive fluorescent properties or colours discovered in this study could be used to identify and authenticate the leaves in their raw state. It could also be used to detect adulteration, with the adulterated samples exhibiting differences in colour emission when compared to the control samples.The presence of diverse phytochemicals in the plants suggests that this plant could be useful in the development of novel medications to treat a variety of illnesses. In the present study, we have found that most of the biologically active phytochemicals were present responsible for the medicinal properties.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGEMENT:

The authors would like to take this opportunity to express our gratitude and thanks to the Hon’ble Former V.C. Prof. Aditya Shastri Banasthali Vidyapith, Banasthali, Rajasthan for providing us the research facilities, encouragement and support. The author’s thank Hon’ble V.C. Prof. Ina Shastri Banasthali Vidyapith, Banasthali, Rajasthan, for the research facilities andencouragement. The author’s take this opportunity to thank Prof. Dipjyoti Chakraborty Head, Department of Bioscience and Biotechnology, and Dean of Research and Development, Banasthali Vidyapith, Banasthali, for establishing the environment and support. The authors would like to acknowledge Bioinformatics Centre, Banasthali Vidyapith supported by DBT for providing computation support. Authors would also like to acknowledge DST for providing networking support through the FIST program at the Department of Bioscience and Biotechnology.

 

REFERENCE:

1.     Majee C.Mazumder R. Choudhary AN. Pharmacognostical Investigation of Trapa natans L Leaves. Research Journal of Pharmacy and Technology. 2021; 14(9):4680-4. doi: 10.52711/0974-360X.2021.00813.

2.     Singh V. Rizvi A. Sara US. Standardization and Phytochemical screening of Carica papaya seeds. Research Journal of Pharmacy and Technology. 2021; 14(9):4540-6. doi: 10.52711/0974-360X.2021.00790

3.     Kadasala V. Ghosh NS. Chinnaiyan SK. Mallik A. Manjunath SY. Bhattacharjee C. Phytochemical and Pharmacological Evaluation of Annona reticulate. Research Journal of Pharmacy and Technology. 2021; 14(9):4881-6. doi: 10.52711/0974-360X.2021.00848

4.     Khan NI. Hatapakki BC.Tamboli AM. Isolation and Characterization of Flavonoids of the Ethanolic extracts of stems of Mimosa hamata (Willd.) by Chromatographic techniques. Research Journal of Pharmacy and Technology. 2021; 14(9):4602-8. doi:10.52711/0974-360X.2021.00800

5.     Gayatri. “11 Amazing Benefits Of Hyacinth Herb For Skin, Hair And Health.” STYLECRAZE, IncNut Digital, 6 June 2019, stylecraze.com/articles/benefits-of-hyacinth-herb-for-skin-hair-and-health/

6.     Ayanda OI. Ajayi T.Asuwaju FP. Eichhornia crassipes (Mart.) Solms: Uses, Challenges, Threats, and Prospects. The Scientific World Journal. 2020, 12, Article ID 3452172.

7.     Thamaraiselvi PL. and Jayanthi P. “Preliminary studies on phytochemicals and antimicrobial activity of solvent extracts of Eichhornia crassipes (Mart.) Solms.,” Asian Journal of Plant Science.

8.     Vijetha P. Kumaraswam K. Prasanna KY. Satyasree N. Siva PK. Biosorption of Cu, Zn and Pb by Eichhornia crassipes: Thermodynamic and isotherm studies. International Journal of Scientific and Technology Research. 2014; 3(3), 439-443.

9.     Bergier I. Salis SM. Miranda CHB. Ortega E.Luengo CA. Biofuel production from water hyacinth in the Pantanal wetland. Ecohydrology and Hydrobiology. 2014; 12(1), 77-84. DOI: 10.2478/v10104-011-0041

10.  Mako AA. Babayemi OJ.Akinsoyinu AO. An evaluation of nutritive value of water hyacinth (Eichhornia crassipes Mart. Solms-Laubach) harvested from different water sources as animal feed. Livestock Research for Rural Development. 2011; 23, 106.

11.  Fileto-Perez HA. Rutiaga-Quinones OM. Sytsma MD. Lorne IM. Luo W. Pankow JF.Rutiaga-Quinones JG. GC/MS analysis of some extractives from Eichhornia crassipes. BioResources. 2015; 10(4), 7353-7360. DOI: 10.15376/biores.10.4.7353-7360.

12.  Nimbekar T. Jain A. Mohanty PK. Phytochemical screening and In-Vitro Antidiabetic Activity of Extracts of some Indian Medicinal Plants. Research Journal of Pharmacy and Technology. 2021; 14(4):2026-0. doi: 10.52711/0974-360X.2021.00359.

13.  Hiremath G. Urmila. Evaluation of physicochemical and Phytochemical parameters of Amaranthus caudatus leaves. International Research Journal of Pharmacy. 2012; 3(2):138-139.

14.  Chaudhri KR. Girase ON. Determination soluble extractives and physico- chemical studies of bark of Sesbania sesban (L) Merr. Journal of Chemical and Pharmaceutical Research. 2015;7(8):657-660.

15.  Rao Y. Xiang B. Determination of total ash and acid insoluble ash of Chinese herbal medicine Prunellae Spica by near Infrared Spectroscopy. Planetary Science Journal. 2009;129(7):881-886

16.  Bhardwaj A. Modi KP. A review on therapeutic potential of Nelumbo nucifera (GAERTN): the sacred lotus. International Journal of Pharmaceutical Sciencesand Research. 2016;7(1): 42-54.

17.  Vasta E. Chandel S. Parashar BN. Physio-chemical and Phytochemical Evaluation of Dendrobium macraeilindl. (Whole plant). International Journal of Pharmcognosy. and Phytochemical Research. 2016; 8(11); 1801-1811 

18.  Ranjith D. Fluorescence analysis and extractive values of herbal formulations used for wound healing activity in animals. Journal of Medicinal Plants Studies. 2018; 6(2): 189-192.

19.  Lara S. Javier S.Rutiaga QO. Miriam Lopez M. Javier; Fileto P. Hector A. Pedraza B. Fabiola E. Rico C. Jose L. Rutiaga Q. Jose G. Physicochemical Characterization of Water Hyacinth (Eichhornia crassipes (Mart.) Solms). BioResources. 2016;11(3).

20.  Sumitra C. Importance of pharmacognostic study of medicinal plants: An overview. Journal of Pharmacognosy and Phytochemistry. 2014; 2 (5): 69-73.www.phytojournal.com

21.  Madhvi SK. Sharma M. Iqbal J. Younis M. Sheikh R. Phytochemical analysis, Total Flavonoid, Phenolic contents and Antioxidant activity of extracts from the Leaves of Rhododendron arboreum. Research Journal of Pharmacy and Technology. 2020; 13(4):1701-1706. doi: 10.5958/0974-360X.2020.00307.8

22.  Rajendran R.Hemachander T.Ezhilarasan C. Keerthana DL. Saroja KV. Saichand, GasimM. Abdullah. Phytochemical Analysis and In-Vitro Antioxidant Activity of Mimosa pudica Lin., Leaves. Research Journal of Pharmacy and Technology. 3(2): April- June 2010; Page 551-555

23.  Thilagavathi T. Kathiravan G. Phytochemical Analysis and Antimicrobial Activity of Ethonolic Leaf Extract of Ficus racemosa Linn. Research Journal of Pharmacy and Technology. 2017; 10(2): 537-540. doi: 10.5958/0974-360X.2017.00107

24.  Magesh A. Lakshmi T. Preliminary phytochemical analysis of Acacia catechu willd Heartwood extract. Research Journal of Pharmacy and Technology. 5(11): Nov. 2012; Page 1393-1395.

 

 

 

Accepted on 22.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1379-1384.