Qualitative and Quantitative Analysis of Phytochemicals in some Medicinal plants of Western Himalayas
Manju*, Aiman Aziz, Shamee Rehman
Department of Life Sciences, RIMT University, Mandi Gobindgarh, Punjab, India.
*Corresponding Author E-mail: manjujaswal90@gmail.com
ABSTRACT:
Plants have been well known for their medicinal applications for ages and traditional medicines are still a major part of habitual treatments in different parts of the world. They are used since ancient times for treating different ailments and providing valuable drugs such as analgesics (morphine), antihypertensives (reserpine), cardiotonic (digoxin) and antimalarials (quinine and artemisinin). The discovery of medicinal plants helps in achieving the new targets in treating various diseases like cancer, malaria and neurological disorders. Herbs proved to be the only solution for treating a large number of health-related problems. Most of the recent drugs are obtained in one or another way from plants. Medicinal plants contain biologically active, naturally occurring chemical compounds known as phytochemicals which provide health benefits and protect the human cell from any type of damage. Phytochemical screenings of medicinal plants have contributed to the discovery of new drugs. In our present investigation, an attempt has been made to screen out the bioactive constituents of three selected plants of western Himalayas and further they had been subjected to quantitative analysis for estimation of different parameters.
KEYWORDS: Medicinal plant, Phytochemicals, Biological Activity, Bioactive constituents, Western Himalayas.
INTRODUCTION:
Plants can synthesize a wide range of bioactive compound that performs important biological functions and provides defense from the attack of herbivores, insects, fungi and mammals1. At least 12000 such components have been isolated so far to heal various ailments2. Medicinal plants are used as a valuable source of ingredients that can be used in drug development3. The products derived from the plants are one of the vital sources to combat serious diseases across the world mostly in the developing countries where traditional medicinal methods play a key role to cover the basic health requirements4.
Most commonly used plants; for the treatment of diseases that acts as anti-inflammatory, anticancer and antioxidant are Cinnamon, Aloe vera, Crocus sativus, Withania somnifera, Gingko biloba, Adhatoda vasica, Catharanthus roseus, Ficus bengalensis,Aegle marmelos etc.5 Medicinal plants are considered as an effective source of Unani, Homeopathic, and modern medicine. Herbal medicines are used by 75-80% of the population in developing countries6. In India, there is a long history of using medicinal plants for curing several diseases. It has also been believed that using fruits/herbs has better potential to heal the suffering caused by cancer diseases than synthetic medicine7.
Phytochemicals are the naturally occurring chemical compounds in plants that possess specific bioactivity and provide health benefits to humans8. These are present in various parts of the plant that works along with the nutrients and fibers to fight against diseases9. They are having an overlapping mechanism of action in the body, including antioxidant effects, stimulation of the immune system, modulation of the enzyme actions, antiviral and anti-bacterial effects10. Due to such bioactivities, they have been used for the prevention and treatment of various diseases such as diabetes, high blood pressure, muscular degeneration, gastric pain, and hay fever11.
The Indian Himalayan Region is a mega hot spot of plant diversity that includes about 1748 species of medicinal plants with various traditional and modern pharmaceutical uses, 675 species of wild edible plants, and 121 rare-endangered plants12. Estimates indicate that at least 90% of medicinal plant species are extracted from wild plants13. Therefore the present study was concerned with three wild plants viz; Artemisia absinthium, Eleusine coracana and Urtica diocia which are mostly grown in the Western region of the Indian Himalayas and used locally as the medicine. These plants are widely grown in Jammu and Kashmir, Uttarakhand and Himachal Pradesh. Artemisia absinthium and Urtica diocia are used extensively for medicinal purposes like indigestion, gastric pain, and chronic inflammation. Only edible parts of the respective plants were used for the analysis.
MATERIAL AND METHODS:
Collection of plant material:
Three selected medicinal plants viz; Artimisia absinthium, Urtica diocia and Eleusine coracana (Fig.1) are harvested and collected from district Anantnag, Kashmir (Latitude- 33.73º N & Longitude-75.15ºE with Elevation - 1,601meters) and Pauri Garhwal, Uttarakhand (Lattitude-. 29.8688º N and Longitude-78.8383º E with Elevation- 3100 meters). The plants were collected from February to September.
Fig. 1: Images of the study plants a) Artemisia absinthium b) Urtica diocia c) Eleusine coracana
Qualitative Phytochemical Screening:
Qualitative screening of aqueous and ethanolic extracts of edible parts of selected medicinal plants was carried out for the presence of various phytochemicals by using standard methods.
Detection of Alkaloids:
The extracts obtained from the selected medicinal plants were dissolved separately in dilute hydrochloric acid for the presence of alkaloids by using Mayer’s reagent (Potassium mercuric iodide) and Hager’s reagent (saturated picric acid solution). The formation of a yellow colour precipitate indicates the presence of alkaloids14.
Detection of Flavonoids:
The detection of flavonoids was carried out with an alkaline reagent test by dissolving the plant extracts in few drops of sodium hydroxide solution. Formation of reddish-yellow colour; which becomes colourless after the addition of dilute acid, indicates the presence of flavonoids15.
Detection of Proteins:
The detection of proteins was carried out by Xanthoproteic acid test by adding the concentrated nitric acid solution to the plant extract. The formation of yellow colour indicates the presence of proteins16.
Detection of Amino acids:
The detection of amino acids was carried out by Ninhydrin test, by adding Ninhydrin reagent to the plant extract and boiled for few minutes. The formation of blue colour indicates the presence of amino acids17.
Detection of phenols:
The detection of Phenols was carried out by using the Ferric chloride test. Extracts of three individual plants were treated with few drops of ferric chloride solution. The formation of bluish-green colour indicates the presence of phenols18.
Quantitative Determination of Phytochemicals:
Quantitative analysis for the detection of protein, amino acid, carbohydrate, phenol, flavonoid, alkaloid, saponins, tannin and minerals was also done after qualitative assessment by using standardized methods mentioned below.
Estimation of Protein Content:
Protein estimation was done by using the standard method of Lowry19 which goes as: 0.1ml of the sample and standard were pipette out into a series of test tubes. Distilled water was added to make it 1ml. A tube with 1ml of distilled water was put as blank. 5ml of 2% sodium carbonate in 0.1% sodium hydroxide was added to each tube. It was mixed well and was allowed to stand for 10 minutes. Then 0.5ml of folin reagent was added, mixed well and incubated at room temperature in dark for 30 minutes. The blue colour was developed. The final readings were taken at 660nm on a UV spectrophotometer.
Total Free Amino Acids:
The amount of total free amino acids in the samples was determined by placing glycine as standard20. One gram of shoots was grounded in 5mL of 80% ethanol. The homogenate was centrifuged at 10,000 r.p.m for 15 minutes. To 0.1ml of amino acid extract, 5ml of Ninhydrin reagent was added. The mixture was vortexed vigorously and then placed in the hot water bath for 12 minutes. The mixture was cooled under running tap water to room temperature and a blue-purple colour developed in the solution. The optical density was measured at 570nm on UV Spectrophotometer against blank which was prepared by adding 0.1ml of 80% ethanol instead of extract.
Total Phenols:
The total phenolic content in the samples was determined by using the Folin-Ciocalteau method21. According to which 1g of sample was extracted with 5 ml of 80% ethanol and put in the water bath for 30 min at 30°C. Centrifugation was done for 10 minutes at 4500 rpm. Supernatants were put in a test tube. 0.1ml of supernatant was transferred to a test tube. 5mL of folin-ciocalteu reagent was added to the above. After 5 to 8 minutes, 3.5ml of sodium carbonate dissolved in water; was added and vortexed. The tubes were incubated for 1 hour at 40°C in a digital water bath. The absorbance was measured at 765nm on a UV spectrophotometer.
Total Flavonoid Content:
The total flavonoid content was measured spectrophotometrically by following the method22 in which, flavonoids in the plant extract reacts with aluminium chloride and potassium acetate present in the reagent giving coloured product and can be measured at 415nm using UV spectrophotometer. The method in brief; about one ml of 2% aluminium chloride in methanol was mixed with one ml of leaf extract in the concentration of one mg per ml. The mixture was incubated at room temperature for an hour and absorbance was measured at 415nm on a UV spectrophotometer.
Total Alkaloids:
Quantification of alkaloids was performed by using the method of Harborn23. About 5mg of plant extract was mixed with 20ml of 10% acetic acid in methanol, covered the beaker and allowed to stand at room temperature for 4 hours. The mixture was concentrated to one-third of its volume by the addition of ammonium hydroxide solution drop by drop in the mixture until complete precipitation occurs and filtered. The absorbance was taken at 415nm on a UV spectrophotometer.
RESULTS AND DISCUSSION:
The present investigation of selected plants reveals the presence of various chemical constituents when examined qualitatively (Table I). It was found that alkaloids are present in all the selected flora in both aqueous and ethanolic extract. These results are in accordance with the findings in Punica grantum, Swietenia macrophylla and Nicotiana tabacum24,25,26. Flavonoids were present in both aqueous and ethanolic extracts of Artemisia absinthium which is similar to the screening results of Zingiber officinale, Emblica officinalis and Datura metel27,28,29. Flavonoids were present only in the aqueous extract of Urtica diocia and Eleusine coracana. These results are parallel to Vitex negunda30, Hibiscus rosa- sinensis31 and Moringa oleifera32. However, flavonoids were absent in ethanolic extract of Urtica diocia and Eleusine coracana as also reported in Cannabis sativa and Gongronema latifolium33,34. Amino acids and phenols were present only in ethanolic extract of Eleusine coracana and Urtica diocia. Similar results were obtained in an aqueous extract of Psidium gujava35. Further, it was also investigated that amino acids are found in both aqueous and ethanolic extracts of Artemisia absinthium. These results accord with Moringa concanensis, Morinda citrifolia and Kedrostis foetidissima36,37,38. Phenols are present in both aqueous and ethanolic extracts of Artemisia absinthium as also reported in Thymus vulgaris39 and Cecropia obtusa40. However, phenols are absent in the aqueous extract of Urtica diocia and Eleusine coracana; in parallel to Bauhinia blakeana41 and Lavandula officinalis42. Proteins are present in both aqueous and ethanolic extracts of all the selected plants. These results are similar to Moringa concanensis43, Cissus quadrangularis44 and Euphorbia cuneata45.
Table I: Qualitative analysis of aqueous and ethanolic extracts of selected medicinal plants for the presence of Phytochemicals.
|
S. No. |
Phytochemicals |
Artemisia absinthium (Leaves) |
Urtica diocia (Leaves) |
Eleusine coracana (Seeds) |
|||
|
Aqueous |
Ethanol |
Aqueous |
Ethanol |
Aqueous |
Ethanol |
||
|
01. |
Alkaloid |
+ |
+ |
+ |
+ |
+ |
+ |
|
02. |
Flavanoids |
+ |
+ |
+ |
_ |
+ |
_ |
|
03. |
Proteins |
+ |
+ |
+ |
+ |
+ |
+ |
|
04. |
Amino acids |
+ |
+ |
_ |
+ |
_ |
+ |
|
05. |
Phenols |
+ |
+ |
_ |
+ |
_ |
+ |
The quantitative composition of different secondary metabolites in Artemisia absinthium, Urtica diocia, and Eleusine coracana have shown in Table II and Fig. 2. Total free amino acid content was observed as highest in Urtica diocia as compared to the two other selected medicinal plants, whereas Eleusine coracana was found to contain the least amount of amino acids. A higher amount of amino acid content was also reported in many plants such as Tournefortia hartwegiana46 and Morinda citrifolia47. Due to the presence of higher content of total free amino acids in the leaves of stinging neetle, it can be used for the treatment of gastrointestinal problems and a genetic disease called McArdle disease48. Total phenolic content was found to be highest in seeds of Eleusine coracana, followed by leaves of Urtica diocia and Artemisia absinthium. These results are parallel to Acacia concina49, Ageratum conyzoides50, Rumex hastatus51 and Berberis lycium52. Phenols are the major plant chemical compounds that contribute towards the antioxidant activity53. Due to the presence of higher levels of phenol, it has various applications such as prevention of cancer, acts as an antioxidant and antimicrobial, reduces the risk of chronic disease, prevents disease progression, increasing antioxidant activity and treats or prevents atherosclerosis54. Total protein content was found to be more in the seeds of Eleusine coracana as compared to the selected parts of the other two flora. Due to the presence of a higher range of protein in finger millet, it provides a wide range of health benefits like a building block of bones, muscles, cartilage, skin and blood55. Alkaloids were found to be highest in Artemisia absinthium followed by Eleusine coracana and Urtica diocia. Plant-based alkaloids are the biggest class of phytochemicals that shows evidence of many therapeutic effects like analgesics, muscle relaxant, antibiotics and also responsible for antiprotozoal, cytotoxic and antimicrobial properties56. Due to the richness of alkaloids, Artemisia absinthium possesses a wide range of medicinal properties like anticancer, antioxidant and anti-inflammatory. A higher quantity of flavonoids was detected in Urtica diocia, while Eleusine coracana was found to have the least flavonoid content. Due to the higher flavonoid content in the leaves of Urtica diocia, it possesses various medicinal properties like the potential of anti-inflammatory, anti-microbial, anti-carcinogenic, anti-HIV and neuroprotective properties57.
Table II: Quantitative Estimation of Phytochemicals present in the edible parts of selected medicinal plants.
|
S. No. |
Medicinal plants |
Total Free Amino acids |
Total Phenolic content |
Total Protein Content |
Flavanoids |
Alkaloids |
|
01. |
Artemisia absinthium |
0.364±0.003 |
0.234±0.001 |
0.178±0.002 |
0.364±0.003 |
0.41±0.001 |
|
02. |
Urtica diocia |
0.414±0.002 |
0.261±0.001 |
0.224±0.003 |
0.414±0.002 |
0.245 ±0.003 |
|
03. |
Eleusine coracana |
0.256± 0.004 |
0.279±0.015 |
0.303±0.0028 |
0.256± 0.004 |
0.324±0.002 |
Fig 2: Comparison of phytochemicals of three selected medicinal plants
CONCLUSION:
Three selected medicinal plants are the source of many important secondary metabolites i.e., alkaloids, flavonoids, amino acids, phenols and proteins. The phytochemical analysis i.e., qualitative and quantitative analysis of these medicinal plants are also important and have a commercial interest in both research institutes and pharmaceuticals companies for the manufacturing of the new drugs. Reported aspects which are known to affect the production of secondary metabolites in plants are physiological differences, climatic changes, topographical features, and hereditary elements, amount of plant material, space and labour needs. Further extraction of individual phytochemicals from these plants may lead to their wider analysis and applications.
ACKNOWLEDGEMENT:
The authors express a deep sense of gratitude to RIMT University, Mandi Gobindgarh, Punjab (India) for all the support, assistance and constant encouragement to carry out this work.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 01.06.2020 Modified on 16.02.2021
Accepted on 19.07.2021 © RJPT All right reserved
Research J. Pharm.and Tech 2022; 15(4):1419-1423.
DOI: 10.52711/0974-360X.2022.00235