PHYSICOCHEMICAL AND PHYTOCHEMICAL EVALUATION OF CHLOROPHYTUM BORVILIANUM AND SAPINDUS MUKORROSSAI
HTML Full TextPHYSICOCHEMICAL AND PHYTOCHEMICAL EVALUATION OF CHLOROPHYTUM BORVILIANUM AND SAPINDUS MUKORROSSAI
Shilpi Mishra * and Mayank Thakur
Advance Institute of Biotech & Paramedical Sciences, Kanpur - 209217, Uttar Pradesh, India.
ABSTRACT: The study is aimed at the development of physicochemical parameters and to investigate the active principle present in Chlorophytum borvilianum and Sapindus mukorrossai. The present work embodies the investigations carried out to establish methods for quality control of drugs as per WHO guidelines: complete botanical evaluation which comprises of macroscopy, quantitative microscopy, powder microscopy, physicochemical parameters like loss on drying, extractive values, saponification value, total ash values, acid insoluble ash value, water-soluble ash value, pH value, loss on drying, bulk density and fluorescence analysis of powdered drug to investigate the phytochemicals present in the extracts in the preliminary level.
Keywords: |
Phytochemical, Chlorophytum borvilianum and Sapindus mukorrossai
INTRODUCTION: Herbal medicines are the promising choice over modern synthetic drugs. These are believed to be safe and having a minimum or no side effects. Generally, herbal formulations involve the use of fresh or dried plant parts. It is essential to have correct knowledge of such crude drugs in preparation, safety and efficacy aspect of the herbal product. Pharmacognosy is a simple and reliable tool, by which complete information of the crude drug can be obtained 1. Medicinal plants are of great importance to the health of individuals and communities. The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body. Chlorophytum borivilianum (Liliaceae) commonly called Safed musli is considered as one of the most important medicinal plant used in our traditional systems of medicines to treat various diseases of human beings. The plant is used for dysuria 2. Leaves are eaten by the tribal people of Western Ghats as an expectorant 3. Root is used as an aphrodisiac, diuretic, and astringent useful in dysentery, as an antidiabetic and as appetizing agent 4. Tubers are used as lactagogue. 5
Sapindus mukorossi is a deciduous tree belongs to the family Sapindaceae grew in upper reaches of Indo-Gangetic plains, Shivaliks and sub-Himalayans tracks at altitudes from 200 m to 1500m. Also known as a soap-nut tree, it is one of the most important trees of tropical and subtropical regions of Asia.
MATERIAL AND METHOD: The roots and fruits of drugs namely Chlorophytum borvilianum and Sapindus mukorrossai were collected from the local market, Nayaganj, Kanpur, U.P. These drugs were authenticated by Dr. Prabodh Shukla, Pharmacognosy, RIMS. The collected roots and fruits were washed, shade dried and pulverized with a mechanical pulverizer for the size reduction. It was then passed with 60 mesh size sieve, and the fine powder was collected and used for the studies.
Pharmacognostic Studies: Macroscopic studies of dried roots and fruit of both Chlorophytum borvilianum and Sapindus mukorrossai were carried out, which comprise of organoleptic parameters 6, viz. color, odor, taste, texture, etc Table 1. Powder microscopy was performed to investigate the microscopical features as per Evans 7 Fig. 1, 2 and 3.
Quantitative Microscopy: In transverse sections it is not possible to study nature of epidermal cells, trichomes, and stomata; stomatal index, vein islet number and vein termination number which play an important role in identifying characteristics of crude drugs and adulterants 8, 9.
But, these can be determined by quantitative microscopy. These quantitative microscopic values are comparatively constant for a particular species and can be used to make a difference in closely related species. For the study of epidermal cells and stomatal index, leaf segments were boiled in chloral hydrate solution, and the epidermis was peeled with forceps and then mounted on a slide with 5% (v/v) glycerin in water and observed under a microscope. Stomatal index was calculated as Number of stomata/Total number of epidermal cells multiplied by 100. Microscopic descriptions of tissue are supplemented with micrographs wherever necessary.
For determination of vein islet number and vein termination number a 1 mm square leaf piece was cleared by boiling in chloral hydrate solution and then observed under a microscope for determining vein islet and terminations 10 Table 4.
Fluorescence Analysis: Crude drugs show their characteristic fluorescence when exposed to ultraviolet radiation and are dependent on its chemical constituents. This analysis is useful to identify adulterants during crude drug evaluation. In the present study, one gram of crude drug was taken in a watch glass and subjected for fluorescent analysis as such and after treatment with different reagents 11, 12, 13 Table 5.
Physicochemical Studies:
Determination of Ash Values: For determining ash content of drug, about 3 g of powder was spread in a pre-ignited and weighed silica crucible. Then the crucible was incinerated gradually to make the crucible free from carbon. After cooling, the crucible was weighed to get the total ash content and then the ash was subjected for determining the acid insoluble and water soluble ash. The percentage of total ash was calculated by taking the air dried sample as standard 14.
Determination of Acid-Insoluble Ash: Twenty- five (25) ml of hydrochloric acid (~70g/l) TS was added to the crucible containing the total ash, covered with a watch-glass and boiled gently for 5 minutes. The watch-glass was rinsed with 5 ml of hot water and this liquid added to the crucible. The insoluble matter was collected on an ashless filter-paper (Whatmann 41) and washed with hot water until the filtrate was neutral.
The filter paper containing the insoluble matter was transferred to the original crucible, ignited by gradually increasing the heat to 550 °C for 3 h in a muffle furnace (Nabertherm) to constant weight. Allowed the residue to cool in a suitable desiccator for 30 minutes, and then weighed without delay. Acid-insoluble ash content was calculated as mg per g of air-dried material.
Determination of Extractive Values: Considering the diversity and chemical nature of the drug, five different solvents viz. water, ethanol, chloroform, ethyl acetate, diethyl ether, and petroleum ether were used for determination of extractive values. About 5 g of powdered leaf material was subjected for cold maceration extraction with 100 ml of above solvents.
Determination of extractive values of a crude drug is beneficial in its evaluation process wherever evaluation of chemical components in drugs is not possible by any other means 14. After extraction, the extracts are concentrated in rota vaporizer and dried in the vacuum desiccator. Then the extractive values are calculated as percentage w/w of solvent soluble extractive with reference to the air dried drug.
Determination of Water-Soluble Ash: Twenty- five (25) ml of water was added to the crucible containing the total ash, covered with a watch-glass and boiled gently for 5 min. Insoluble matter was collected on an ashless filter-paper. Washed with hot water and ignited in a crucible for 15 min at a temperature not exceeding 450 °C in a muffle furnace. Allowed the residue to cool in a suitable desiccator for 30 min, and then weighed without delay. The weight of the residue was subtracted in mg from the weight of total ash. Water - soluble ash content was calculated as mg per g of air-dried material.
Determination of Moisture Content: Moisture content was determined by loss of weight on drying (LOD) method 14. For this 5 gm of drug (powdered leaf material) was taken and kept in an oven at 105 °C till a constant weight was obtained. Amount of moisture present in the sample was calculated as a reference to the air-dried leaf material.
Determination of pH Range: The pH of different formulations in 1% w/v (1g: 100ml) and 10% w/v (10g: 100ml) of water-soluble portions of whole powder of drugs, were determined using standard simple glass electrode pH meter 15 Table 2.
Preliminary Phytochemical Screening: Table 3 The preliminary phytochemical screening of the water (hot) extracts of plant powder of drugs were carried out using standard laboratory procedures, to detect the presence of different secondary metabolites (phytochemical constituents) such as alkaloids, flavonoids, saponins, tannins, steroid glycosides, phenols, coumarins, reducing sugars, protein, anthraquinones, quinines, Fixed oils and fats 16-19.
Determination of phenolic compounds two to three drops of 1% ferric chloride (FeCl3) solution was added into 2 ml portions (1%) of each extract. Phenolic compounds produce a deep violet color with ferric ions.
Determination of Tannins:
Ferric Chloride Test: A small quantity of the extract was boiled with water and filtered. Two drops of ferric chloride were added to the filtrate, formation of a blue-black or blackish green color in the presence of ferric chloride precipitate was taken as evidence for the existence of tannins.
Determination of Flavonoids:
Shinoda Test: The extract was dissolved in methanol (50%, 1-2 ml) by heating. To an alcoholic solution of each of the extract, three pieces of magnesium chips were added followed by a few drops of concentrated hydrochloric acid. The appearance of orange, pink or red to purple color indicates the presence of flavonoids.
Determination of Steroid Glycosides:
Libermann Burchard’S Test: Extract was dissolved in equal volumes of anhydrous acetic acid and chloroform (CHCl3) and cooled to 0 °C. The mixture was transferred to a dry test tube, and concentrated sulfuric acid (H2SO4) was introduced to the bottom of the tube. Formation of a reddish brown or violet-brown ring at the interface of the two liquids indicates the presence of steroids.
Determination of Alkaloids:
Mayer‟s Test: One ml portions of each extract was acidified with 2-3 drops of 1M hydrochloric acid and treated with 4-5 drops of Mayer‟s regent (Potassium mercuric iodide) formation of a yellow or white colored precipitate or turbidity indicates the presence of alkaloids.
Dragendroff‟s Test: Extracts were dissolved individually in dilute hydrochloric acid and filtered. Filtrates were treated with Dragendroff‟s reagent (solution of potassium bismuth iodide). Formation of red precipitate indicates the presence of alkaloids.
Detection of Proteins:
Xanthoproteic Test: The extracts were treated with a few drops of conc. nitric acid. Formation of yellow color indicates the presence of proteins.
Determination Saponins:
Foam Test: 0.5 g of extract was shaken with 2 ml of water. If foam produced persists for ten minutes, it indicates the presence of saponins.
Detection of Reducing Sugar:
Fehling‟S Test: To a test tube 1 ml each a Fehling‟s A, and B solutions were added and mixed. To this, ~2 ml of plant extract was added and heated on a boiling water bath for ~10 minutes. Formation of brick red or orange precipitate indicates the presence of reducing sugar/ carbohydrates.
RESULTS: The organoleptic characters of the dried roots and fruit of both Chlorophytum borvilianum and Sapindus mukorrossai powder are tabulated as Table 1.
TABLE 1: ORGANOLEPTIC EVALUATION PARAMETERS
S. no. | Organoleptic parameters | Inference | |
Chlorophytum borvilianum | Sapindus mukorrosai | ||
1 | Colour | Pale white | Brown |
2 | Odour | Characteristics | Characteristics |
3 | Taste | Mucilaginous | Bitter |
4 | Nature | Crystalline | Crystalline |
5 | Texture | Smooth | Rough |
6 | Solubility | Forms colloidal solution in water &
insoluble in alcohol & chloroform |
Soluble in water |
TABLE 2: PHYSICOCHEMICAL STUDY OF POWDERED DRUGS
S.
no. |
Parameters | Percentage (%w/w) | |
Chlorophytum borvilianum | Sapindus mukorrosai | ||
1 | Ash Value | ||
Total ash | 7.19 | 3.60 | |
Acid-insoluble ash | 3.66 | 0.24 | |
Water soluble ash | 2.05 | 14.69 | |
2 | Successive extractive values | ||
Pet. ether | 1.4 | 1.1 | |
Diethyl ether | 1.3 | 1.4 | |
Chloroform | 1.1 | 1.8 | |
Ethyl acetate | 2.1 | 2.2 | |
Ethanol | 2.7 | 3.2 | |
Aqueous | 5.8 | 4.8 | |
3 | Loss on drying | 12.66 | 9.6 |
4 | pH values | ||
1% water solution | 5.4 | 5.9 | |
10% water solution | 4.5 | 5.3 | |
5 | Bulk density | 0.8 | 1.2 |
6 | Saponification values | 182.3 | 148.66 |
Note: values are the average of three experiments.
TABLE 3: PRELIMINARY SCREENING OF MAJOR PHYTOCHEMICALS
S.
no. |
Chemical constitutents | Tests /
reagent |
Chlorophytum borvilianum
(aqueous extract) |
Sapindus mukorrosai
(aqueous extract) |
1 | Alkaloids | Dragendorff’s | - | - |
Wagners | - | - | ||
Mayer’s | - | - | ||
2 | Carbohydrate | Molisch’s | + | + |
Fehling’s | - | + | ||
Benedict’s | + | + | ||
3 | Flavonoids | Mg ribbon and dil. HCl | - | + |
4 | Glycosides | NaOH test | + | + |
5 | Tannins/Phenols | Ferric chloride | + | + |
Liebermann’s | + | + | ||
Lead acetate | + | + | ||
6 | Protein’s | Xanthoproteic | + | - |
Biuret | + | - | ||
7 | Starch | Iodine | + | + |
8 | Saponins | Frothing with NaHCO3 | + | + |
9 | Steroids / Terpenes | Salkowski | - | + |
10 | Amino acid | Ninhydrin solution | + | - |
11 | Resin | Acetic anhydride | + | + |
Indication: (-) Absent & (+) Present
FIG. 1: CALCIUM OXALATE CRYSTALS OF SAPINDUS MUKOROSSAI
FIG 2: CALCIUM OXALATE CRYSTALS ATTACHED WITH FIBER IN SAPINDUS MUKOROSSAI POWDER
FIG. 3: FIBER IN SAPINDUS MUKOROSSAI POWDER
FIG. 4: STONE CELL WITH CALCIUM OXALATE CRYSTALS
FIG. 5: POWDER MICROSCOPY OF CHLOROPHYTUM BORVILIANUM
TABLE 4: DETERMINATION OF PHYTOCONSTANTS
S.
no. |
Leaf constant | Report (/mm2) | |
Chlorophytum borvilianum | Sapindus mukorrosai | ||
1 | Vein islet number | 12.4 | 11.2 |
2 | Vein termination number | 11.6 | 12.9 |
3 | Stomatal index (upper epidermis) | 7 | 5.4 |
4 | Stomatal index (lower epidermis) | 18 | 15 |
TABLE 5: FLUORESCENCE ANALYSIS OF CRUDE DRUGS WITH DIFFERENT CHEMICAL REAGENTS
S. no. | Powdered drug + Chemical reagent | Daylight | UV Short | UV long |
1 | Safed musli + Conc. HCl | Brown | Green | Black |
Reetha + Conc. HCl | Dark brown | Dark brown | Black | |
2 | Safed musli + Dil. HCl | Brown | Green | Black |
Reetha + Dil.HCl | Dark brown | Dark green | Black | |
3 | Safed musli +Iodine solution (2%) | Dark brown | Dark green | Black |
Reetha + Iodine solution (2%) | Dark brown | Dark green | Black | |
4 | Safed musli + Glacial acetic acid | Yellow | Green | White |
Reetha + Glacial acetic acid | Dark brown | Brown | Black | |
5 | Safed musli +Conc. HNO3 | Dark brown | Dark green | Black |
Reetha + Conc. HNO3 | Dark brown | Black | Black | |
6 | Safed musli + Dil. HNO3 | Dark brown | Dark green | Black |
Reetha + Dil. HNO3 | Light brown | Light green | Grey | |
7 | Safed musli + Glacial acetic acid +HNO3 | Light brown | Light green | Green |
Reetha + Glacial acetic acid +HNO3 | Dark brown | Dark green | Black | |
8 | Safed musli + Dil. H2SO4 | Light brown | Light green | Grey |
Reetha + Dil. H2SO4 | Dark brown | Dark green | Black | |
9 | Safed musli + Conc. H2SO4 | Dark brown | Black | Black |
Reetha + Conc. H2SO4 | Dark brown | Dark green | Black | |
10 | Safed musli + Fehling’s reagent | Light brown | Light green | Grey |
Reetha + Fehling’s reagent | Dark brown | Dark brown | Black | |
11 | Safed musli + Picric acid solution (Hager’s reagent) | Yellow | Light green | Black |
Reetha + Picric acid solution (Hager’s reagent) | Brown | Light brown | Black | |
12 | Safed musli +NaOH (10%) | Light brown | Green | Black |
Reetha + NaOH (10%) | Brown | Dark green | Black | |
13 | Safed musli + KOH (10%) | Light brown | Light green | Dark brown |
Reetha + KOH (10%) | Dark brown | Light brown | Black | |
14 | Safed musli +Wagner’s reagent | Black | Dark brown | Black |
Reetha + Wagner’s reagent | Dark brown | Brown | Black | |
15 | Safed musli + CuSO4 (5%) | Bluish | Light green | Black |
Reetha + CuSO4 (5%) | Bluish | Light green | Black | |
16 | Safed musli + Lead acetate | Brown | Light green | Black |
Reetha + Lead acetate | Light brown | Light green | Black |
Powder microscopy of Sapindus mukorrossai reveals the presence of calcium oxalate crystals, fibers, stone cells Fig. 1, 2, 3, 4 whereas powder microscopy of Chlorophytum borvilianum stone cells, starch granules and raphides Fig. 4. The Average physicochemical parameters of the Chlorophytum borvilianum and Sapindus mukorrossai powder are tabulated as Table 2. The Preliminary phytochemical screening for various functional groups is tabulated as Table 3. The list of phytoconstituents parameters is summarized in Table 4. Fluorescence analysis of powdered drugs is presented in Table 5.
DISCUSSION: Physicochemical parameters like ash values, moisture content and fluorescence analysis help determine the physiological and non-physiological ash, the possibility of microbial growth or contamination and presence of impurities respectively. The relative low acid insoluble ash value 3.66% of Chlorophytum borvilianum and 0.24% of Sapindus mukorrossai and a high ratio of water-soluble ash content 2.05% of Chlorophytum borvilianum and 14.69% of Sapindus mukorrossai indicates that the crude drug contains more physiological ash or contents (i.e. related to plant tissue) than the non- physiological content. The relative low moisture content indicates that the drugs give an equal possibility for microbial growth and contamination. The preliminary phytochemical investigations in Chlorophytum borvilianum and Sapindus mukorrossai aqueous extract indicated the presence of glycosides, tannins, flavonoids, saponins and triterpenoids in more quantity whereas the sterols and proteins in less quantity, but there are no fats and oils. Presence of such phytochemical constituents may be responsible for various pharmacological activities of this elite medicinal plant. Medicinal plants are considered as living factories as they produce various phytochemicals viz., alkaloids, flavonoids, glycosides, phenols, saponins, sterols, etc in the form of secondary metabolites.
These serve as life-saving drugs. Due to this, day by day demand for crude drugs is increasing and also adulteration of crude drugs. Hence, standardization of a crude drug is essential to avoid and identify adulteration. In standardization of a crude drug, macroscopic and microscopic evaluation is the primary step. According to WHO, standard botanical investigations like epidermal cells, stomatal index, vein islet and termination values, etc are mandatory for the diagnosis of the crude herbal drug.
CONCLUSION: In the present investigation, a set of pharmacognostical standardization parameter studies were conducted on Chlorophytum borvilianum and Sapindus mukorrossai as per pharmacopeia and WHO guidelines. These studies revealed the presence of various important bioactive compounds and proved that these plant drugs are also medicinally important. These results may help in standardization, identification and in carrying out further research in Chlorophytum borvilianum and Sapindus mukorrossai based drugs which are used in Ayurveda and modern pharmacopeia.
ACKNOWLEDGEMENT: Nil
CONFLICT OF INTEREST: Nil
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How to cite this article:
Mishra S and Thakur M: Physicochemical and phytochemical evaluation of Chlorophytum borvilianum and Sapindus mukorrossai. Int J Pharmacognosy 2016; 3(4): 184-91. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.3(4).184-91.
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