Pharmacognostic and Phytochemical Characteristics of the Colchicum luteum Baker (Suranjan) growing wild in Kashmir Valley, India
Regional Research Institute of Unani Medicine, Naseem Bagh,
University of Kashmir Hazratbal, Srinagar, J&K - 190006, India.
*Corresponding Author E-mail: myounis985@gmail.com
ABSTRACT:
Colchicum luteum (Liliaceae) belongs to genus Colchicum. The Liliaceae family consists of 280 genera and 4000 species, with diverse geographical locations. In the Kashmir valley, they are widely distributed. Colchicum luteum is a perennial plant with corms and starchy rhizomes. This drug holds a prime position in the Unani system of medicine due to presence of alkaloid colchicine, which is found to be effective in arthritis, gout, rheumatism, and used as a carminative, laxative, and aphrodisiac. Its hydro-alcoholic extract was screened for the presence of several phytoconstituents using preliminary phytochemical methods. The microscopy of the Colchicum luteum corm revealed the presence of Fragment of Vessels with Spiral and Annular Thickening, Parenchyma and Part of a Spirally Thickened Vessel, Calcium-Oxalate Crystal, Starch-Granule and Single-Fibre. Proximate analysis revealed that Colchicum luteum corm dry plant powder has a 2.6% Total Ash Value, 1.0% Acid Insoluble Ash Value, and 1.8% Water Soluble Ash Value. 10.6 percent was determined to be the loss on drying. Post treatment with various chemical agents, fluorescence examination of the powdered plant material was performed under UV light at 254 nm and visible light. The current study showed the microscopical characters, the preliminary phytochemical screening and the proximate analysis of the Colchicum luteum corm. Information collected from such studies can be used as benchmark in the quality control of this plant as herbal medicine for treatment of various diseases.
KEYWORDS: Colchicum luteum, Suranjan, Microscopical Characters, Proximate Analysis, Fluorescence Analysis and Phytochemical Screening.
INTRODUCTION:
The C. luteum is listed as rare and threatened species by the Department of Ecology, Environment, and Remote Sensing in Jammu and Kashmir1. Colchicum luteum L., referred to as Suranjan-e-Talkh in Urdu, belongs to the Liliaceae family. The Liliaceae family includes around 280 genera and 4000 species of perennial plants with starchy rhizomes, corms, or bulbs. Based on when they bloom, there are two groups of colchicum species. A "monocot plant," C. luetum is harvested from the meadows of Kashmir. It has dark, oval-shaped tuberous roots. The leaves have length of 6 to 12 inches. The flowers are between one and two inches long and about half an inch wide.
Between the middle of February and April, the flowering season starts, and between April and June, the seeds reach maturity2. From Kashmir to Chamba, at elevations ranging from 700 to 2800m, Suranjan-e-Talkh is extensively dispersed on the periphery of the forest or in open grasslands and temperate western Himalayas. It stretches in to the neighboring nations of the Nepal, Afghanistan, Pakistan, and the Hindu Kush Mountains 1, as well as the other Indian states of Punjab, Himachal Pradesh, and Sikkim. A substantial amount of starch, a tiny amount of oily resinous material, and the bitter alkaloid colchicine are present in the Colchicum luteum corm3. The other important alkaloids present in C. luteum are 3-demethyl-N-deacetyl-N-formylcolchicine, 3-demecolchicine, β and ɣ Lumicolchicines, 3-demethylcolchicine, N-desacetyl-N-desacetyl-N-formylcolchicine, N-formyl-desacetylcolchicine and kesselringine3. The primary alkaloid found in all Colchicum species is colchicine 4.
Colchicine is well known for its ability to treat inflammatory diseases including acute gout and Mediterranean fever5. Colchicine was first used to disrupt mitosis and induce polyploidy in 1937, and it is still widely used in a variety of plant breeding projects today 5. Additionally, it has been observed Demecolcine, one of the colchicine derivatives, isused to treat myeloid leukaemia6, while the allocolchinoid phosphate derivative ZD6126 is being studied as a potential cancer treatment7.
The dried Colchicum luteum corm Baker (Suranjan) was collected from registered vendor of Jammu & Kashmir. This plant material was authenticated by Dr. Akhtar H. Malik, Curator Centre for Biodiversity and Taxonomy (CBT), Department of Botany, University of Kashmir under specimen voucher No. 4293-KASH. The collected material was also deposited in herbarium as a specimen for future references.
The reagents used during the course of this work were of analytical grade purchased from Central Drug House (P) LTD., Bombay, India.
Macroscopical and Microscopical study of the Colchicum luteum corm Baker (Suranjan) were performed following the methods mentioned in Trease and Evans Pharmacognosy8. The study was necessary to check adulteration and inaccuracy in selection procedures of raw medicinal material from genus Colchicum.
The freshly obtained Colchicum luteum corm Baker (Suranjan) was first air dried and then finely crushed into powder form. The powdered form of the plant material was then subjected to hydroalcoholic extraction (cold extraction) through maceration for 72hours. The extract so obtained was filtered and concentrated under reduced pressure using rotary vacuum evaporator. After extraction, the dried hydroalcoholic extract was sequentially fractionated with different organic solvents such as Hexane, DCM, Ethylacetate and Butanol in the increasing order of polarity by using separating funnel. The dried fractions were then preserved in air tight glass containers for further use.
Using the powdered Colchicum luteum corm, proximate analysis revealed physicochemical parameters, including extractive value (hot and cold), loss on drying, acid insoluble ash value, total ash value, sulphated ash value, and pH determination of drug (for solution of 1% and 10% drug)9. To determine the change in colour under UV at 254 nm (low UV), 366 nm (high UV), and under day light, the fluorescence analysis of the powdered drug was also carried out10. Colchicum luteum corm hydroalcholic extract underwent preliminary phytochemical screening to identify the presence of several phyto constituents.
The sample (Colchicum luteum) was predigested in microwave digester and detected for following metals viz. Lead, Cadmium, Arsenic and Mercury using Atomic Absorption Spectrophotometer (AAS)11.
The determination of microbial load in the sample was performed following the guidelines prescribed by the World Health Organization. The analysis included Bacterial Count (Enterobacteriaceae, Escherichia coli, Salmonella Spp, Staphylococcus aureus and Pseudomonas aeruginosa) and Total Fungal Count12.
The sample (Colchicum luteum) was processed as per guidelines given in VICAM instruction manual for quantitative measurement of aflatoxins in many drugs. The instrument used was VICAM Afla Test Fluorometer. The sample was mixed with an extraction solution, blended and filtered. The filtrate was then passed through Afla test column where antibodies bind to aflatoxin, if present. The column was then washed with water to remove the impurities. After this,methanol was passed through the column to remove the bound aflatoxin from the antibody. The collected methanol solution was added with equal volume of developer (Fluorescent dye), shaken well and measured in Fluorometer13.
RESULTS:
Macroscopical evaluation: It has deep brown colour, tastes bitter, odorless, elliptical in shape with smooth texture. Powder microscopy of Colchicum luteum corm: The powdered Colchicum luteum corm was inspected using microscope. The various microscopic images obtained are given in Figure1.
a) Fragment of vessels with Sprial and Annular Thckenings b) Parenchyma and part of a Spirally Thickened Vessel
c) Calcium-Oxalate Crystal d) Strach-Granule e) Single Fibre
Figure1: Depicts the microscopic image of powdered Colchicum luteum corm with (a) Fragment of Vessels with Spiral and Annular Thickenings
(b) Parenchyma and Part of a Spirally Thickened Vessel (c) Calcium-Oxalate Crystal (d) Starch-Granule and (e) Single-Fibre.
Physicochemical Analysis and Phytochemical Screening of C. Luteum Corm:
The proximate analysis was done for the evaluation of Pharmacognostic parameters of the Colchicum luteum corm as shown in Table-1. Table-2 shows the presence of various Phyto-constituents present in the hydroalcholic extract of the Colchicum luteum corm. The fluorescence analysis was also studied with different reagents under visible light and UV light as shown in Table3.
Table1: Physicochemical analysis and extractive value of Colchicum luteum (Baker) corm
|
Physicochemicalparameters |
Results |
|
Total ash value (%w/w) |
2.6 |
|
Acid insoluble ash value (%w/w) |
1.0 |
|
Water soluble ash value (%w/w) |
1.8 |
|
Loss on drying (%w/w) |
10.6 |
|
Foreign matter (%w/w) |
0.018 |
|
pH of 1% soution |
7.19 |
|
pH of 10% solution |
6.05 |
|
Swelling index |
0.00 |
|
Foaming index |
0.00 |
|
Extractive values (%w/w) |
Cold extractive / value Hot Extractive value |
|
Ethanolic |
11.2/14.4 |
|
Aqueous |
21.6/39.2 |
Table2: Phytochemical Screening of hydroalcoholic extract of Colchicumluteum (Baker) corm
|
Tests |
Inference |
Hydroalcoholic extract |
|
Carbohydrates |
|
|
|
Molish’s test |
Violet ring |
+ |
|
Fehling’s test |
Brick red ppt |
+ |
|
Benedict’s test |
Brick red ppt |
+ |
|
TANNINS |
|
|
|
FeCl3 test |
Black colour |
+ |
|
Lead acetate |
White ppt |
+ |
|
Potassium dichromate test |
Red ppt |
- |
|
Saponins |
|
|
|
Foamtest |
Foaming |
- |
|
Flavonoids |
|
|
|
Shinoda test NaOH test |
Pink colour Yellow colour to colourless |
- - |
|
PHENOLICS |
|
|
|
FeCl3test |
Bluish colour |
+ |
|
Anthraquinone Glycosides |
|
|
|
Borntrager’s test |
Pink Colour |
- |
|
Cardaic Glycosides |
|
|
|
Keller killiani Test |
Brown ring at Junction |
+ |
|
Legal test |
Pink colour |
+ |
|
Terpenoids |
|
|
|
Salkowski’s test |
Golden yellow ring at Junction |
+ |
|
Phytosterols |
|
|
|
Libermann’s test |
Brown ring at junction |
+ |
|
Alkaloids |
|
|
|
Dragendroff’s reagent |
Orange ppt |
+ |
|
Mayer’s reagent |
Cream ppt |
- |
|
Hager’s test |
Yellow ppt |
+ |
|
Wagner’s test |
Red brown ppt |
+ |
|
Proteins |
|
|
|
Millons test |
Brick red colour |
- |
|
Biuret test |
Violet colour |
- |
(+) sign stands for the presence and (-) sign for absence, (ppt) stands for precipitation.
Table 3: Fluorescence Analysis of Colchicum luteum (Baker) corm:
|
Treatment |
DayLight |
UV(254nm) |
UV(366nm) |
|
Powder as such |
Cream colour |
Cream white |
Cream white |
|
Powder treated with distilled water |
Light brown |
Parrot colour |
Turbid white |
|
Powder treated with GAA |
Golden |
Green |
White |
|
Powder treated with conc.HCl |
Dark golden |
Green |
Light violet |
|
Powder treated with conc.HCl+ H20 |
Light golden |
Green |
Light golden |
|
Powder treated with Pet. ether |
Light brown |
Green |
Milky white |
|
Powder treated with ethanol |
Golden |
Green |
Pinkish |
|
Powder treated with methanol |
Cream colour |
Green |
Light violet |
|
Powder treated with ethyl acetate |
Golden |
Green |
Violet |
|
Powder treated with conc. H2SO4 |
Black |
Greenish Black |
Black |
|
Powder treated with conc. H2SO4+H2O |
Black |
Black |
Black |
|
Powder treated with picric acid |
Yellow |
Parrot green |
Black |
|
Powder treated with 5% FeCl3 |
Dark amber |
Dark green |
Black |
|
Powder treated with chloroform |
Light brown |
Green colour |
Milky white |
|
Powder treated with HNO3 |
Brown |
Dark green |
Black |
|
Powder treated with HNO3+H2O |
Brown |
Green |
Black |
|
Powder treated with Benzene |
Light golden |
Green |
White |
Table 4: Heavy metal analysis of Colchicum luteum (Baker) corm:
|
Heavy Metal |
Heavy Metal in Drug (ppm) |
Permissible limit of heavy metals in crude drugs (ppm) as per WHO guidelines. |
|
Arsenic |
0.000 |
NMT2.0 ppm |
|
Mercury |
0.000 |
NMT1.63 ppm |
|
Cadmium |
0.0101 |
NMT0.3 ppm |
|
Lead |
0.0991 |
NMT1.0 ppm |
Table 5: Determination of Microbial load of Colchicum luteum
|
S. No. |
Parameters |
Results |
Remarks |
|
1 |
Total bacterial count (TBC) |
8x103 cfu/gram |
Pink and white colonies are seen in Macconkey agar plates |
|
2 |
Total Fungal count (TFC) |
3x103cfu/gram |
|
|
3 |
Enterobacteriaceae |
2x103cfu/gram |
|
|
4 |
Escherichiacoli |
Absent |
|
|
5 |
SalmonellaSpp |
Absent |
|
|
6 |
Staphylococcus aureus |
Absent |
|
|
7 |
Pseudomonas aeruginosa |
Absent |
CFU: Colony Forming Unit
Table 6: Determination of Aflatoxin of Colchicum luteum
|
S. No. |
Parameters |
Method/Refrence |
Results |
|
1 |
Total Aflatoxin B1+B2+G1+G2 |
Vicam Af latest Fluorometer |
8ppb |
Colchicum luteum corm's quality, safety, and efficacy for usage as a possible drug candidate were determined by the findings of physicochemical analysis, phytochemical screening, heavy metal detection, microbiological load, and aflatoxin detection. The quality control evaluation study presented here may be a useful tool for identifying and standardizing the plant in terms of quality control of the raw materials used in the manufacture of nutraceuticals and herbal remedies. It will prevent its adulteration from drug of same or other genus having low potency.
The authors have no conflicts of interest regarding this investigation.
All the authors express their heartiest gratitude to DG-CCRIUM for encouragement, support and providing good lab facilities to carry out this research work.
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Received on 07.10.2022 Modified on 22.05.2023
Accepted on 10.10.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(12):5728-5731.
DOI: 10.52711/0974-360X.2023.00926