Pharmacognostic Profile of Trigonella corniculata L. Seeds and effect of its Aqueous Extract on Growth Inhibition of Cancer Cells

 

Pardeep Kaur¹, Sanjeev Kumar Kataria², Balbir Singh³, Saroj Arora^1*

¹Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar-143005

²Drug Testing and Research Laboratory, Herbal Health Research Consortium Pvt. Ltd, Amritsar

³Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005

 

ABSTRACT:

Trigonella corniculata L. (kasuri methi) is used as a pot herb especially in Northern parts of India. The seeds of the plant hold immense therapeutic potential but, the reports on its pharmacognostic validation are not available. Thus, the present investigation provides a detailed description of pharmacognostical characteristics viz., macroscopic and microscopic features, ash values, extractive values, fluorescence behavior and preliminary estimation of phytochemicals in the seeds. Macroscopic analysis showed elliptical shaped seeds with dimensions 1.5×1.0×0.15 (length, breadth and thickness). The microscopic transverse section of seeds showed two cotyledons enclosed in a mucilaginous endosperm with semilunar radicle. The powder microscopy showed the presence of epidermal and palisade cells of testa, aleurone grains and oil globules. The seeds exhibited characteristic fluorescence in response to reaction with different chemical reagents. The presence of alkaloids, phenolic compounds, flavonoids, carbohydrates, proteins, amino acids and phytosterols were recorded in preliminary phytochemical screening. To the best of our knowledge, the present study establishes the first pharmacognostic documentation of Trigonella corniculata L. seed to validate its authentication and purity. Furthermore, the aqueous seed extract was prepared and evaluated for its effect on cell lines viability of five malignant (MG-63, IMR-32, HeLa, A549, HepG2) and one non malignant (293T) cells of human origin. Among all human cancer cell lines, it was observed that seed extract inhibited the growth of the malignant cells with the best activity against HepG2 cells with IC₅₀ of 232.80 µg/ml. The extract was found to be ineffective against non-malignant 293T cells with very high IC₅₀ of 4.10×10¹³ µg/ml. Thus, the seed extract showed the therapeutic potential against cancer cells which can be exploited in further investigation against carcinogenesis.

 

 

INTRODUCTION:

Trigonella corniculata L. (family: Fabaceae), commonly known as ‘kasuri methi’ is used as a green vegetable (pot herb) especially in Northern parts of India. Its origin of cultivation is Kasur (a district of Punjab province), Pakistan and is a geographical indicator of that region¹. The leaves and seeds of kasuri methi are used as spice and condiments and as flavoring agents due to their characteristic pleasant odor.

 

The leaves are rich source of iron, proteins, calcium, magnesium, potassium, phosphorus and zinc². The seeds are considered as dry and hot in nature and have been traditionally employed as household remedial measure to heal swellings, backache, bruises and post-partum infections in women³. These have also been reported to contain choline, bataine, diosgenin, tigogenin, yamogenin, gitogenin, 6-8 di-b-D glucopyranosyl acacetin, triacontane and 22,23-dihydrostigmasterol⁴. However, the major alkaloid of Trigonella foenum-graecum i.e. trigonelline has not been found in T. corniculata³. The seeds of Trigonella spp. possess numerous pharmacological properties viz., expectorant, aphrodisiac, carminative, hepatoprotective, anti-microbial, anti-cancer, anti-inflammatory and anti-lithigenic^4,5,6,7,8,9. Although, much consideration has been given to T. foenum-graecum, but the seeds of T. corniculata have been reported to exhibit better antioxidant efficacy as compared to T. foenum-graceum¹⁰.

 

The potential of kasuri methi seeds towards beneficial health promoting characteristics has not been fully explored in the area of nutraceuticals and pharmaceuticals. Furthermore, as per our literature survey, no reports are accessible on the pharmacognostic and phytochemical analysis of Trigonella corniculata L. seeds. Also, no reports were found on the effect of its aqueous seed extract on cell viability of cancer cells of human origin. Thus, the present study was aimed at the morphological and microscopical, physico-chemical investigation and preliminary phytochemical analysis of kasuri methi seed as per recommendations of World Health Organization (WHO) and Ayurvedic Pharmacopoeia of India. Furthermore, the aqueous seed extract of Trigonella corniculata L. was assessed for its effect on cell viability of five cancer cell lines (MG-63, IMR-32, HeLa, A549, HepG2) and one non-malignant cell line (293T).

 

MATERIAL AND METHODS:

Procurement of herbal raw material:

The well-authenticated and validated dried samples of Trigonella corniculata L. (kasuri methi) seeds were procured from Herbal Health Research Consortium (HHRC) Pvt. Ltd. Amritsar (India), a Government of India approved Ayurvedic, Siddha and Unani Drug Testing Laboratory.

 

Pharmacognostical studies:

Macroscopical observations:

The organoleptic characterstics of kasuri methi seeds such as shape, size and color were investigated with naked eye and with the aid of a magnifying lens for morphological identification. Further, the dried seed powder was subjected to organoleptic estimation for taste and odor¹¹.

 

Microscopical observations:

Section preparation:

The kasuri methi seeds were soaked in water to obtain sufficient moistening for section cutting. The free hand thin transverse sections of seeds were prepared and collected in a borosilicate petri plate filled with water. The best sections were preferred, mounted on glass slide using glycerine, covered with cover slip and observed under light microscope. The presence of some anatomical characters and features were noted and photographed¹¹.

 

Powder microscopy:

The seed powder was prepared by grinding seeds in an electric grinder. The powder was then placed on a glass slide, mounted in glycerine and observed under microscope for the investigation of microscopic features¹¹.

 

Physico-chemical parameters:

The various physico-chemical parameters of seeds viz., loss on drying, total ash, acid insoluble ash, water soluble ash, ether soluble extractive value, chloroform soluble extractive value, alcohol soluble extractive value, water soluble extractive value and fluorescence behavior pattern were analyzed according to the standard methods^11,12,13.

 

Determination of loss on drying:

5 g of seed powder was weighed in a tared evaporating dish and dried at 105 °C for 3 h. The drying and weighing was done at an interval of 30 min until difference between two consecutive weighing corresponded to not more than 0.25 percent.

 

Loss on drying (% w/w)= 100 × [(Loss in weight of sample)/(Weight of crude sample)]

 

Determination of total ash:

2 g of seed powder was incinerated in a tared crucible at 450 °C in a muffle furnace until the powdered seed material was free from carbon. The carbon free ash was then cooled in a desiccator for 30 min and weighed. The percentage of ash with reference to the air-dried drug was then calculated.

 

Total ash value (% w/w)= 100 × [(Weight of total ash)/(Weight of crude sample)]

 

Determination of acid-insoluble ash:

25 ml of dilute hydrochloric acid was added drop wise to the crucible containing total ash and the insoluble matter was collected on an ashless filter paper (Whatman 41) and washed with hot water. Then the filter paper containing the insoluble matter was transferred to the original crucible and ignited to a constant weight. The residue was cooled, weighed and the content of acid-insoluble ash was estimated with reference to the air-dried drug.

 

Acid insoluble ash (% w/w)= 100 × [(Weight of acid insoluble ash)/(Weight of crude sample)]

 

Determination of water-soluble ash:

The total ash was boiled with 25 ml of water for 5 min and the insoluble material was collected on an ashless filter paper and ignited at 450 °C. The difference in the weight of insoluble matter and the weight of ash represented water-soluble ash and percentage of water-soluble ash with reference to the air-dried drug was noted.

Water soluble ash (% w/w)= 100 × [( (Weight of total ash-Weight of water insoluble ash)/(Weight of crude sample)]

 

Determination of ether soluble extractive value: About 5 g of coarsely powdered kasuri methi seed sample was macerated with petroleum ether (100 ml) in a stoppered flask for 24 h. The sample was shaken on an orbital shaker for first 6 hours and then allowed to stand for 18 h. The obtained extract was filtered and 25 ml of the filtrate was evaporated to dryness in a tared dish on a water bath followed by drying at 105 °C to acquire a constant weight. The percentage of ether soluble extractive with reference to the air dried powdered seed material was then calculated.

 

Ether soluble extractive value (% w/w)= 100 × [(Weight of extract)/(Weight of crude sample )×(Volume of petroleum ether used)/(Volume of filtrate)]

 

Determination of chloroform soluble extractive value:

The coarsely powdered seed sample (5 g) was macerated with 100 ml chloroform for 24 h. 25 ml filtered extract was evaporated to dryness in a shallow dish till a constant weight was obtained. The percentage of chloroform soluble extractive was then determined.

 

 

Chloroform soluble extractive value (% w/w)= 100 × [(Weight of extract)/(Weight of crude sample )×(Volume of chloroform used)/(Volume of filtrate )]

 

Determination of alcohol soluble extractive value:

The maceration of powdered seed material was carried out with 100 ml ethanol. The solution thus, obtained was filtered through filter paper and filtrate was evaporated to dryness. The residue was dried at 105 ºC, weighed and extractive value was determined with respect to air dried sample.

 

Alcholol soluble extractive value (% w/w)= 100 × [(Weight of extract)/(Weight of crude sample)×(Volume of alcohol used)/(Volume of filtrate )]

 

Determination of water soluble extractive value:

The seed powder sample was macerated with 100 ml chloroform water in a closed conical flask for 24 h. After maceration, the 25 ml of filtrate was allowed to dry on a water bath and then at 105 ºC to obtain a constant weight. The percentage of water soluble extractive value was determined with reference to air dried seed material.

 

 

 

Water soluble extractive value (% w/w)= 100 × [(Weight of extract)/(Weight of crude sample)×(Volume of water used)/(Volume of filtrate )]

 

Analysis of fluorescence behavior of T. corniculata seed:

A pinch of finely powdered seed sample was placed on a clean, grease free microscopic glass slide and few drops of reagent were added. The slide was gently tilted to mix the material. The color obtained on the addition of different chemical reagents was viewed and recorded in day light, short (254 nm) and long (365 nm) wavelength using UV chamber.

 

Determination of pH value:

The pH of the freshly prepared 1% and 10 % seed powder suspension in distilled water was determined using an electronic pH meter (Lab India).

 

Determination of bulk density, tap density and Hausner’s ratio:

The bulk density of kasuri methi seed powder was determined by introducing known weight of powdered drug into a graduated cylinder carefully. The tap density was observed by tap density meter (Lab India TD1025) and then compressibility index and Hausner’s ratio were calculated.

 

Bulk density (g/ml)=(Mass of crude drug)/(Volume occupied by crude sample in a graduated cylinder)

 

Tap density (g/ml)=(Weight of powder)/(Volume occupied after tapping)

 

Compressibilty index=(Tap density-Bulk density)/(Tap density)×100

 

Hausner’s ratio=(Tap density)/(Bulk density)

 

Preparation of aqueous extract:

100 g of crude seed sample was soaked overnight in a stainless steel vessel containing potable water in the ratio 1:16 (w/v) at room temperature under aseptic conditions. The sample was heated at boiling temperature on a mild flame to reduce it to one quarter of original volume¹⁴. The decoction was cooled, filtered and then evaporated to dryness on water bath. The extract thus, obtained was stored at 4 °C till further use.

 

Preliminary phytochemical screening:

As shown in Table 1 the phytochemical examination of aqueous seed extract was carried out as per standard methods^15,16

 

 

 

Table 1: Standard procedures for pre phytochemical screening of aqueous seed extract

S. No.	Phytochemical test	Confirmatory observation
1.	Detection of Alkaloids: 0.5 g of extract was stirred in 10 ml of dil. HCl (0.1 N) and filtered. This filtrate was then used to test the presence of alkaloids.
(i)	Dragendorff’s test: Few drops of Dragendorff’s reagent were added to 2 ml of filtrate.	Reddish brown precipitates
(ii)	Mayer’s test: Few drops of Mayer’s reagent were added to 2 ml of filtrate.	Cream colored precipitates
2.	Detection of Phenolic compounds: Approximately 1 g of seed extract was dissolved in 5 ml distilled water and filtered. The filtrate was used to carry out the following tests:
(i)	Ferric chloride test: 2 ml of 1% ferric chloride solution was added to 2 ml of filtrate in a test tube.	Bluish black color
(ii)	Lead acetate test: Few drops of 10% lead acetate solution were added slightly to 2 ml filtrate.	Bulky white precipitates
3.	Detection of Flavonoids:
(i)	Alkaline reagent test: 100 mg of extract was mixed with few drops of NaOH solution.	Intense yellow color on addition of NaOH solution and its disappearance on adding dilute acid.
(ii)	Lead acetate test: Extract was treated with few drops of lead acetate solution.	Yellow precipitates
4.	Detection of Carbohydrates: 100 mg of extract was stirred in distilled water and filtered. The prepared filtrate was used to test the presence of carbohydrates.
(i)	Molisch’s Test: To the 1 ml of above filtrate added 2 drops of Molisch’s reagent followed by 2 ml of conc. sulphuric acid gently along the sides of the test tube.	Violet color at the interface
(ii)	Fehling’s Test: 4 ml of Fehling’s reagent was added to above filtrate and heated on a water bath for few minutes.	Red precipitates
5.	Detection of Glycosides: Seed extract was subjected to hydrolysis with dil. HCl and then used to test the following:
(i)	Modified Borntrager’s test: Ferric chloride solution was added dropwise to the aqueous seed extract and kept on water bath for few minutes, cooled and extracted with equal volume of benzene. The benzene layer was then separated and treated with ammonia solution.	Rose-pink colour in ammoniacal layer indicates the presence of anthranol glycosides
(ii)	Legal’s test: The aqueous seed extract was treated with sodium nitroprusside in pyridine and sodium hydroxide.	Pink to blood red color
6.	Detection of Proteins and Amino acid: 100 mg of extract was dissolved in water and filtered.
(i)	Ninhydrin test: Few drops of Ninhydrin reagent were added to 2 ml of above filtrate and boiled for 2 min.	Blue color
(ii)	Biuret test: The filtrate was stirred with 10% sodium hydroxide solution and heated followed by addition of few drops of 7% copper sulphate solution.	Violet color
7.	Detection of Phytosterols: Approximately 0.5 g of extract was shaken with 10 ml chloroform and filtered. The filtrate was used to perform:
(i)	Libermann’s test: The filtrate was mixed with acetic anhydride and then few drops of conc. Sulphuric acid were added carefully along the sides of test tube.	Brown ring at the junction
(ii)	Salkowski test: Few drops of concentrated sulphuric acid were added to the filtrate.	Red color at interface.

 

Resazurin cell viability assay:

The human cancer cell lines (MG-63, IMR-32, HeLa, A549 and HepG2) of different tissue origin and non-malignant (293T) cell line were procured from National Centre for Cell Science (NCCS), Pune (India). The cell lines were grown in DMEM or RPMI-1640 growth medium in tissue culture flasks. The cancer cells were centrifuged at 1000 rpm for 5 min at 4 °C and the cell pellet thus, obtained was resuspended in complete growth medium to get 1 to 2×10⁵ cells/ml. The cell suspension (100 µl/well) was then seeded in 96 well culture plate. The adhered cells were treated with different concentrations (ranging from 31.25 to 1000 µg/ml) of aqueous seed extract followed by incubation in CO₂ incubator at 37 °C and 5% CO₂ with 90% relative humidity for 24 h. Thereafter, 20 µl of freshly prepared deep blue colored resazurin solution was added to each well. It was thoroughly mixed by gentle stirring and further incubated for 4 h. Then the fluorescence was recorded at excitation/emission wavelength of 560/590 nm employing ELISA plate reader (Synergy HT, BioTek)¹⁷.

RESULTS AND DISCUSSION:

The crude herbal material comprises unprocessed plant parts viz., stem, bark, root, leaves, flower, seed, fruit or whole herb. These are used in the form of fragments, powder or as entire herb during the preparation of single or multi-component herbal extracts, medicines and other botanical preparations¹⁸. However, the botanical preparations lack consistency and exhibit variations in composition and bioactivities. This may be due to misidentification of crude drug material, genetic variability, seasonal and geographical variations, and different methods of harvesting and processing of extracts¹⁹. Thus, the appropriate quality control of the raw material is extremely crucial to sustain the standardization of medicinal botanicals of immense therapeutic potential^20,21. The standardization procedure deploys specific characteristics, constant parameters, qualitative and quantitative values to strengthen efficacy, safety, reproducibility and quality of herbal products²².

 

 

Macroscopic features:

The foremost step to establish the identity of a botanical is the physical examination of crude drug. This is based on the analysis of morphological features (shape, size, texture) and sensory profile (color, odor, taste) of whole drug and its powder²³. In the present study, T. corniculata L. seeds are solid with elliptical appearance (Fig. 1a). A deep furrow extends along the length and divides the seed into two unequal halves. The seeds have a plain and smooth texture, approximately 1.5 mm in length, 1 mm in breadth and 0.15 mm thick. The seeds are dull yellowish brown and become mucilaginous when dipped in water. The seed powder exhibited characteristic odor and bitterness (Fig. 1b).

 

 

(a) T. corniculata L. seeds                 (b) T. corniculata seed powder

Fig. 1: Morphological features of Trigonella corniculata L. seeds and its powder

 

Microscopic characteristic features:

The morphological examination is followed by microscopic investigation to ensure right identification and purity of the crude drug. The accurate and detailed description of diagnostic botanical characteristics is observed and illustrations are then visually documented²⁴. In the current study, both the transverse section and powder microscopic analysis were carried out to establish the identity of T. corniculata seed. The observations recorded are described below.

 

Transverse section of seed:

T.S. of T. corniculata L. seed is represented in Fig. 2a. The section clearly illustrates the presence of embryo comprising two large sized cotyledons facing each other. The cotyledons are enclosed in a translucent mucilaginous endosperm. The radicle is semilunar in shape positioned in a spherical pocket extending from the testa. The outermost seed layer consists of palisade like cells with conical projections covered externally with thick cuticle (Fig. 2b).

 

Seed powder analysis:

The seed powder shows the presence of epidermal and palisade cells of testa, fragments of hypodermal cell of testa, parenchymatous cells of cotyledons and radicle, seed fibers, prism type crystals, aleurone grains and oil globules (Fig. 3a to 3h).

 

 

(a) Transverse section of T. corniculata L. seed

(b) Outermost layer representing palisade like cells with conical projection

Fig. 2: Transverse section of Trigonella corniculata L. seed and cellular arrangement of outermost layer

 

 

(a) Epidermal cells of testa                 (b) Palisade cells of testa

 

(c) Hypodermal cells                            (d) Parenchymatous cells

 

(e) Seed fibre                                          (f) Prism type crystal

 

(g) Aleurone grains                               (h) Oil globules

Fig. 3: Powder microscopy of Trigonella corniculata L. seeds

 

Physico-chemical parameters:

The physico-chemical parameters such as loss on drying, ash value, extractive value and fluorescence analysis were executed as per WHO guidelines and the results are represented in Table 2 and 3. The loss on drying was found to be 6.73±0.002% which is quite less. This low percent content of moisture i.e. weight loss on drying is required to maintain the stability of seed sample and to prevent its decomposition by inhibiting the fungal, bacterial or yeast growth during storage²⁵. The total ash, acid insoluble ash and water soluble ash were determined to be 7.17±0.04%, 3.80±0.03% and 1.69±0.10% respectively. The ash values indicate the presence of earthy (carbonate, silicate, oxalate) or inorganic or any other impurities and highlight the authenticity, purity and quality of the drug²⁶. In the present study, ash values were observed to be reasonably low indicating less adulteration and contamination. The percentage of extractive value was observed to be maximum in water (21.23±0.39%) followed by alcohol soluble extractive value (10.27±0.07%). The ether soluble extractive value was seen to be lowest among four extraction solvents i.e. ether, chloroform, alcohol and water. The extractive values provide the preliminary indication towards the presence of definite chemical constituents in the crude drug soluble in a particular solvent²⁷. Thus, in the present study, the extractive values pointed towards the presence of more water-soluble contents in the seed sample.

 

Table 2: Physico-chemical constants of Trigonella corniculata L. seed

S. No.	Physico-chemical Parameter	Values (% w/w)
1.	Loss on Drying	6.73±0.002
2.	Total Ash	7.17±0.04
3.	Acid Insoluble Ash	3.80±0.03
4.	Water Soluble Ash	1.69±0.10
5.	Ether Soluble Extractive Value	4.31±0.12
6.	Chloroform Soluble Extractive Value	5.68±0.20
7.	Alcohol Soluble Extractive Value	10.27±0.07
8.	Water Soluble Extractive Value	21.23±0.39

 

The fluorescence evaluation was carried out by treating the seed powder with various standard chemical reagents to determine the chemical nature and type of constituents present in kasuri methi seeds²⁸. The observations recorded in visible (day) and ultra violet light (short and long wavelengths) are displayed in Table 3.

 

Table 3: Behaviour pattern of Trigonella corniculata L. seed powder to different chemicals reagents under fluorescence analysis

S.No	Seed powder + Reagent	Day Light	254nm	366nm
1.	Seed powder as such	Light brown	Grey	Browinsh orange
2.	Seed powder + Distilled water	Light brown	Light blue	Purple
3.	Seed powder + Methanol	Light brown	Brownish yellow	Light brown
4.	Seed powder + Hydrochloric acid	Yellowish brown	Dark brown	Light brown
5.	Seed powder + Nitric acid	Light brown	Greenish	Dark green
6.	Seed powder + Sulphuric acid	Brownish	Blue	Blue
7.	Seed powder + Glacial acetic acid	Light brown	Dark green	Light green
8.	Seed powder + Chloroform	Light brown	Light brown	Light pink
9.	Seed powder + Petroleum ether	Light brown	Dark brown	Light brown
10.	Seed powder + Acetone	Light brown	Greenish	Light green
11.	Seed powder + Diethyl ether	Light brown	Purple	Purple
12.	Seed powder + Ethyl acetate	Light brown	Brown	Light pink
13.	Seed powder + Dichloromethane	Light brown	Dark brown	Light brown
14.	Seed powder + Toulene	Light brown	Black	Light brown
15.	Seed powder + Butanol	Light brown	Brown	Light brown
16.	Seed powder + Dimethyl sulfoxide	Dark brown	Dark brown	Light brown

 

The pH of powder was observed to be 5.87±0.03 and 5.90±0.06 at 1 and 10% w/v suspension in distilled water (Table 4). The observed pH value of seed powder indicates its suitability for human consumption.

 

Table 4: pH of Trigonella corniculata L. seed powder

S. No.	Concentration (% w/v)	pH value
1.	1 %	5.87±0.03
2.	10 %	5.90±0.06

 

The bulk density, tap density and Hausner’s ratio are presented in Table 5. The inter-particulate void volume i.e. spatial arrangement of particles determines the compressibility characteristics of a powder²⁹. These physical parameters determine the flow ability of a powder which is a crucial necessity in pharmaceutical industries during packaging, mixing with other herbal ingredients in a composite formulation and transportation³⁰. Based on the physical parameters obtained in this work, kasuri methi seed powder is considered as slight difficult to flow due to high compressibility index and Hausner’s ratio as shown in the table.

 

Table 5: Powder flow analysis of Trigonella corniculata L. seed powder

Bulk density (g/ml)	Tap density (g/ml)	Compressibility index	Hausner’s ratio
0.50±0.01	1.34±0.17	61.78±3.88	2.67±0.27

 

The aqueous seed extract of T. corniculata was subjected to systematic preliminary phytochemical screening to estimate the presence of various phytochemicals. The extract exhibited the positive reactions for alkaloids, phenolic compounds, flavonoids, carbohydrates, proteins, amino acids and phytosterols as shown in Table 6. This initial scrutinization of secondary metabolites is important to detect bioactive constituents present in T. corniculata seed to explore its various biological and therapeutic properties³¹.

 

Table 6: Preliminary phytochemical investigation of Trigonella corniculata L. aqueous seed extract

S. No.	Aqueous seed extract	Result
1.	Alkaloids	+
2.	Phenolics	+
3.	Flavonoids	+
4.	Carbohydrates	+
5.	Glycosides	-
6.	Proteins and amino acids	+
7.	Phytosterols	+

 

Resazurin reduction assay:

The growth inhibitory efficacy of kasuri methi aqueous seed extract was scrutinized against a panel of malignant cancer cells by reduction of resazurin as a fluorometric marker for the estimation of cellular viability. The percentage inhibition and IC₅₀ values demonstrated by seed extract is represented in Table 7. The extract inhibited the growth of MG-63, IMR-32, HeLa, A549 and HepG2 cells in a concentration dependent manner. Moreover, the extract was observed to the most active against HepG2 cells with the percentage inhibition of 81.06± 0.14% at 1000 µg/ml exhibiting lowest IC₅₀ value (232.80 µg/ml). The efficacy presented by seed extract to suppress the proliferation of different cancer cells based on IC₅₀ values is: HepG2 ˃ A549 ˃ IMR-32 ˃ MG-63 ˃ HeLa. The extract was seen to be ineffective in suppressing the growth of non-malignant 293T cells as indicated by the relatively high value of IC₅₀ Table 7. This may ensure its safety and non toxic nature towards non-malignant normal cells also. This data underscores that potential of the extract to suppress the growth of malignant cells might be attributed to the presence of secondary metabolites as reported in preliminary screening of possible phytoconstituents as shown in Table 6. The present investigation is the first report of the effect of aqueous seed extract of T. corniculata on viability of cells employing resazurin reduction assay against MG-63, IMR-32, HeLa, A549 and HepG2 cell lines.

 

 

 

 

 

Table 7: Percentage growth inhibition and IC₅₀ values exhibited by aqueous seed extract of Trigonella corniculata L.

Conc. (µg/ml)	Inhibition (%±SE)
	Malignant					Non-malignant
	MG-63	IMR-32	HeLa	A549	HepG2	293T
31.25	17.26± 0.88	11.66±0.76	08.81±0.37	14.95±0.43	19.24±0.15	3.18±2.15
62.5	17.86± 0.81	20.79±0.44	10.73±01.13	25.24±0.65	26.45±0.17	5.36±3.58
125	19.54± 1.94	25.38±1.76	12.98±2.03	33.70±0.54	33.66±0.07	6.45±1.73
250	25.50± 1.25	32.18±0.84	17.25±1.08	52.17± 0.64	52.61±0.10	7.72±6.37
500	31.90± 0.59	45.45±0.59	23.35±2.09	58.72±0.03	58.36±0.08	8.63±1.46
1000	52.27± 0.40	54.37±1.68	44.14±0.10	77.46±0.02	81.06±0.14	8.99±1.44
Regression equation	y = 0.036x+ 15.55	y = 12.1ln(x)- 31.1	y = 0.035x+ 7.98	y = 17.8ln(x)-48.3	y = 17.5ln(x)-45.2	y = 1.65ln(x)-1.84
R²	0.994	0.979	0.992	0.983	0.959	0.948
IC50 (µg/ml)	956.94	803.68	1200.74	252.10	232.80	4.10×1013
Camptothecin (% inhibition at 10 µM)	81.86	15.68	71.60	68.07	51.35	22.62

 

 

CONCLUSION:

Pharmacognostic and physico-chemical parameters are important evaluation parameters for confirming identity and purity of a crude drug. As there is no pharmacognostic documentation of Trigonella corniculata L. seed, the present study was undertaken to lay down the standard parameters for the first time which could be useful for establishing the authenticity of this medicinally potent plant. Furthermore, the aqueous seed extract represented the greatest growth inhibition activity against HepG2 cells and thus, the results demand for further studies on this extract to probe its therapeutic potential in hepatocellular carcinoma.

 

 

ACKNOWLEDGEMENT:

The first author duly acknowledges the University Grants Commission, New Delhi for providing the scholarship under the MANF scheme (vide grant no. 201213-MANF-2012-13-SIK-PUN-16650). The Director, Herbal Health Research Consortium Pvt. Ltd. is also duly acknowledged for providing necessary facilities to carry out this work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1        Erum S, Anwar R, Masood S. Evaluation of kasuri methi Trigonella foenum-graecum L. var. to establish GI right of Pakistan. Pakistan Journal of Agricultural Sciences. 24; 2011: 1-4.

2        Pasricha V and Gupta, RK. Nutraceutical potential of Methi (Trigonella foenum-graecum L.) and Kasuri methi (Trigonella corniculata L.). Journal of Pharmacognosy and phytochemistry. 3(4); 2014: 47-57.

3        Atal CK, Sood SP. A phytochemical investigation of Trigonella corniculata Linn. Journal of Pharmacy and Pharmacology. 16(9); 1964: 627-629.

4        Jain SC, Agrawal M, Sharma RA. The genus Trigonella–phytochemistry and biology. Ancient Science of Life. 16; 1996: 108-117.

5        Mehrafarin A, Rezazadeh SH, Naghdi Badi H, Noormohammadi GH, Zand E, Qaderi A. A review on biology, cultivation and biotechnology of fenugreek (Trigonella foenum-graecum L.) as a valuable medicinal plant and multipurpose. Journal of Medicinal Plants. 1; 2011: 6-24.‎

6        Yadav UC, Baquer NZ. Pharmacological effects of Trigonella foenum-graecum L. in health and disease. Pharmaceutical Biology. 52; 2014: 243-254.

7        Skaltsa H. Chemical constituents. In Fenugreek: The genus Trigonella, Edited by Petropoulos, GA, Taylor and Francis, New York. 2002; pp. 132-161.

8        Rayyan S, Fossen T, Andersen ØM. Flavone C-glycosides from seeds of fenugreek, Trigonella foenum-graecum L. Journal of Agricultural and Food Chemistry. 58; 2010: 7211-7217.

9        Bin-Hafeez B, Haque R, Parvez S, Pandey S, Sayeed I, Raisuddin S. Immunomodulatory effects of fenugreek (Trigonella foenum graecum L.) extract in mice. International Immunopharmacology. 3; 2003:.257-265.

10      Semalty M, Semalty A, Joshi GP, Rawat MSM. Comparison of in vitro Antioxidant Activity of Trigonella foenum-graecum and T. corniculata Seeds. Research Journal of Phytochemistry. 3; 2009: 63-67.

11      WHO. Macroscopic and microscopic Examination: Quality Control Methods for Medicinal Plant Materials. World Health Organisation, Geneva. 1998.

12      Lohar DR. Protocol for Testing of Ayurvedic, Siddha and Unani medicines. Government of India, Department of AYUSH, Ministry of Health and Family Welfare: Pharmacopoeial laboratory for Indian medicines, Ghaziabad. 2007; pp. 47-52.

13      The Ayurvedic Pharmacopoeia of India, Part-I, Vol. IX. Government of India, Ministry of AYUSH, Pharmacopoeia Commission for Indian Medicine and Homoeopathy, Ghaziabad. 2016.

14      The Ayurvedic Pharmacopoeia of India, Part-I, Vol. I. Government of India, Ministry of Health and Family Welfare, Department of Indian System of Medicine and Homeopathy (ISMandH), New Delhi. 1990.

15      Trease GE, Evans WC. Textbook of Pharmacognosy, 12^th edition. Tindall and Co., London. 1983.

16      Harborne JB. Phytochemical methods - a guide to modern techniques of plant analysis, 2^nd edition. Chapman and Hall, London. 1984.

17      Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, Minor L. Cell viability assays. In Assay Guidance Manual [Internet], Edited by Sittampalam GS, Coussens NP, Brimacombe K, et al., Eli Lilly and Company and the National Center for Advancing Translational Sciences, Bethesda (MD). 2013 (Updated: July 1, 2016); pp. 355-385.

18      Shinde VM, Dhalwal K, Potdar M, Mahadik KR 2009. Application of quality control principles to herbal drugs. International Journal of Phytomedicine. 1(1); 2009: 4-8.

19      Tilton R, Paiva AA, Guan JQ, Marathe R, Jiang Z, van Eyndhoven W, Bjoraker J, Prusoff Z, Wang H, Liu SH, Cheng YC. A comprehensive platform for quality control of botanical drugs (PhytomicsQC): a case study of Huangqin Tang (HQT) and PHY906. Chinese medicine. 5(1); 2010: 30.

20      Donald MM, Arthur PG. Botanical medicines the need for new regulations. New England Journal of Medicine. 347(25); 2002: 2073-2076.

21      Sahoo N, Manchikanti P, Dey S. Herbal drugs: standards and regulation. Fitoterapia. 81(6); 2010: 462-471.

22      Folashade O, Omoregie H, Ochogu P. Standardization of herbal medicines-A review. International Journal of Biodiversity and Conservation. 4(3); 2012: 101-112.

23      Nikam PH, Kareparamban J, Jadhav A, Kadam V. Future Trends in Standardization of Herbal Drugs. Journal of Applied Pharmaceutical Science. 02(06); 2012: 38-44.

24      Zhao ZZ, Hu YN, Wong YW, Wong WCG, Wu K, Jiang ZH, Kang T. Application of microscopy in authentication of Chinese patent medicine-Bo Ying compound. Microscopy Research and Technique. 67(6); 2005: 305-311.

25      Garg V, Dhar VJ, Sharma A, Dutt R. Facts about standardization of herbal medicine: a review. Zhong Xi Yi Jie He Xue Bao. 10(10); 2012: 1077-1083.

26      Patwekar MSL, Suryawanshi Arvind B, Gaikwad Manoj S, Pedewad Snehal R. Standardization of herbal drugs: An overview. The Pharma Innovation Journal. 4(9); 2016: 100-104.

27      Chandel HS, Pathak AK, Tailang M. Standardization of some herbal antidiabetic drugs in polyherbal formulation. Pharmacognosy Research. 3(1); 2011: 49.

28      Mandal M, Misra D, Ghosh NN, Mandal V. Physicochemical and elemental studies of Hydrocotyle javanica Thunb. for standardization as herbal drug. Asian Pacific Journal of Tropical Biomedicine. 7(11); 2017: 979-986.

29      Mukhi S, Bose A, Panda P, Rao MM 2016. Pharmacognostic, physicochemical and chromatographic characterization of Samasharkara Churna. Journal of Ayurveda and Integrative Medicine. 7(2); 2016: 88-99.

30      Shah RB, Tawakkul MA, Khan MA. Comparative evaluation of flow for pharmaceutical powders and granules. AAPS PharmSciTech. 9(1); 2008: 250-258.

31      Doss A. Preliminary phytochemical screening of some Indian medicinal plants. Ancient science of life. 29(2); 2009: 12.

 

 

Accepted on 07.03.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(5):2022-2029.