Table 1. Geographic and climatic conditions of the places from where the plants collected

S. No	Districts and place	Latitude (˚N)	Longitude (˚E)	Soil type	Altitude (m)	Rainfall per annum (mm)	Climate	Temperature (℃)
1	Ariyalur	11.2398619	79.2902133	Red loamy, alluvial	76	485	Sub-tropical	24.0-38.0
2	Coimbatore (Nallurpalayam)	11.0168445	76.9558321	Red loamy, black soil,	411	612	Tropical wet and dry	22.1-34.7
3	Cuddalore (Neyveli)	11.7480419	79.7713687	Red loamy, alluvium soil	1	1252	Pleasant warm days and cool nights	22.5-37.0
4	Dharmapuri (Jittandahalli)	12.1065271	78.1361409	Non-calcareous red, non-calcareous brown, calcareous black soil	457	850	tropical	26.5-30.1
5	Dindigul (Palani)	10.3673123	77.9802906	Red loamy, black soil	268	717	Sub-tropical	24.8-30.4
6	Erode (Sennimalai)	11.341036	77.717164	Red loamy, black soil	183	700	Steppe climate	25.4-31.2
7	Salem (Chinnapunalvasal)	42.519540	70.896716	Non-calcareous red, non-calcarous brown, calcareous black soil	278	1036	Warm and temperate	23.0-38.0
8	Namakkal (Kumarapalayam)	11.219439	78.167724	Red loamy, black soil	218	764	Norst western and western agro climatic zone	28.0-38.0
9	Madurai (Palamedu)	9.925201	78.119775	Coastal alluvial, black, red to sandy soil, deep red soil	101	857.6	Sub-tropical	23.7-34.2
10	Trichy	10.790483	78.704673	Red loamy, alluvium	88	860	Cauvery delta agro climatic zone	25.0-32.0

11	Karur (Kodumudi)	10.960078	78.076604	Red loamy, black soil	101	775	Semi arid climate	25.3-31.7
12	Thanjavur	10.784684	79.136400	Red loamy, black soil	88	938	Cauvery delta agro climatic zone	25.2-31.6
13	Perambalur	11.240984	78.866573	Red loam, black soil	143	894	Semi-arid climate	24.9-31.7
14	Vellore	12.8465	78.9288	Gravelly,stony,sandy loam soil	30	971	tropical	23.4-32.3
15	Kanchipuram	12.834174	79.703640	Red loam, clay loam, saline coastal, alluvium	83.2	1083	Hot and humid	24.1-32.7
16	Theni (Uttamapalaiyam)	10.010366	77.476811	Red loam, black soil	100	869	Western agro climatic zone	24.9-29.4
17	Viluppuram (Mugaiyur)	11.936890	79.487262	Red sandy loam, clay loam, saline coastal, alluvium	44	1046	tropical	24.6-32.0
18	Tirunelveli (Sankarankovil)	8.713913	77.756652	Coastal alluvium, black, red to sandy soil, deep red soil	47	752	tropical	26.3-31.2
19	Tiruppur (Kanjampalayam)	11.11541	77.35456	Red loam, black soil	304	605	Local steppe climate	24.8-30.0
20	Krishnagiri	12.518611	78.213736	Non-calcareous red, non-calcareous brown soil, calcareous black	429	789	tropical	22.8-30.1
21	Pudukkottai (Mallaiyur)	10.379663	78.820845	Red loam, alluvium	88	910	tropical	23.4-32.3

Preparation of plant extracts for HPTLC:

The entire plant was washed, cleaned with water; shade dried for 10 days under controlled temperature, the leaves were separated from the collected plant and powdered, passed through a # 40 mesh sieve and stored in an air tight container for further use. From coarsely powdered dried leaves, three g was weighed and extracted by cold maceration in 20ml of methanol for 48 h. The methanol extract was filtered through Whatman filter paper No.1 and then concentrated by evaporation and the residue obtained was utilized for HPTLC studies. The percentage yield methanol was 6.32%.

Preliminary Phytochemical Investigations:

Quality control tests for the crude leaf powder of the plants were carried out as per IP 1996⁸ and WHO guidelines⁹ to assess purity.

Proximate analysis:

Extractive Value:

Three g of powdered leaves from each district was taken and extracted by cold maceration with 20ml methanol. After evaporation, the residue obtained was weighed and extractive values were calculated.

Moisture Content:

Two g of powdered leaves was weighed, heated under hot air oven at 105°C for 20 min till there is no difference in weight on subsequent heating. Difference in weight was used to calculate moisture content.

Ash Value:

One g of powdered leaves was weighed in crucible. The powder along with crucible was ignited in muffle furnace at the temperature 480°C. After ignition cooled to room temperature under desiccators and weighed. The differences in the weight present the total ash value.

Estimation of Apigenin content by HPTLC:

Preparation of standard apigenin and sample solution:

Ten mg of standard apigenin was accurately weighed and transferred into 10ml volumetric flask, and it was dissolved and was made up to 10ml with methanol. From this stock solution of apigenin further dilutions were made for estimation. Sample solution was prepared was diluted with methanol to produce a concentration of 10mg/mL.

Selection of detection wavelength:

The sensitivity of HPTLC method depends upon the proper selection of wavelength by UV detector. Shimadzu 1700 Pharma Spec UV-Vis spectrophotometer was used to determine the maximum absorption of standard apigenin solution (1mg/ml) in methanol.

Chromatographic Condition:

Standard apigenin and sample solutions were spotted on a Precoated TLC aluminium sheets silica gel 60 F254 (10 x 10cm, 0.2mm thickness) as 6mm wide band width by using automatic TLC applicator Linomat V, 10mm from the bottom. Toluene: Ethyl acetate: Formic acid: methanol (3:6:1.4:0.4 v/v) was used as mobile phase. The plates were then developed in twin trough chamber after for saturation for 15 min. After development the plates were dried in air and scanned at 254nm for Apigenin by using CAMAG Scanner III. The plates were photographed at 254nm by using CAMAG Reprostar instrument.

Calibration Curve for Standard Apigenin:

The standard apigenin solutions (200-1000ng/spot) were applied on TLC plate and developed and scanned as per the chromatographic conditions mentioned above. The peak areas were recorded. Calibration curve of apigenin was prepared by plotting peak area against concentration of apigenin applied.

Optimization of chromatographic conditions:

Application port: Linomat V

Detector : Camag TLC scanner

Stationary phase: precoated aluminium sheets silica gel 60 GF₂₅₄

HPTLC plate: 10cm x10cm x 0.2mm

Mobile phase: Toluene: ethyl acetate: formic acid: methanol (3:6:1.6: 0.4)

Chamber saturation: 10 min

Band length:5 mm

Application position: 10mm

Solvent front position: 80mm

Instrumental parameters

Number of tracks: 10

Position of first track: 10.0mm

Distance between tracks: 10.0mm

Scan start position: 5.0mm

Scan end position : 75.0mm

Slit dimensions: 5.00 x 0.45mm, Micro

Optimize optical system: Light

Scanning speed: 20mm/sec

Data resolution: 100µm/steps

Measurement table:

Wavelength: 254nm for Apigenin

Lamp: D2& W

Measurement type: Remission

Measurement mode: Absorption

Optical filter: Second order

Detector mode: Automatic

Estimation of Apigenin content in methanol extracts of leaves of Cardiospermum halicacabum collected from 21 districts of Tamil Nadu:

The methanol extracts of the leaves of Cardiospermum halicacabum and standard apigenin were spotted on precoated HPTLC aluminium sheets silica gel 60 GF₂₅₄ (10 x 10cm, 0.2mm thickness) as 4mm wide band positioned 10mm from the bottom of the plate and 10mm from side of the plate by using a automatic TLC applicator Camag Linomat V with nitrogen flow providing a delivery speed of 150nl/s by Camag microliter syringe. These conditions were kept constant throughout the analysis of samples. The linear ascending development was performed in a Camag twin trough glass chamber 10cm x 10cm which is previously saturated with the mobile phase for 10 min. The mobile phase used was Toluene: Ethyl acetate: formic acid: methanol (1:6:1.6:0.4 v/v). The plates were developed to 80mm from the bottom of plate and after the development plates were dried first in air followed by drying in hot air oven at 60^oC for five min. The quantification of apigenin was done by using Camag TLC Scanner Model III equipped with winCATS software. The applied scan conditions were 5.00mm x 0.45mm slit width, wavelength 254nm, and absorption mode. The plates were photographed at 254nm. The amount of apigenin present in the extracts was calculated from the densitogram.

RESULTS:

Proximate composition in dried leaf powders of Cardiospermum halicacabum collected from 21 districts of Tamil Nadu:

The leaf powders of Cardiospermum halicacabum complied with IP 1996 and WHO guidelines for the quality control tests.

Estimation of apigenin content by HPTLC:

The estimation of apigenin from Cardiospermum halicacabum was done by using three precoated HPTLC densitogram and the values are reported in % w/w in Table 2. The standard apigenin and samples at the concentration of 800ng/spot gives the well resolved band in chromatogram as shown in Figure 1.

Standard Apigenin (Rf: 0.86) showed single peaks in HPTLC chromatogram (Figure 2). The amount of apigenin present in the methanol extract was computed from the above Calibration curve. The linearity of apigenin was found to be 100-1000ng/spot. The correlation coefficient of apigenin was found to be 0.9962 and exhibited good linearity between concentration and peak area. The absorbance maximum (λmax) of standard apigenin was obtained at 254nm and hence all the detections of apigenin were carried out at 254nm. The solvent system of toluene: ethyl acetate: formic acid: methanol (3:6:1.6:0.4) gave a good resolution of the standard apigenin and single band at Rf 0.86 was obtained. The method was found to be specific as the overlay spectrum (Figure 3) of standard apigenin spot and the sample spots were found to be similar.

Table 2: Proximate analysis and percentage of Apigenin content in Cardiospermum halicacabum leaves collected from 21 districts of Tamil Nadu

S. No	Place of collection	Moisture content (%w/w)	Extractive value (%w/v)	Ash value (g)	Apigenin (%w/w)
1	Ariyalur	2.41	2.62	12.41	9.733
2	Coimbatore	1.92	4.46	10.33	5.059
3	Tirunelveli	1.47	3.85	11.28	0.918
4	Cuddalore	3.38	9.13	10.34	10.074
5	Thanjavur	2.88	5.33	10.32	4.1090
6	Kanchipuram	0.99	8.14	9.75	9.167
7	Namakkal	0.96	4	9.22	7.377
8	Perambalur	0.98	13.17	9.58	2.884
9	Dharmapuri	1.96	2.10	7.43	3.309
10	Vellore	1.44	19.08	9.51	8.426
11	Trichy	1.45	2.40	11.86	8.690
12	Salem	1.49	7.83	9.63	3.921
13	Theni	0.95	8.60	10.14	9.166
14	Dindigul	1.43	1.29	10.59	9.323
15	Madurai	0.98	6.26	12.26	2.985
16	Villupuram	0.97	4.59	9.46	3.828
17	Tirupur	0.99	3.46	7.72	0.530
18	Erode	0.97	5.25	13.42	2.742
19	Karur	1.47	9.77	11.36	2.485
20	Pudukottai	1.94	8.52	10.11	2.946
21	Krishnagiri	1.99	15.08	11.45	1.681

Figure 1: HPTLC chromatogram at 254 nm for methanol leaf extracts of Cardiospermum halicacabum collected from 21 districts of Tamil Nadu.

Figure 2. Densitogram of apigenin and MLECH collected from Cuddalore Figure 3. Overlay spectra of apigenin and MLECH collected in 21 districts

DISCUSSION:

The present study was undertaken as there is no report on the variation of apigenin content for the Cardiospermum halicacabum plants collected from different regions of Tamil Nadu. Developed chromatograms revealed that active major constituent apigenin was well resolved from the methanol leaf extracts of Cardiospermum halicacabum collected from 21 districts of Tamil Nadu. R_f values are in the range of 0.80 to 0.89. The variation in content of apigenin may be due to the variation in the geographical and climatic conditions. From 21 samples collected from different districts of Tamil Nadu the maximum content of apigenin was found in the leaves of samples collected from Cuddalore, Ariyalur, Dindigul, Kanchipuram, and Theni. The study results also revealed that the plant samples collected in the months of February to April showed more apigenin content (10.074 % w/w to 9.167 % w/w) than plant samples collected in the months of January (0.530 % w/w to 3.921 % w/w). Thus the time of collection had influence in the contents which may be due to the seasonal/climatic changes and geographical features of the regions.

CONCLUSION:

A study was conducted for the first time to determine the quantity of apigenin in Cardiospermum halicacabum in 21 districts of Tamil Nadu. Variation of apigenin content in Cardiospermum halicacabum plants collected from 21 districts of Tamil Nadu was estimated by developing a HPTLC method. Apigenin content in methanol leaf extracts of the plants collected from 21 districts of Tamil Nadu shown significant variations and proved the influence of geographical and seasonal variation in the content of phytoconstituents. Plants collected from Cuddalore, Ariyalur, Dindigul, Kanchipuram, Theni showed more apigenin content. Thus the study provided the scientific evidence for the plants collected in specific regions of Tamil Nadu to be used for the development of Ayurvedic, Siddha and herbal formulations. This quantification technique can also be used as an important quality control tool for the standardization of the herbal formulation containing this herb.

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Received on 03.03.2020 Modified on 14.04.2020

Research J. Pharm. and Tech. 2021; 14(5):2613-2618.

DOI: 10.52711/0974-360X.2021.00460