HPTLC Fingerprinting of Quercetin and comparative assessment of Anthelmintic potency against Eudrilus eugeniae of Schleichera oleosa (Lour) Oken and Tagetes eretca Linn

Shambaditya Goswami¹*, Ravindra Pal Singh², Ritu M Gilhotra³

¹Research Scholar, Suresh Gyan Vihar University, Jaipur, Rajasthan.

²NIMS Institute of Pharmacy, NIMS University, Rajasthan, Jaipur.

³School of Pharmacy, Suresh Gyan Vihar University, Jaipur, Rajasthan.

*Corresponding Author E-mail: shambampharma@gmail.com

ABSTRACT:

Helminthiasis is a great burden in current era as it causes enormous health threats. Schleichera oleosa (Lour.) Oken and Tagetes erecata Linn are the medicinal plants which are traditionally used for the treatment of helminthiasis. They are commonly known as ‘Kusum’ and ‘Genda’ respectively. The present research focused on the evaluations of anthelmintic potency of both the plant extracts against an earthworm, Eudrilus eugeniae. The successive extraction method with different solvents (petroleum ether, chloroform, acetone, ethanol and water) is used to prepare the extracts. The extracts were evaluated for the anthelmintic potency at 25, 50 and 100 mg/ml concentrations. Albendazole was used as standard drug. All the solutions were prepared in 3% tween 80 in normal saline solution and alone this solution was considered as control. All the earthworms (n=6) were taken in separate petri-dishes containing extracts, standard and control solution. Paralysis and death time were noted as result in minutes. Standard HPTLC technique (using CAMAG LINOMAT 5) was used to isolate and identify of quercetin from the ethanolic extracts of both the plants. High extractive yields were observed for ethanolic extracts in both the plants. Significant outcome was observed for the evaluation of anthelmintic activity for all the extracts. S. oleosa ethanolic extract showed maximum potency than T. erecta extracts at all concentrations. Dose responsive results were noted. At 100 mg/ml concentration ethanolic extracts of both plants were significant against E. eugeniae. Quercetin was identified in both ethanolic extracts by HPTLC analysis. Structural elucidation of the quercetin and identification of other constituents should be the future prospect.

KEYWORDS: Helminthiasis, Schleichera oleosa (Lour.) Oken, Tagetes erecata Linn, Eudrilus eugeniae, anthelmintic potency, HPTLC, quercetin.

INTRODUCTION:

The use of herbal and traditional plants to treat different ailments is a history of thousand years¹. The majority of the populations of rural area rely on traditional treatments because of the plenty of availability of herbal medicinal plants in India. The interest towards the use of herbal plants is growing more and more in the world².

Schleichera oleosa (Lour.) Oken is commonly known as “Kusum” or “Lac Tree” which belongs to Sapindaceae family. In India West Bengal, Orissa, Madhya Pradesh and sub-Himalayan range are the main habitat of this plant. The expansive biological actions were reported for the plant like anti-inflammatory³, anticancer⁴, antimicrobial⁵ and antioxidant⁶ activities. The plant was evaluated against several earthworms and nematodes earlier for the support of its folkloric practice⁷.

Tagetes erecta Linn, belongs to Asteraceae family, is a common plant and known as “Marigold” or “Genda” and available in almost all parts of South East Asian region. The researchers significantly proved this decorative plant as a versatile therapeutic agent used in many diseases. The plant was studied earlier for analgesic,antioxidant⁸, anthelmintic⁹, antimicrobial¹⁰ and many more.

Helminthiasis is very common in India which affects children more frequently than adults¹¹. Open defecation, un-hygienic conditions, lacking of awareness among the rural people make the condition very severe in the tropical areas. The effect of worm infection is not only restricted to gastro intestinal disturbances, but also it can cause filariasis, schistosomiasis, ascariasis, nutritional status alteration, anaemia etc¹². Ayurveda uses the term “krimiroga” for helminthiasis. The use of herbal plants in Ayurveda to treat “udarakrimi” (intestinal worms) was mentioned in previous literature¹¹. The synthetic drug treatment has limited use due to their side effects^[9], and anthelmintic resistance¹³.

Eudrilus eugeniae is an earthworm, commonly known as “African Nightcrawler” and belongs to the family eudrilidae. This reddish-brown colour worm is composed of state and prostomium is distinct at the anterior part. At the 8-12^th segments the spermathecal opening is observed. In female worm, the pair of ovaries is present at 13^th segment¹⁴.

As, anthelmintic evaluation of the extracts of both the plants against E. eugeniae have not been scientifically reported, so, the present research is aimed for the comparative in-vitro evaluation of anthelmintic potency of S. oleosa and T. erecta against E. eugeniae along with the high-performance thin layer chromatography (HPTLC) fingerprinting of quercetin from the ethanolic extracts of both the plants.

MATERIALS AND METHODS:

Plant Material collection and authentication:

Schleichera oleosa (Lour.) Oken (Voucher No: 02/2017) was collected from the forest area of Nichlaul (District- Maharajganj, Uttar Pradesh) and the collection of Tagetes erecta Linn (Voucher No: 03/2017) was done from Gorakhpur Industrial Development Authority (GIDA, District- Gorakhpur, Uttar Pradesh) area. Both the plants were authenticated and identified by Indian Council of Agricultural Research, Kamla Nehru Vigyan Kendra, Sultanpur, Uttar Pradesh, India.

Plant Extraction:

The fresh plants were shade dried and grounded. The plants powders were subjected for the successive extraction process with the solvents of increasing polarity. The extraction with petroleum ether, chloroform, acetone and ethanol were performed with continuous hot percolation process, whereas, the aqueous extracts of both the plants were prepared by cold maceration process for 3 days followed by filtration using muslin cloth. The different extracts of the plant Schleichera oleosa (Lour.) Oken were mentioned as SOPE, SOCE, SOAcE, SOEE and SOAE and similarly for the plant Tagetes erectaLinn the extracts were named as TEPE, TECE, TEAcE, TEEE and TEAE.

Collection of worms:

Eudrilus eugeniae (Voucher No: 09-Zoo-17) of nearly equal sizes were collected in the nearby area of GIDA (Sector-7) and the worms were cleaned to remove dirt and kept in saline water. The worms were authenticated by the department of zoology, H.R.P.G. College, Uttar Pradesh.

Anthelmintic potency against Eudrilus eugeniae:

The in-vitro anthelmintic activity was evaluated for both the plant extracts separately as per the recommended procedures¹⁵^,¹⁶. The earthworm, Eudrilus eugeniae, was selected for the evaluation for their anatomical and physiological resemblance¹⁷ with the intestinal worms. The three following groups were selected for the study design.

Group I: 3% Tween 80 in normal saline solution was taken as control treatment.

Group II: Albendazole, standard treatment, was diluted in Tween 80 (3%) in normal saline solution to different concentrations (25, 50 and 100 mg/ml).

Group III: All the extracts were dissolved in Tween 80 (3%) in normal saline solution to produce 25, 50 and 100 mg/ml concentration.

For the experiment, the worms were taken almost in equal sizes (n=6) in each petri-dishes in a group of 6 (n=6). Observations were made for the time taken for paralysis and death of the worms. Paralysis time (PT) was the time when the worms lost their motility even after vigorous shaking and death time (DT) was the time when there was no movement of the worms even after dipping in hot water (50ᵒC)⁹. All the observations were noted in minutes.

Statistical analysis of anthelmintic activity:

One-way ANOVA was performed to analyze the statistical data in Graph Pad Prism 7.4 (n=6) and p<0.05wasconsidered as significant. All values were expressed as ± SEM of the mean. The graph was prepared in Origin 6.1.

Isolation of quercetin by HPTLC of the SOEE and TEEE:

CAMAG LINOMAT 5 instrument (Camag; Muttenz, Switzerland, WINCATS software) was used for the fingerprinting analysis. The fingerprinting was evaluated according to the standard procedures¹⁸^,¹⁹. The ethanolic extracts of both the plants (100 mg of each) were dissolved in 1 ml of ethanol (HPTLC grade). The mobile phase was selected as toluene, ethyl acetate and formic acid in the ratio of 13:11:2. The samples and standard were applied automatically by LINOMAT 5 with 100µl syringe on Silica gel 60F₂₅₄ TLC plates after the saturation of twin trough TLC chamber.

Figure 1: Percentage yield of the extracts of S. oleosa and T. erecta. PE: Petroleum ether extract, CE: Chloroform extract, AcE: Acetone extract, EE: Ethanol extract and AE: Aqueous extract.

The plates were kept in the chamber. The chromatogram was developed in the chamber. CAMAG Reprostar 3 captured the images of the plate at different wavelengths (254 and 366 nm). Densitometric scanning was done by CAMAG TLC Scanner 3.

RESULTS:

The percentage yield of the various extracts of both the plants was evaluated. The highest yield was observed for SOEE (14.3% w/w), SOAE (12.2 % w/w), TEEE (12.1 % w/w) and TEAE (10.1 % w/w). The comparative overview was displayed in figure 1.

The in-vitro evaluations of all the extracts were determined as per the recommended procedure against the earthworms. The effect of S. oleosa extracts was given in Table 1, and the effect of T. erecta was tabulated in Table 2. The results were mentioned in minute and paralysis along with death time were noted which was observed in dose dependent fashion. Among all the extracts of S. oleosa, SEEE (DT 70.21±3.21 min) and SOAE (DT 92.34±4.1 min) were found more potent than other extracts.

Table 1: Anthelmintic potency of Schleichera oleosa (Lour.) Oken on Eudrilus eugeniae.

	25mg/ml		50mg/ml		100mg/ml
	PT (min)	DT (min)	PT (min)	DT (min)	PT (min)	DT (min)
STD	60.2±3.1	82.54±3.22	53.1±4.1	66.2±2.4	42.21±2.47	55.1±3.3
SOPE	160.4±1.24*	180.1±2.2*	153.4±2.33*	180.24±3.1*	145.2±2.3*	173.24±4.1*
SOCE	154.17±3.1*	175.1±2.1*	143.4±3.8*	167.2±4.01*	144.4±1.4*	162.41±2.1*
SOAcE	150.2±1.2*	167.2±3.33*	142.01±1.2*	162.3±2.1*	140.21±3.01*	153.24±4.25*
SOEE	75.3±1.7**	92.22±2.5**	63.21±3.1**	78.4±3.4**	47.7±0.9***	70.21±3.21****
SEAE	92.14±2.77**	124.7±2.71**	80.47±3.4**	97.22±4.56**	69.21±1.77***	92.34±4.1***

All Values represent Mean± SEM; n=6 in each group; p<0.05 was considered significant.

Table 2: Anthelmintic potency of Tagetes erecta Linn on Eudrilus eugeniae.

	25mg/ml		50mg/ml		100mg/ml
	PT (min)	DT (min)	PT (min)	DT (min)	PT (min)	DT (min)
STD	60.2±3.1	82.54±3.22	53.1±4.1	66.2±2.4	42.21±2.47	55.1±3.3
TEPE	164.14±3.10*	209.14±1.28*	161.01±4.2*	193.5±2.13*	151.4±1.14*	190.04±3.1*
TECE	159.71±2.17*	181.21±2.7*	156.14±4.34*	173.51±1.47*	152.14±2.54*	169.06±2.7*
TEAcE	160.14±1.24*	181.1±3.1*	151.12±5.14*	176.4±1.31*	149.44±3.22*	171.1±0.7*
TEEE	81.12±2.01**	112.22±3.08**	70.17±1.7**	91.11±3.74**	63.12±3.74***	81.24±1.4****
TEAE	98.04±2.4**	139.07±3.47**	89.04±2.4**	121.21±3.87**	76.11±0.05***	101.15±2.14***

All Values represent Mean± SEM; n=6 in each group; p<0.05 was considered significant.

In similar fashion, the effect of TEEE (DT 81.24±1.4 min) and TEAE (DT 101.15±2.14 min) was more significant than other extracts of T. erecta. Moreover, at high dose (100 mg/ml) the effect was more prominent than the lower doses (25 and 50 mg/ml). Standard drug (albendazole) was effective in all doses against the worms, at 100 mg/ml the DT was noted as 55.1±3.3 min.

The order of potency of each extracts of S. oleosa was SOPE<SOCE<SOAcE<SOAE<SOEE in this fashion, whereas, in case of T. erecta the order of activity was found as TEPE<TEAcE<TECE<TEAE<TEEE.

The comparison of DT of ethanolic extracts (SOEE and TEEE) of both the plants at different doses was given in the figure 2.

Figure 2: Comparative anthelmintic activity of ethanolic extracts of S. oleosa and T. erecta at 25, 50 and 100 mg/ml. One way ANOVA was performed, p<0.05 was considered significant (n=6).

HPTLC chromatogram of SOEE and TEEE were evaluated with appropriate solvent system. The chromatogram of SOEE, TEEE and standard quercetin were given in figure 3 and 4.

Figure 3: HPTLC chromatogram A) SOEE; B) TEEE; C) Quercetin at 254 nm.

Figure 4: HPTLC chromatogram A) SOEE; B) TEEE; C) Quercetin at 366 nm.

In the densitogram 12 bands were found in SOEE in which 4^th band was denoted as quercetin, whereas, out of 15 bands in TEEE, 6^th band was found as quercetin while compared with the standard. All the densitograms were given in figure 5, 6 and 7. The details of R_f values were tabulated in table 3.

Figure 5: HPTLC desitogram of SOEE.

Figure 6: HPTLC desitogram of TEEE.

Figure 7: HPTLC desitogram of standard quercetin.

Table 3: HPTLC parameters of SOEE, TEEE and standard quercetin.

Track name	Peak No	Start position (Rf)	Max position (Rf)	End position (Rf)	Start Height (AU)	Max Height (AU)	Max %	Area %	Assigned substance
SOEE	4	0.46	0.48	0.49	97.9	113.8	3.66	2.43	Quercetin
TEEE	6	0.44	0.45	0.46	178.3	179.1	3.62	1.83	Quercetin
Standard	1	0.41	0.49	0.51	6.0	700.4	100	100	Quercetin standard

DISCUSSION:

The widespread problem of current era in the developing countries which causes morbidity is soil transmitted helminthiasis (STH). WHO has considered this as a neglected tropical disease (NTD)²⁰ and it has also been reported that required preventive chemotherapy (PC) for the pre-school age children (Pre-SAC) and school age children (SAC) is more than 225 million in 2017 which rises from 220 million from 2014. The situation of the disease is also alarming in South East Region (SER), where the number for required PC gets a hike from 354 million (2014) to 370 million (2017)²¹. Albendazole and mebendazole are the recommended treatment for the de-worming programme. But these drugs, upon prolonged use, have several side effects like elevation level in hepatic enzyme and bone marrow depression⁹.

The importance of herbal drugs in the treatment of helminthiasis is an alternative approach and a long practice. In the present study, the anthelmintic potency of the plants S. oleosa and T. erecta extracts were evaluated against E. eugeniae. Both ethanolic extracts (SOEE and TEEE) were represented as significant and among them S. oleosa was established as more potent candidate than Tagetes erecta Linn extracts. All the responses were recorded as dose dependent fashion. In the previous literature, both the plants were evaluated against earthworms. S. oleosa was reported as potent anthelmintic against Pheritima posthum, Eisenia fetida and Perionyx excavates and among all the extracts ethanolic showed the maximum inhibition at 100 mg/ml concentration⁷. Tagetes species were also evaluated earlier against earthworms. Methanolic extract of the bark of T. erecta Linn was reported as potent anthelmintic at 300 mg/ml²². T. erecta ethanolic and aqueous extracts were earlier evaluated against earthworms and nematodes, in which dose dependent response was observed and ethanolic extract was reported as more significant than others⁹.

Quercetin is a type of flavonoids which have enormous biological actions²³. It causes energy generation process in earthworms resulting in paralysis and death²⁴. Earlier study reported the effect of quercetin as a potent anthelmintic²⁵. HPTLC is an accurate and precise chromatographic technique which helps to isolate and identify the plant constituents from the plant extracts. This modern technique can be utilized as a quality control parameter for the crude extracts²⁶. The present study isolated and identified quercetin by HPTLC in both plant ethanolic extracts.

Hence, both the plants can be considered as an alternative treatment of helminthiasis and the study can be extended by isolating other constituents like alkaloids and tannins which are also associated with anthelmintic activity. The underlying mechanism at molecular level should be the future interest for the researchers.

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Received on 19.11.2019 Modified on 27.01.2020

Research J. Pharm. and Tech. 2020; 13(12):5699-5704.

DOI: 10.5958/0974-360X.2020.00992.0