Flavonoid Separation from different extracts of aerial parts of Convolvulus arvensis by HPLC and Screening of Anthelmintic Activity

ManbirKaur Richa^{* *}Correspondence: ManbirKaur Richa, Department of Pharmacy, Global Group of Institutes, Amritsar, India, Email:

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Introduction

Convolvulus arvensis is a weed (Whitson TD, et al., 2006) and it is found in 32 different crops in 54 countries (Holm LG, et al., 1977). Convolvulus arvensis is native to Europe and Asia, and it grows in temperate, tropical and Mediterranean climates (Lyons KE, 1998; Gubanov I, et al., 2004).

A perennial vine (0.4-2 inches in height), field bindweed has deep, persistent roots that spread widely. Possibly reflecting soil moisture and fertility variability, this species has extremely variable leaves. Petals are generally white to very pale pink. This plant has whitish roots and fleshy, cord-like rhizomes. Taproots form from the primary root, which can penetrate soil to depths of 2-10 feet. At the stem base, adventitious buds and taproot buds form lateral roots. (Arora M and Malhotra M, 2011; Mehrafarin A, et al., 2009).

According to an analysis of its phytochemical constituents, Convolvulus arvensis contains alkaloids, phenolic compounds, flavonoids, sugars, carbohydrates, mucilage, sterols, tannins, unsaturated sterols, triterpenes, lactones, and proteins (Krzaczek T, et al., 2004; Hilal SH, et al., 1983).

Historically, the plant has been used as a medicine since the 1730s. Convolvulus arvensis aerial parts were used as laxatives, wound healers, anti-spasmodic, anti-haemorrhagic, antiangiogenetic, and for treatment of parasites and jaundice (Alkofahi A, et al., 1996). Additionally, it was used to treat skin disorders such as furunculosis, dandruff, and spider bites (Leporatti ML, et al., 2003). In addition, C. arvensis was traditionally used for pain relief, inflammation, and swelling in coughs and flu (Ali M, et al., 2013).

Materials and Methods

Plant materials

The aerial parts of the plant were collected from the waste land of Amritsar region in the month of October-November and authenticated by Dr. B.K. Kapahi, Taxonomist, Department of Botany, IIIM, Jammu. A voucher specimen was retained and deposited at the crude drug repository of the herbarium of IIIM, Jammu. (Vide CDR accession No. 21583).

Preparation of extracts

The collected aerial parts were shade dried, coarsely powdered and the powder was exhaustively extracted with both ethanol (95%), ethanol (50%) separately using percolator for 16 hours. The extract was drained, filtered and solvent was removed using rotary evaporator. Moreover the hydro alcoholic was lyophilized. To prepare the stock solutions, 20 mg of each extract was dissolved in respective concentration of Dimethyl Sulfoxide (DMSO) and 100 µl Roswell Park Memorial Institute (RPMI)-1640 media was added. pH was adjusted to 7.2, penicillin was added and sterilized by filtering through 0.2 µ microfilter and kept in refrigerator (2°C-8°C).

Standards and chemicals

HPLC grade methanol and other chemicals (Acetonitrile) of analytical reagent grade were procured from Merck. The authentic standards of the studied flavonoids were taken from of IIIM, Jammu.

Development of protocol for HPLC method using the extracts of Convolvulus arvensis

Preparation of standards: 1.2 mg of each standard (rutin, kaempferol, quercetin and quercitrin) was taken in 5 ml of methanol (HPLC grade). From which 5, 10, 15, 20, 25 μl were injected in HPLC system for making standard curve.

Preparation of extract solution: 20 mg of dry alcoholic, hydro-alcoholic and aqueous extract was dissolved in 10 ml extraction solvent (HPLC grade) to get 2 mg/ml solution, centrifuged and filtered through 0.45 μm and was injected to HPLC (water) system.

Quantification

The compounds exhibited linear responses in the calibration curves, which were prepared by using the multipoint calibration curve method. Working solutions as such and after mixing were injected in different amounts. Calibration curves were obtained for rutin, quercetin and kaempferol. Calibration curves were determined on the basis of fine amount of each standard.

Calibration plots

Calibration plots were prepared in order to find out the range of marker concentration, which shows a linear relation with respect to response of analytical technique. Once this range of response was established, concentrations of all test samples were so adjusted as to give the established linear range (Figure 1).

Figure 1: Calibration plots (A): Quercetrin; (B): Kaempferol and (C): Rutin

Standardization and calculation

The chromatograms were acquired using the analyst software and the data was processed by peak area method. The unknown concentration of the samples were calculated from the following equation using regression analysis of spiked calibration standard with the reciprocal of the ratio of the extract to the Internal Standard (IS), concentration as a weighing factor (1/ concentration²)

y=mx+b

Where, y is peak area ratio of the extract over IS, m is slope of the calibration curve, x is concentration ratio of extract over IS, b is y-axis intercept of the calibration curve

Anthelmintic activity

While studying the literature review of plant it was found that ethanolic extract of plant possess various pharmacological activities and most of its chemical constituents are soluble in ethanol. So, ethanolic extract of Convolvulus arvensis was selected for the activity and percentage yield of that extract was found to be 18.6%. Further the prepared ethanol extract was evaluated by anthelmintic activity against fetida worms (diseases/infections caused by helminths) at three concentrations (50, 100 and 150 mg/ ml) (Figure 2). The study showed that ethanolic extract of Convolvulus arvensis was effective as anthelmintic. Results interpret dose dependent reduced motility of worms with reference to the standard (Albendazole). Therefore, current investigation leads to conclusion that the roots of Convolvulus arvensis have potent anthelmintic activity when compared with the conventionally used drugs and hence can be treatment of helminthic infections.

Figure 2: Graph for anthelmintic activity of ethanolic extract of Convolvulus arvensis

Results and Discussion

The RP-HPLC behavior of the flavonoids on the reversed phase column was tested sequentially varying the proportion of the acetonitrile-water elution mixture. The separation was achieved by modifying the mobile phase with small amount of trifluoroacetic acid. HPLC fingerprints of alcoholic extract of Convolvulus arvensis given in Figure 3and their retention times and amounts were calculated as shown in Table 1.

Table 1: Retention time and amount calculated of all standards

Figure 3: Retention time of standards rutin, kaempferol, quercetrin and quercetin

Results of HPLC analysis of Convolvulus arvensis alcoholic, hydroalcoholic and aqueous extract at 254 nm had shown the presence of abundant constituents as evidenced by chromatogram obtained at various retention times shown in Figures 4, 5 and 6respectively. Kaempferol had shown the highest retention time followed by quercetin. These constituents detected were present in ng amounts. Rutin was present in very high amount as compared to other constituents. The retention time of standards rutin, quercetrin, quercetin and kaempferol appeared at 17.644, 24.471, 38.193 and 39.533 as shown in Table 1.

Figure 4: Chromatogram for the alcoholic extract of Convolvulus arvensis

Figure 5: Chromatogram for 50% alcoholic extract of Convolvulus arvensis

Figure 6: Chromatogram for the aqueous extract of Convolvulus arvensis

On the basis of quantitative HPLC for flavonoids content (Table 2), it was found that maximum percentage of flavonoids was present in alcoholic and aqueous extracts as compared to 50% hydro-alcoholic of aerial parts of the Convolvulus arvensis. On the basis of above experiment performed, the ethanolic extract of Convolvulus arvensis with a concentration of 50 mg/ml shows significant anthelmintic activity (Table 3).

Table 2: Percentage amount of some flavonoids present in Convolvulus arvensis extract

Table 3: Anthelmintic potency of ethanolic extract of Convolvulus arvensis

Conclusion

Based on HPLC studies of different extracts of C. arvensis, it is concluded that rutin is present in higher concentration in all extracts, although other flavonoids like kaemferol, quercetin and quercetrin are also present in extracts. These flavonoids are responsible for various therapeutic activities of the plant. The ethanolic extract of this plant exhibits promising anthelmintic activity, which can treat infections of animals with parasitic worms.

References

1. Whitson TD, Larry C, Burrill, Steven A, Dewey, David W, et al. Weeds of the West, 9th ed. 2006: 630.
2. Holm LG, Plucknett DL, Pancho JV, Herberger JP. The world's worst weeds. Distribution and biology. University Press of Hawaii. 1977.
3. Lyons KE. Element stewardship abstract for Convolvulus arvensis L. Field bindweed. Arlington: The Nature conservancy. 1998: 1-21.

   [Google Scholar]

4. Gubanov I. An Illustrated identification book of the plants of Middle Russia: Angiosperms (dicots: archichlamydeae). Moscow: Institute of Technological Researches, 2004; 3: 520.
5. Arora M, Malhotra M. A review on macroscopical, phytochemical and biological studies of Convulvulus arvensis (Field Bindweed). Pharmacologyonline. 2011; 3: 1296-1305.

   [Google Scholar]

6. Mehrafarin A, Meighani F, Baghestani MA, Mirhadi MJ, Labbafi MR. Investigation of morphophysiological variation in field bindweed (Convolvulus arvensis L.) populations of Karaj, Varamin, and Damavand in Iran. Afr J Plant Sci. 2009; 3(4): 064-73.

   [Google Scholar]

7. Krzaczek T, Gogucka-Kocka A, Ryn D. Chromatographical analysis of phenolic compounds in herb Convolvulus arvensis L. Herba Pol. 2004;3(50):1-5.

   [Google Scholar]

8. Hilal SH. Phytochemical study and biological screening of Convoivulus lanatus vabl. Egypt Pharm J. 1983;24(3-4):139-148.

   [Google Scholar]

9. Alkofahi A, Batshoun R, Owais W, Najib N. Biological activity of some Jordanian medicinal plant extracts. Fitoterapia (Milano). 1996; 67(5): 435-442.

   [Google Scholar]

10. Leporatti ML, Ivancheva S. Preliminary comparative analysis of medicinal plants used in the traditional medicine of Bulgaria and Italy. J Ethnopharmacol. 2003; 87(2-3): 123-142.

    [Crossref] [Google Scholar] [Pubmed]

11. Ali M, Qadir MI, Saleem M, Janbaz KH, Gul H, Hussain L, et al. Hepatoprotective potential of Convolvulus arvensis against paracetamol-induced hepatotoxicity. Bangladesh J Pharmacol. 2013; 8(3): 300-304.

    [Crossref] [Google Scholar]