Pulicaria laciniata (Coss and Kral): Effects of Different Extraction Solvents on Phytochemical Screening and Antioxidant Activity

Kamilia Bireche*, Hocine Dendougui, Asma Abid, Abdeldjabbar Messaoudi, Mohamed Hadjadj

University of Kasdi Merbah, Faculty of Mathematics and Material Sciences, Department of Chemistry, Valorization and Promotion of Saharan Resources (VPRS) Laboratory, 30 000 Ouargla, Algeria.

*Corresponding Author E-mail: kamiliab053@gmail.com

ABSTRACT:

This study aims to investigate phytochemical constituents of Pulicaria laciniata extracts and determine their antioxidant activity using three methods; Phosphomolybdate, Reducing Power, and Metal Chelating. The phytochemical investigation showed various secondary metabolites such as Phenols, Glycosides, Flavonoids, Alkaloids, Tannins, and Terpenoids.  The N-butanol extract exhibited the highest antioxidant activity comparing with the other extract in all methods (0.51 and 0.65 mg/ml as A0.5 values of Phosphomolybdate, reducing power) and (1.65mg/ml for IC50 value of metal-chelating). In contrast, all the extracts showed week activity against the metal-chelating method.

KEYWORDS: Pulicaria laciniata, Antioxidant, Phytochemical screening, Secondary metabolites, Extractions.

1. INTRODUCTION:

Recently, worldwide is exciting about natural products that we use in many domains, such as medical and pharmaceutical. We can find these products; one of the choices is the medicinal plants used in traditional to treat many diseases naturally. Our study chose a local plant named Pulicaria Laciniata (Coss. and Kral.)¹. Pulicaria laciniata belong to the Asteraceae family, which is one of the most important families of angiosperms. It designates a family of compounds belonging to dicotyledons comprising more than 1500 genera and more than 25000 species described, including 750 endemics. In Algeria, this family has about 109 genera and more than 408 species². The Pulicaria species are used in traditional medicine, such as Pulicaria crispa used as diuretic³, to treat inflammations, against insects⁴, Wash hemorrhoids, and treat vaginal cancer⁵. Pulicaria odora L used against inflammation⁶, back pain, intestinal disorder, menstrual cramps⁶. Pulicaria dysenterica used as a treatment of dysentery⁷, an antidiarrheal agent⁸, Pulicaria mauritanica is used to treat different inflammation disorders⁹. also as antihistaminic¹⁰. In our study investigate the phytochemical fingerprint and Phytochemicals screening using different tests. Also, it will evaluate the antioxidant activity of Pulicaria laciniata different extracts using three different methods.

2. MATERIALS AND METHODS:

2.1. Chemicals

Chemicals and reagents of analytical grade used in this experiment were Hexane, Ethyl acetate, Chloroform, Butanol, Methanol, Petroleum ether, Trolox, Gallic acid (GAE), Quercetin (Q.E.), Catechin (C.E.), Ascorbic Acid (A.A.), Sulfuric acid, Hydrochloric Acid, and Iron (III) chloride were purchased from Sigma-Aldrich Company, Algeria.

2.2. Plant material

The aerial parts of Pulicaria laciniata were collected in April 2016 from Zelfana Ghardaia, Algeria. The plants were identified and authenticated (201604zel/pullac) by Dr. Iddoude, Faculty of Science of Nature and Life, Department of Biological Sciences, Kasdi Merbah University, Ouargla, Algeria. The fresh aerial parts were cut into small pieces and were air-dried at room temperature for 20 days. The final weight was 3 kg.

2.3. Sample preparation

The dried and powdered plants were macerated with Petroleum ether for 24 hours. After the filtration, the plants left to dry at room temperature, and then the plant macerated again with a mixture of methanol: water (7:3) for 48h at room temperature three times. The extract obtained after the filtration concentrated under pressure to get a very concentrated extract that presents the crud extract. The crud extract was extracted successively with different organic polarity solvents to produce petroleum-ether, chloroform, ethyl acetate, and n-butanol extracts (Figure 1).

Figure 1. Extraction protocol

2.4. The phytochemical fingerprint

The phytochemical fingerprint profiles of the different extracts were performed by vanillin solution, methanolic KOH solution (5% m/v), and Lieberman-Bürchard ^11,12.

2.5. Screening of Phytochemicals

The phytochemical fingerprint profiles were followed using several phytochemical analyses described by Boulenouar ^13-16.

Phenols Test: A mixture of a small amount of the extract was placed in a test tube with 1 ml of water and one to two drops of Iron (III) chloride. Blue, green, red, or purple color formation indicates the presence of phenols.

Glycosides Test: An extract was mixed with 1 ml of water and a few drops of aqueous sodium hydroxide. Yellow coloration indicates the presence of glycosides.

Flavonoids Test: an amount of extracts was mixed with five drops of concentrated hydrochloric acid. Primary production of red color indicates the presence of flavonoids.

Alkaloids Test: 1ml of the extracts was added in a test tube with 0.2 ml of diluted hydrochloric acid. After a few seconds, 1 ml of dragendorff’s reagent was mixed. The appearance of white precipitation indicated the presence of alkaloids.

Tannin Test: Five ml of each extract was placed in a test tube, and then added 2 ml of iron (III) chloride (5% w/v) solution. A greenish-black coloration means the presence of tannins existence.

Terpenoids Test: A mixture of 0.5 ml of an extract with 2 ml of chloroform and 3 ml of concentrated sulfuric acid giving the brown-reddish interface coloration indicates the presence of terpenoids.

2.6. Antioxidants activities

2.6.1. Metal ions Chelating

The samples were (0.5 mL) combined with two milliliters of 0.1 M sodium-acetate-pH 4.9 buffer and 50 μL of 2 mM iron (II) chloride. 0.2 mL of 5 mM ferrozine had been added after a 30 min incubation at room temperature then the absorption was recorded at 562 nm. As control and standard, distilled water and EDTA have been utilized. The percentage prevention of complex formation of ferrozine has been estimated as [(A0-A1)/A0 ×100] where A0 was the control absorbent, and A1 was the extract/EDTA absorption. The results were expressed as IC50 ¹⁷ with, certain adjustments.

2.6.2. Phosphomolybdate

The procedure is to add 200 μl of extracts with 2ml of a reagent consisting of H2SO4 (0.6 m), Na2PO4 (28 mM), and ammonium molybdate (4 mM). after 90 minutes of incubation at 95°C. The absorbance was measured at 695 nm after it has been cooled. The control consists of methanol instead of extracts ¹⁸. The results were given as A0.5 (mg/ml), corresponding to the concentration indicating 50% absorbance intensity.

2.6.3. Ferric reducing power ability

FRAP reagent was prepared by mixing sodium acetate buffer (300 mM, pH 3.6), 10 mM TPTZ solution in 40 mM HCl and 20 mM FeCl₃. 200 μl of each extract were added to 3 ml of FRAP reagent. After incubation in the dark at 37 ° C for 30 minutes, the absorbance was measured at 593 nm against the blank^{19, 20}.

2.7. Statistical analysis

All the experiments were carried out at least triplicate and presented as mean ± standard deviation.

3. RESULTS AND DISCUSSIONS:

3.1. Phytochemical fingerprint

The results of the phytochemical screening by thin-layer chromatography (TLC) of the extracts are indicated in Figure 2. Orange, yellow, blue, green, and purple spots were observed on the chromatogram under UV 254 and 365 nm corresponding to the several classes of secondary metabolites. To specify the nature of these compounds, the chromatograms were developed in different solvent systems such as hexane/ethyl acetate 1/0.1 (v/v). For the chloroform extract, chloroform/ethyl acetate/ methanol 2/0.5/0.250 (v/v/v). For the ethyl acetate extract and chloroform/ methanol/ water 40/9/1 (v/v/v) for the n-butanol extract and revealed with specific reagents using Libermann-Bürchard reagent for sterols and terpenes, potassium hydroxide, and vanillin solution for coumarin. The chromatograms were used to purify chemical compounds to establish phytochemical sample fingerprints. Figure 2 showed that the structural diversity of compounds reacted the chromatograms indicated various secondary metabolites found in different extracts. The sterols generally beamed fluoresce in blue, yellow, and green under U.V./366 nm while terpenes in blue, yellow, green, and purple. If the spots have an invisible orange-yellow fluorescence after spraying Libermann-Bürchard reagent on the chromatogram, that’s indicated the presence of lupine-type triterpenes. Suppose the spots colored with red, oleanane-and ursane type triterpenes exist ²¹. The majority of coumarins fluorescent under UV at 366 nm in blue, purple, pink, green, yellow, purple colors. Some coumarins are colored with yellow invisibly due to their structure based on daphnetin, and their color intensifies after treatment with a methanolic solution of KOH (5%, m/v)²¹. Depending on this previous research, our extract certainly contains lupane-type triterpenes and coumarins. Also, other active compounds react with the vanillin solution which could be sterols according to (Kotze M. Eloff JN. Houghton PJ)²². More analyses are needed to identify the rest of other active substances needed to be determined.

Figure 2 Phytochemical fingerprint Chromatograms showing separated compounds in the extracts. The chromatograms were developed in the different solvents.

3.2. Phytochemical screening

Various phytoonstituents were screened in (Table1), showing the different subsequent pharmacological in the extracts.

Table 1. Phytochemical screening of the extracts.

	N-butanol	Ethyl Acetate	Chloroform
Phenols	+	+	+
Glycosides	++	+	-
Flavonoids	++	++	+
Alkaloids	+	++	++
Tannins	+	+	+
Terpenoids	-	+	+++
(+) = positive, (-) = negative

3.3. Antioxidants activities

The Antioxidants activities were evaluated using three different methods where the results were summarized in table 2.

Chelation of metal ions are necessary for the functioning of processes biochemical and physiological cellular, but in some cases and when their mechanism action is not well controlled, these same ions can be the cause of peroxidation lipid, oxidative stress, or tissue injury, as an example Cu²⁺ is a stimulator peroxidation of Light density lipoproteins (LDL)²³. The Fe2+chelating activity was determined by measuring the Fe²⁺ formation of the ferrozine complex. Although iron is essential for oxygen transport, respiration, and enzyme activity, it is a reactive metal that catalyzes oxidative damage in living tissues and cells ²⁴. Ferrozine can quantitatively form complexes with Fe2+. In the presence of chelating agents, the complex formation is disrupted, resulting in a decrease in the blue color of the complex. However, those extracts exhibited the following order: EDTA > ethyl extract > n-butanol > chloroform. None of the extracts appeared to better chelators of iron (II) ions than the positive control. These results suggested that our extracts are very weak ferrous chelators. The Phosphomolybdate (PPM) is based on the reduction of molybdate Mo (VI) to molybdate Mo (V) in the presence of the extract or an agent antioxidant. This reduction is materialized by forming a greenish complex (phosphate /Mo (V) at acidic pH)¹⁷. The increase in the color of the molybdate (VI) complex in the presence of antioxidants. Unlike other tests, this test quantifies the antioxidant activity of polyphenols and other antioxidant compounds such as vitamins (C, E). These extracts showed exceptional values at the absorbance of 0.5 nm, where the ethyl acetate exhibited the highest activity with A_0.5 0.48±0.09mg/ml, followed by the n-butanol extract than chloroform extract.

While the Ferric Reducing Ability Power (FRAP) based on (Fe³⁺to Fe²⁺) increased with the concentration of the extracts, the ferric reducing ability (FRAP) is a novel method for assessing antioxidant activity, in this ferric reduce to ferrous ion at low pH caused the formation of ferrous complex ²⁵. In the present study, ferric ion (Yellow) is changed into a ferric-tripyridyl triazine complex (green colored) during the reducing activity. In the complex mixture, the absorption change was associated with the total reducing power20 the highest FRAP activity, also signifying richer flavonoids and phenolic compounds²⁶. As shown in Table 2, the n-butanol extract was the highest effective with A_0.5 equal to 0,65 ±0.01 mg/ml, close to the standard Trolox with A0.5 equal to 0.40±0.01. In our extract, the butanol and the ethyl acetate extracts showed a very approximate antioxidant activity in all methods compared with the Chloroform extract, which may be explained by containing the n-butanol and the ethyl acetate extracts more polar bioactive compounds. The antioxidant activity of Pulicaria laciniata extracts could be explained by the presence of active secondary metabolite compounds. The different behavior of these extracts on the antioxidant activity may be explained by the difference in composition of extracts regarding the quantity quality of active secondary metabolites. Various studies have stated that phenolic compounds exhibited remarkably antioxidant properties in vitro and vivo ^27,28.

Table 2. Antioxidants activities of Pulicaria laciniata extracts

Extract / standards	Metal Chelating  (IC50µg)	Reducing power (A0.5mg/ml)	Phosphomolybdate (A 0.5 mg/ml)
N-butanol	1,65 ± 0.44	0.65±0.06	0.51±0.02
Ethyl-acetate	1.40± 0.44	1.06±0.06	0.48±0.09
Chloroform	˃ 2	4.66±0.79	0.63±0.03
BHT	--	0.23±0.03	--
Trolox	--	0.40±0.01	--
Ascorbic acid	--	--	0.23±0.01
EDTA	0.002±0.0001	--	--
IC50 and A 0.50 values represent the means ± SEM of three parallel measurements .

4. CONCLUSION:

This preliminary study investigated the n-butanol, ethyl acetate, and chloroform extracts of Pulicaria laciniata. The obtained results of the screening and the fingerprint of Pulicaria laciniata organic extracts revealed the essential compounds of Phenols, Glycosides, Flavonoids, Alkaloids, Tannins, and Terpenoids, with some variabilities between butanol and ethyl acetate extracts. These extracts exhibit a considerable antioxidant activity in Phosphomolybdate and reducing power methods. These results suggest that the polar extracts from this endemic plant species from Algeria may play a therapeutic role with oxidative stress and suggest the potential application use of Pulicaria laciniata extracts in the field of pharmaceutical industries, in particular as oxidative inhibitory. However, preclinical and clinical studies along with phytochemical studies are required.

5. REFERENCES:

1. Ozenda P. Flora and vegetation of the Sahara. Paris. 1991.

2. Quezel P. Santa S. Nouvelle Flore de l’Algerie et Des Regions Desertiques Meridionales, Paris 1963.

3. Al-Saleh FS. Ali HH. Mirza, M. Chemical constituents of some medicinal plants growing in Bahrain. Fitoterapia-Milano-. 1993; 64: 251.

4. Stavri M. Mathew KT. Gordon A. Shnyder SD. Falconer RA. Gibbons S. 2008. Guaianolide sesquiterpenes from Pulicariacrispa (Forssk.) Oliv. Phytochemistry. 2008; 69: 1915–1918.

5. Watt JM. Breyer-Brandwijk MG. The Medicinal and Poisonous Plants of Southern and Eastern Africa being an Account of their Medicinal and other Uses, Chemical Composition, Pharmacological Effects and Toxicology in Man and Animal. Med. Poisonous Plants South. East. Africa being an Acc. their Med. Other Uses, Chem. Compos. Pharmacol. Eff. Toxicol. Man Anim. 1962.

6. Hanbali FEL. Akssira M. Ezoubeiri A. Mellouki F. Benherraf A. Blazquez AM. Boira H. Chemical composition and antibacterial activity of essential oil of Pulicaria odora L. J. Ethnopharmacol. 2005; 99: 399–401.

7. Williams CA. Harborne J.B. Greenham J. 2000. Geographical variation in the surface flavonoids of Pulicaria dysenterica. Biochem. Syst. Ecol. 2000; 28: 679–687.

8. Nickavar B. Mojab F. Antibacterial activity of Pulicaria dysenterica extracts. Fitoterapia. 2003; 74: 390–393.

9. Znini M. Cristofari G. Majidi L. Paolini J. Desjobert JM. Costa J. Essential oil composition and antifungal activity of Pulicaria mauritanica Coss., against postharvest phytopathogenic fungi in apples. LWT-Food Sci. Technol. 2013; 54: 564–569.

10. Mahfouz M. Ghazal A. El-Dakhakhny M. Ghoneim MT. Pharmacological studies on the active principle isolated from Pulicaria dysenterica. J. Drug Res. 1973; 5: 151–172.

11. S. C. Verma, S. Subhani, E. Vashishth, R.K. Tiwari, R. Singh, P. Pant, M. M. Padhi, K.S Dhiman. Comparative phytochemical study of root versus small branches of Desmodium gangeticum using High Performance Thin Layer Chromatographic UV detection Method. Asian J. Research Chem 8(4): April 2015; Page 318-323. doi: 10.5958/0974-4150.2015.00052.8

12. Ragya Eslavath, V. Harikrishna, N. Kosuru, Goli Venkateshwarlu, Manoranjan Sabat, Kanakaiah K.. Phytochemical Screening and TLC, UV-Spectrophotometer Study of Bougainvillea glabra. Asian J. Pharm. Ana. 3(3): July-Sept. 2013; Page 83-85. Boulenouar N. Marouf A. Cheriti A. Antifungal activity and phytochemical screening of extracts from Phoenix dactylifera L. cultivars. Nat. Prod. Res. 2011; 25: 1999–2002.

13. Batra Neha, Jain Honey, Bairwa Ranjan, Bachwani Mukesh. Pharmacognostical and preliminary phytochemical investigation of Acorus calamus linn. Asian J. Pharm. Res. 2(1): Jan.-Mar. 2012; Page 39-42.

14. Ch. Madhu, J. Swapna, K. Neelima , Monic V. Shah. A Comparative Evaluation of the Antioxidant Activity of Some Medicinal Plants Popularly Used in India. Asian J. Res. Pharm. Sci. 2(3): July-Sept. 2012; Page 98-100.

15. Valli G, Jeyalakshmi M.. Preliminary Phytochemical and Antioxidant Study of Odina woodier Leaf Extract. Asian J. Pharm. Res. 2(4): Oct. - Dec. 2012; Page 153-155.

16. Samydurai P , M. Saradha. Effects of Various Solvent on the Extraction of Antimicrobial, Antioxidant Phenolics from the Stem Bark of Decalepis hamiltonii Wight and Arn. Asian J. Res. Pharm. Sci. 2016; 6(2): 129-134. doi: 10.5958/2231-5659.2016.00018.7

17. Nirmala Shinde, R.A. Saindane, S.K. Bhosale, Shreenivas Deshpande. In Vitro Antioxidant Activity of Ethanolic and Chloroform Extracts of Tagetes erecta Flowers. Research J. Pharmacognosy and Phytochemistry 2010; 2(4): 324-326.

18. Priyanga Rangasamy, Vadakkenchery Salimudheen Hansiya, Palanisamy Uma Maheswari, Thamburaj Suman, Natesan Geetha. Phytochemical Analysis and Evaluation of In vitro Antioxidant and Anti-urolithiatic Potential of various fractions of Clitoria ternatea L. Blue Flowered Leaves. Asian J. Pharm. Ana. 2019; 9(2): 67-76. doi: 10.5958/2231-5675.2019.00014.0

19. Patel D.S., Shah P. B., Managoli N. B. Evaluation of In-vitro Anti-oxidant and Free Radical Scavenging activities of Withania somnifera and Aloe vera. Asian J. Pharm. Tech. 2(4): Oct. - Dec. 2012; Page 143-147.

20. SB Datir, AM Patel, AK Patel, PP Patil, FI Rohit, VD Thorat, DK Bharti, AB Ganjare. Evaluation of Antioxidant Activity of the Aerial Parts of the Abutilon indicum (Linn) Sweet (Malvaceae). Research J. Pharmacology and Pharmacodynamics. 2010; 2(5): 324-327.

21.  Kotze M. Eloff JN. Houghton PJ. Extraction of antibacterial compounds from Combretum microphyllum (Combretaceae). South African J. Bot. 2002; 68: 62–67.

22. Guy BK. Akhanovna MBJ. Odette DD. Jonathan GS. Yves-Alain B. Sur la Composition Phytochimique Qualitative des Extraits bruts Hydrométhanoliques des Feuilles de 6 Cultivars de Manihot Esculenta Crantz de Côte d’Ivoire. Eur J Sci Res. 2010; 45: 200–211.

23. Carter P. Spectrophotometric determination of serum iron at the submicrogramlevel with a new reagent (ferrozine). Anal. Biochem. 1971; 40: 450–458.

24. Tiwari.A.K. Imbalance in antioxidant defence and human diseases: Multiple approach of natural antioxidants therapy. Curr. Sci.2001;81: 1179-1187.

25. Benzie IFF. Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 1996; 239: 70–76.

26. Akinola AA. Ahmad S. Maziah M. Total antioxidant capacity, flavonoid, phenolic acid and polyphenol content in ten selected species of Zingiberaceae rhizomes. African J. Tradit. Complement. Altern. Med. 2014; 11: 7–13.

27. Pang Y. Ahmed S. Xu Y. Beta T. Zhu Z. Shao Y. Bao J. Bound phenolic compounds and antioxidant properties of whole grain and bran of white, red and black rice. Food Chem. 2018; 240: 212–221.

28. Yuwang P. Sulaeva I. Hell J. Henniges U. Böhmdorfer S. Rosenau T. Chitsomboon, B. Tongta S Phenolic compounds and antioxidant properties of arabinoxylan hydrolysates from defatted rice bran. J. Sci. Food Agric. 2018; 98: 140–146.

Received on 15.08.2021                    Modified on 17.09.2021

Accepted on 02.11.2021                   ©AJRC All right reserved

Asian J. Research Chem. 2021; 14(6):425-430.

DOI: 10.52711/0974-4150.2021.00073