Comparative Phytochemical Analysis of Hemi Parasite Loranthus longiflorus Desr; on Host Azadiracta indica by GCMS Analysis

Comparative Phytochemical Analysis of Hemi Parasite Loranthus longiflorus Desr; on Host Azadiracta indica by GCMS Analysis

Ch. Uday Kumar^* , Lakshmi Bhavani

Department of Botany, University College of Science, Saifabad Campus, Osmania University, Hyderabad-India

Corresponding Author Email: uddu.ch@gmail.com

DOI : http://dx.doi.org/10.5281/zenodo.7282162

Abstract

Keywords

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Introduction

Some plants acquire their food or water from other plants, which are known as parasites, and the source providing plants are known as hosts. The stem parasitic plants are commonly named mistletoes. Mistletoes are derived from the Anglo-Saxon word mistelian which means,” mist” for dung and “tan” for twig [1]. Loranthus longiflorus Desr. commonly known as Mistletoe, all-heal, devils fuge, belongs to Loranthaceae family with 76 Genera and 1000 species. The synonym for Loranthus is Dendrophthoe falcata.The semi-parasites possess leaves and synthesize the carbohydrates portion of their food. These parasites establish relations with host vascular elements to draw nutrients [2-3]. Hemi parasites have a parasitic relationship with more than 300 host plants [4]. Unlike other parasites, Loranthus seeds do not require a germination stimulant from the living host [5]. Numerous hemi parasites do not synthesize  or modify the secondary compounds taken up from their hosts [6], thus the presence of certain secondary compounds varies within and among the populations of hemi parasitic plants, depending on the host association of individual parasites [7], it has been used in traditional medicine and found to have antimicrobial, anti-diabetic, antioxidant, anticancer,  hypertensive and antiviral properties [8-10]. They are used as a health food for enhancing immunity and used as a pain reliever, aphrodisiac, narcotic and diuretic [11-12]. The aim of the study was comparative analysis of host-Azadirachta indica and parasite-Loranthus longiflorus for phytochemicals.

Preparation of plant material powder

The fresh leaves of parasite-Loranhus longiflorus and host-Azadirachta indica were collected from Hyderabad. The plant material was washed thoroughly first with tap water and then distilled water to remove the surface contaminants. Later they are shade dried separately at room temperature (30°C±5°C) for about two weeks to get constant weight and then pulverized into a dry powder using an electric blender. Until needed, the powdered leaf samples were kept in an airtight container.

Preparation of extract

The dried plant materials of L. longiflorus and Azadirachta indica leaves samples were extracted with Methanol in soxhlet apparatus for 3hrs. The extract was cooled, filtered, and concentrated using a vacuum flask evaporator. Finally, this extract was dissolved in 1ml methanol and centrifuged at 3000 rpm for 5min.

Phytochemical Analysis

Phytochemical analysis of methanol leaves extract was carried out following certain prescribed methods adopted by Srinivas R. et al., 2014.

Test for Tannins

In 2ml of distilled water, a few drops of ferric chloride solution were added to roughly 2ml of extract. The appearance of green-tinted precipitate confirms the presence of tannins.

 

Test for Saponins

In a test tube, 3 ml of the extract was mixed with 3 ml of distilled water and vigorously shaken for a few moments. The test tube was heated, and creating stable foam indicates the presence of saponins.

Test for Flavonoids

1ml of 10 percent lead acetate solution was added to 1 ml of extract in a test tube. The appearance of the yellow precipitate can determine the presence of flavonoids.

Test for Alkaloids

On a hot water bath, 3 ml of the extract was mixed with 3 ml of 1% HCl. Then 1ml of the liquid was split between two test tubes.

Dragendroff’s Test

1 ml of Dragendroff’s reagent was added to the first test tube. The presence of orange-red precipitate was regarded as a good sign.

 Mayer’s Test

1ml of Mayer’s reagent was added to the second test tube. The formation of a buff-colored precipitate can confirm the presence of alkaloids

Test for Terpenoids

2 ml extract was dissolved in 2 ml CHCl3 and evaporated to dryness. After that, 2 ml of concentrated sulphuric acid (H₂SO₄) was added and heated for about 2 minutes. The emergence of a grayish color indicates the presence of terpenoids.

Test for Steroids

Two tests were carried out to perceive the presence of steroids in the extract, and those were the Salkowski’s test and the Liebermann test.

Salkowski’s test: 2ml of the organic extract was dissolved in 2ml of chloroform. 2ml concentrated H₂So₄ was added to this. The presence of steroids is shown by the appearance of red color in the chloroform portion.

Test for Phenols

5ml distilled water was added to 1gm of extract, and the solution was then treated with a 5% ferric chloride solution. The presence of phenols is confirmed by forming a dark green color.

 GC-MS Analysis

The GC-MS analysis of themethanolic leaves extract was done at the Central Analytical Facility, University College of Technology,  Osmania University using the standard GCMS model.  The  parasite plant Loranthus longiflorus and host Azadiracta indica leaves extracts were analyzed using a GCMS-QP2010 SHIMADZU, Japan, fused with an Optima 5 ms capillary column (30 0.25 mm) 0.25 m film thickness, as described by Iheagwamet al (2019). The initial column oven temperature (60°C) is planned to climb to 160°C at a rate of 10°C/min, then to 250°C with hold duration of 2 min/increment. An injection volume of 1.0 L in the splitless mode with a split ratio of 1:1 and injector temperature set at 200° C with minor adjustments. The following were the mass spectrophotometer settings: 230°C for the ion source, 250°C for the interface, 4.5 minutes for the solvent delay, and 50–700 amu for the scan range. The multiplier voltage and electron ionization mode were modified to 70 eV and 1859 V. For compound identification, the retention time, fragmentation pattern, and mass spectral data of unknown components in the extracts equated to those in the Wiley and National Institute of Standards and Technology (NIST) libraries [14-24].

Results and Discussion

The phytochemical analysis of the methanol extract of two plant leaves Loranthus longiflorus –parasite and host-Neem revealed the presence of tannins, phenols, anthraqinones and the absence of saponins and alkaloids. Flavonoids and steroids absent in the parasite are present in host (Table-1 and Fig-3). The GC-MS  chromatographic profile of the host and parasite plant leaves extracts showed various significant constituents like  Difluorine monoxide; Nitrosyi  chloride; 1-Propyne,3-chloro; Pyridazine, 3-methoxy; N,N Dinitro 1,3,5,7 tetrazabicyclononane in diverse compositions. Host plant contains the compounds of 2-Nonynoic acid (15.26%), Methane, chloro Trinitron (13.04%) that were absent in parasite plant followed by  Difluorine  monoxide (11.28%), Nitrosyi chloride (2.11%), 1-Propyne, 3-chloro- (0.60%) N,N Dinitro1,3,5,7 tetrazabicyclononane (0.41%), Pyridazine, 3-methoxy (0.28%). Parasitic plant  contains highest level of Phenylethylene.3-methoxy-2,2-dinitro (62.40%) followed by Difluorine  monoxide (0.40%), Nitrosyi  chloride (0.38%), Pyridazine, 3-methoxy (0.13%), 1-Propyne, 3-chloro-(0.13%), N, N Dinitro1, 3, 5, 7 tetrazabicyclononane (0.34%). 2-Nonynoic acid, Methane, chloro trinitron that were absent in host plants are absent inparasite plants.

Fig: (1) Chromatogram Neem Parasite E:/2022/OCT/10-uday-GCMS/Neem parasite.

Fig:(2)  Chromatogram Neem Host E:/2022/OCT/10-uday-GCMS/Neem Host.

Table: 1: Phytochemical analysis of parasite-L.longiflorus and Host- Azadiracta indica

 

Sno	Constituents	Host- Azadiracta indica	Parsite- L.longiflorus
1	Test for Alkaloids	Absent	Absent
2	Test for tannins	Present	Present
3	Test for saponins	Absent	Absent
4	Test for flavonoids	Present	Absent
5	Test for terpenoids	Absent	Present
6	Test for steroids	Present	Absent
7	Test for Phenols	Present	Present
8	Test for Anthraquinones	Present	Present

Fig: (3) Phytochemical analysis of Host and Parasite plants.

 

(A)Tannins (B) Saponins (C) Flavonoids (D) Alkaloids  (E) Terpenoids  (F) Steroids (G) Phenol (H) Anthroquinones

Table 2: Phyto compounds identified by GC-MS analysis.

 

S.NO	COMPOUND NAME	RETENTION TIME			MOLECULAR WEIGHT	FORMULA	CAS NO	%AREA HOST	%AREA PARSITE
		(H.N)	(P.L)
1	Difluorine  monoxide	0.108		3.060	54	F2O	7783-41-7	11.28%	0.40%
2	Nitrosyi  chloride	0.595		3.875	65	ClNO	2696-92-6	2.11%	0.38%
3	1-Propyne,3-chloro-	2.180		3.505	74	C3H3Cl	624-65-7	0.60%	0.13%
4	Pyridazine, 3-methoxy	2.180		3.505	110	C6H6N2O	19064-65-4	0.28%	0.13%
5	N,N Dinitro 1,3,5,7 tetrazabicyclononane	2.545		3.584	218	C5H10N6O	0-00-0	0.41%	0.34%
6	2-Nonynoic acid	0.059		0	154	C9H14O2	1846-70-4	15.26%	0
7	Phenylethylene,3-methoxy-2,2-dinitro	0		2.429	224	C9H8N2O5	16990-0-2	0	62.40%
8	Methane, chloro trinitro	0.200		0	185	CClN3O6	1943-16-4	13.04%	0

CONCLUSION

Host and parasite plant leaves extracts depicts similar  Bioactive compounds like Difluorine monoxide, Nitrosyi chloride, 1-Propyne,3-chloro, Pyridazine, 3-methoxy, N,N Dinitro 1,3,5,7 tetrazabicyclononane with varied composition. The analysis can be used as a useful tool to pharmacognacy. Finally, the present reports points towards the need of further investigations on the possible role of transferred secondary compounds either as mediators of host defense, host defense compounds utilize as cues of identification of host by the hemi parasite, or compounds taken up by the parasite to support their defense against rejection by the host.

Acknowledgment

Authors are thankful to the Department of Botany, UCSS Saifabad and University College of Technology,  Osmania University, Hyderabad, and Telangana for their valuable help during the time of Ph.D. work

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