Phytochemical profiling of the Methanolic root extract of Lavatera` cashmeriana using GC- MS and Evaluation of its Potential Antimicrobial Activity

Mohmmad Ashaq Sofi¹, Anima Nanda¹*, Ezekiel Raj², Mohd Abass Sofi³

¹Department of Biomedical Engineering Sathyabama Institute of Science and Technology,

Deemed to be University, Chennai, 600119, Tamil Nadu India.

²Biocontrol and Microbial Metabolites Lab, Centre for Advanced Studies in Botany,

University of Madras, Guindy Campus, Chennai – 600025.

³Department of Chemistry Sathyabama Institute of Science and Technology,

Deemed to be University, Chennai, 600119, Tamil Nadu India.

*Corresponding Author E-mail: animanandabiomed@gmail.com, animananda72@gmail.com

ABSTRACT:

Medicinal plants found worldwide are vast repositories of diverse bioactive phytoconstituents that owe their broad range of medicinal applicability in the treatment, prevention, and diagnostics of diseases. Researchers in the past have successfully tried and are determined in the extraction and purification of phytocompounds which are active agents in treating various alarming to simple diseases and serve as lead compounds in the manufacturing or formulation of novel drugs. The present aim of the research was to conduct the GC-MS analysis of the methanolic root extract of the medically significant plant Lavatera cashmeriana. Through this research analysis, arctigenin is a new compound identified to be present in Lavatera cashmeriana, which was earlier reported and considered to be present in some plant families (Asteraceae, Convolvulaceae and Oleaceae). Meanwhile, the present research was further aimed at evaluating the antimicrobial properties of root extract of this plant on some pathogenic micro-organisms like Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The results obtained revealed significant antimicrobial activity of the plant's root extract.

KEYWORDS: Lavatera cashmeriana, Kashmir Himalayas, Gas chromatography-mass spectrometry (GC-MS), Arctigenin, Antimicrobial activity.

INTRODUCTION:

Lavatera cashmeriana, commonly known as suzkol¹, is a traditional medicinal plant belonging to the family Malvaceae². The genus Lavatera has almost twenty-five species. These are commonly known as mallow trees. The Lavatera cashmeriana inhabit forests, sunny rocky slopes, and grasslands of the Kashmir Himalaya, Pakistan Gilgit regions, and Iran 3. It is an endemic species of the Kashmir Himalaya and is reported to have significant medicinal importance⁴. It is used to treat various diseases or ailments in the form of Unani medicinal preparations or its crude parts ⁵.

It is reported to have antiviral⁶, antiproliferative ⁷, and anti-inflammatory⁸. It is also used to prevent and treat cardiovascular diseases and throat infections ^3,
9.

Modern synthetic and chemical medications are often explored with hesitate since they have adverse effects, while traditional herbals are gaining popularity because they are more natural, eco-friendly, and free of side effects. Hence, people continue to favour plant-based medicines over synthetic medicines^10,11

The preliminary screening of medicinal plants using chromatographic techniques offers valuable information on chemical and pharmacological properties, assisting in selecting biologically active plants. In recent years GC–MS has been widely used for identifying numerous bioactive therapeutic chemicals found in plants. GC–MS is one of the best and most accurate methods for detecting a wide range of chemicals, including long-chain hydrocarbons, esters, alcohols, steroids, organic acids, alkaloids, and amino acids, and it only requires a small amount of plant extract. ^{12, 13, 14}

Hence, in the present study, gas chromatography-mass spectrometry technique was used to detect and identify phytochemical compounds present in the medicinal plant Lavatera cashmeriana. We have also evaluated the antimicrobial efficacy of the medicinal plant.

MATERIALS AND METHODS:

Collection and processing of the sample:

Figure 1: Lavatera cashmeriana Plant

Figure 2: Collection site Daksum Anantnag Jammu and Kashmir, India. Latitude 33°36'43"N and longitude 75°26'6"E

The Lavatera cashmeriana plant (Figure1) was collected from the Daksum area of Anantnag, Jammu and Kashmir, India, at an altitude of 2438 meters above sea level (Figure 2). The identification and authenticity process of the plant was completed in the Kashmir University (CBT-botany) vide voucher number 3070-(KASH) Herbarium. The plant's root part was shade dried in hygienic conditions for at least fifteen days. After that, the roots were crushed into a coarse powder in an electrical grinder machine and packaged carefully for further processing.

Extraction process:

A simple maceration process was employed for the extraction of the plant material. 10 grams of coarse powder was mixed with 100 ml of desired quality grade methanol in a 250 ml flask, and the mixture was kept on a shaker for 24 hours. This was followed by filtration of the reaction mixture using Whatman filter paper No.1. The filtrate so collected was left for evaporation of the solvent to obtain a concentrated mass. The procedure was repeated three times to get the desired quantity and quality of the sample for further analysis.

GC-MS analysis:

The phytochemical profiling was done using GC-MS Shimadzu –QP2010 Ultra analyzer, with ion source temperature and interface temperature as 240 ⁰C and 25⁰C^., respectively. The column oven temperature was initially programmed from 80⁰C to 200⁰C^.at a rate of 3⁰C/minute, then to 260⁰C at a rate of 10⁰C/minute, maintained for 5 minutes. The sample was injected in a split mode into the capillary column, which contains helium as a carrier gas, with a column flow rate of 1 ml/minute and run time was 50 minutes.

Antimicrobial activity:

Methanolic extract of the plant was randomly tested on five pathogenic microbial species viz: fungus Candida albicans ATCC 10231, two Gram-negative bacteria: Pseudomonas aeruginosa ATCC 15442 and Escherichia coli ATCC 11229, and two Gram-positive bacteria: Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923. The antimicrobial assay was performed using the agar well diffusion method. Five Petri plates with solidified MHA agar medium (M173 HiMedia, Mumbai) were taken, and in each Petri plate, six wells were made, as shown in figure1. The cultured microbial species were uniformly spread on plates separately and left for 24 hours to enhance the uniform growth of microbes. Four of the six wells in each plate were filled with different volumes (25 µL, 50 µL, 75 µL and 100 µL) from 10 mg/mL of prepared extract from Lavatera cashmeriana roots. The remaining 5th and 6th wells were filled with positive and negative controls as shown in Figure 3.

Determination of the MIC:

The experiment was conducted in 96 well plates’ flat-bottom well, according to (Gabrielson et al.; 2002)¹⁵. The first ten wells containing 100 µL of serially diluted different concentrations of Lavatera cashmeriana root extract (512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 µg/mL) were added 5 µL 12 hours old test pathogens. The plates were placed in an incubator at 37⁰C for 24 hours. After incubation, the 10μL 5 mg/mL of freshly prepared MTT was added to all the wells and incubated for 2 hours. After that, 100 μL of DMSO (TC185 HiMedia, Mumbai) solution was added as the solubilizing agent, and color change was observed visually. The color change from yellow to purple was considered positive. The minimum inhibitory concentration (MIC) was described as the lowest concentration that fully inhibited bacterial growth. The experiment was done in triplicate.

RESULTS AND DISCUSSIONS:

Analysis of the mass Spectra of the obtained phytocompounds through gas chromatographic separation by comparing with the references in the Wiley and NIST led to the identification of different bioactive phytocompounds (as shown in Figure 3 and Table 1), which include flavonoids, lignin, polyol, saturated/unsaturated essential/nonessential fatty acids, esters, medium and long-chain hydrocarbons and steroids.

Figure 3: Chromatogram of Lavatera cashmeriana methanolic root extract showing peaks of number of compounds.

Table 1: Phytocompounds identified in the methanolic root extract of Lavatera cashmeriana by GC-MS.

Peak#	R.Time	Area	Area%	Name
1	3.877	360672	4.06	Glycerin
2	12.165	284122	3.20	Nonanoic acid
3	37.679	541975	6.10	n-Hexadecanoic acid
4	40.806	181663	2.04	Methyl 2-octylcyclopropene-1-heptanoate
5	41.820	1857430	20.89	9,12-Octadecadienoic acid (Z,Z)-,
6	41.940	369814	4.16	Essigsaeure, 2-Nitro-2-(2,7-Octadien-1-YL)-, Ethylester (Isomer 2)
7	42.087	349243	3.93	Hexatriacontane
8	42.214	213782	2.40	Octadecanoic Acid, Methyl Ester
9	42.307	292701	3.29	9,12-Octadecadienoic acid (Z,Z)-
10	42.403	556849	6.26	9,12,15-Octadecatrienoic acid, (Z,Z,Z)-
11	44.635	160103	1.80	Dihydrofuran-2-one, 4-(3,4-dimethoxybenzyl)-3-(4-hydroxy-3-methoxybenzyl)-
12	45.304	174568	1.96	Tetrapentacontane
13	46.870	178001	2.00	Cyclopropaneoctanoic Acid, 2-[[2-[(2ethylcyclopropyl)Methyl]Cyclopropyl]Methyl]-, Methyl Ester
14	47.797	169005	1.90	Tetrapentacontane
15	48.291	294220	3.31	gamma.-Sitosterol
16	49.235	348881	3.92	3-N-Nitroso-solanocapsine
17	49.325	657153	7.39	Docosanoic acid, docosyl ester
18	49.385	372640	4.19	Stigmastane, 23,24-epoxy-, (5.alpha.)-
19	49.473	630192	7.09	Propanoic acid, 3,3'-thiobis-, didodecyl ester
20	49.560	239937	2.70	Decyl .alpha.-d-galactoside, 2,4,6-detrioxy-3-O-benzyl-4,6-S-dibenzylthio
21	49.580	140067	1.58	Docosanoic acid, docosyl ester
22	49.605	516460	5.81	Propanoic acid, 3,3'-thiobis-, didodecyl ester
		8889478	100.00

Twenty-two peaks are shown in the chromatogram, which are from eighteen different compounds. Additionally and importantly, this research led to the presence and identification of the well-known and highly researched compound "Arctigenin" (peak#11). This lignin compound was reported to be present in only some plant families (Asteraceae, Convolvulaceae and Oleaceae). This research analysis gives us confirmation that arctigenin is present in the Lavatera cashmeriana (family Malvaceae). Arctigenin is reported and researched to have a lot of therapeutic medicinal properties such as anticancer¹⁶, antiviral ¹⁷, anti-inflammatory, antibacterial ¹⁸, and so on. Besides, as shown in the chromatogram, other compounds have notable biomedical applications. Compounds {5#}, {17#}, {10#}, {3#}, {7#}, {8#}, {2#}, {1#} have been reported to have either one or more among the following uses: cosmetics, emollients, creams, skin moisturizers, soaps, deodorants, hair creams, wound or burn healing, against soreness of throat, toothpaste and mouthwashes. ^{19, 20,
21}

Compound: {18#} stigmastane,23,24-epoxy-,(5-alpha) is a steroid cardiac glycoside that can be useful in the prevention and treatment of arrhythmia and congestive heart failure (CHF). ²²

Compound: {10#} 9-12-15-octadecatrienoic acid (Z, Z, Z) or alpha-linolenic acid helps in the treatment of atherosclerosis, lowering of cholesterol, and in the prevention of heart attack. ^{23, 24, 25}

Compound: {15#} gamma-sitosterol acts as antidiabetic²⁶, hypolipidemic²⁷, antiviral, and antibacterial ²⁸, anticancer ²⁹ agents.

Compounds: {19#} Propanoic acid, 3, 3’-thiobis-, didodecyl ester ³⁰ and {3#} n-hexadecanoic acid ³¹are reported to have antioxidant property.

Compounds: {2#} Nonanoic acid ³², {1#} Glycerol ³³ H and {3#} n-hexadecanoic acid ³¹ are also antimicrobials.

All the compounds mentioned above, obtained through GC-MS analysis of methanolic root extract of Lavatera cashmeriana, have notable therapeutic and other uses. Many of the reported biomedical properties of Lavatera cashmeriana, like anticancer, antiviral and antioxidant, can be certainly justified by the presence of phytocompounds reported in this research analysis.

The alarming rise in the incidence of bacterial infections is presently posing a severe threat to global public health. Antibiotic resistance and the emergence of new pathogens with the potential for fast worldwide transmission exacerbate the situation, fueling the hunt for new bioactive agents. There are undoubtedly a plethora of drugs currently available today to treat all bacterial infections. Still, unfortunately, they all have substantial adverse side effects, limiting their usage in specific population sectors. As a result, there is an ongoing and pressing need to develop novel antibacterial agents with a high safety index. ^{34, 35, 36, 37}

Here, the Lavatera cashmeriana medicinal plant used for different remedies by local communities was also tested against ATCC microbial cultures to determine and investigate their antimicrobial potential. We observed that the crude methanolic root extract of Lavatera cashmeriana showed significant antimicrobial activities against Enterococcus faecalis, Staphylococcus aureus, and Escherichia coli at different ranges of concentrations of extract, with 18 ± 0.57, 15 ± 0.63, and 17 ± 0.45 mm zones of inhibition, respectively. For Pseudomonas aeruginosa, the extract has comparatively intermediate action at defined 100 µL volume, showing a 13 ± 0.73 mm zone of inhibition. However, no antifungal activity was observed on Candida albicans at the defined range of extract concentrations, as shown in (Figure 4, Table 2 and Table 3) with zones of inhibition and MIC, respectively.

Thorough investigations of the above analytical compounds present in this plant reveal tremendous medical significance, owing to the presence of varied phytoconstituents, which can treat various diseases and can potentially act as lead compounds that can be harnessed for many more medicinal and other uses.

E. faecalis

S. aureus

C. albicans

E. coli

P. aeruginosa

Figure 4: Antimicrobial activity of root extract of Lavatera cashmeriana using well diffusion method

The numerals depicted on each petriplate in the Fig 4 represent the wells introduced with different micro volumes of Lavatera cashmeriana root extract (10 mg/mL stock solution), standard drugs 25µl (1mg/mL) and negative control.

1: 25µL; 2: 50µL; 3: 75µL; 4: 100µL; 5: 25µL Standard drug positive control, Ampilox for Gram-positive: E. faecalis and S. aureus,

Ciprofloxacin for Gram-negative: P. aeruginosa and, E. coli, Fluconazole for fungi: C. albicans. 6: DMSO as a negative control.

Table 2: Antibacterial activity of methanolic root extract of Lavatera cashmeriana against human pathogens using well diffusion method.

S. No	Pathogen Name	Zone of Inhibition in mm
S. No	Pathogen Name	25 µL	50 µL	75 µL	100 µL	Standard drug`	Negative control
1	E. faecalis	-	12 ± 0.83	15 ± 0.53	18 ± 0.57	20 ± 0.56	-
2	S. aureus	-	11 ± 0.63	13 ± 0.72	15 ± 0.63	15 ± 0.45	-
3	E. coli	11 ± 0.54	15 ±0 .54	17 ± 0.84	17 ± 0.45	35 ± 0.75	-
4	P. aeruginosa	-	-	-	13 ± 0.73	32 ± 0.67	-
5	C. albicans	-	-	-	-	-	-

(-) means no activity. The results represent the mean of zones of inhibition ± SD of three separate experiments, carried out in triplicate. (n=3)

Table 3: Minimum inhibitory concentration of the Lavatera cashmeriana root extract in methanol against human pathogens

Minimum inhibitory concentration (MIC) (µg/mL)
Gram-Positive Bacteriu	Gram-negative Bacteriu	Fungal Pathogen
*E. faecalis*	*S. aureus*	*E. coli*	*P. aeruginosa*	*C. albicans*
128	128	128	128	ND

ND: Not determined as they did not show antimicrobial activity in the well diffusion assay.

CONCLUSION:

GC-MS analysis of methanolic root extract of Lavatera cashmeriana led to the identification of many vital compounds. However, the primary purpose was to identify the compounds that were not reported earlier and justify the reported and traditional uses of the plant claimed to be associated with it. Successfully, one of the highly researched and essential compounds, Arctigenin, has been found in the Lavatera cashmeriana. A successful attempt has also been made to investigate the antimicrobial properties of the plant with promising results. There have been given the proper justifications for the claimed uses of this plant by separately analyzing the obtained phytocompounds supported by their relevant research works and consulting the authenticated research literature data. More research is needed in the future regarding the medicinal usages of Lavatera cashmeriana.

CONFLICTS OF INTEREST:

The authors declare that there are no conflicts of interest.

SOURCE OF FUNDING:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

ACKNOWLEDGMENTS:

The authors are thankful to the Department of Biomedical Engineering, Sathyabama Institute of Science and Technology and CAS Botany University of Madras, Tamil Nadu, for providing laboratory facilities to carry out the experiment.

ABBREVIATIONS:

CBT= Centre for Biodiversity and Taxonomy

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