Preliminary Phytochemical Analysis and Isolation and Identification of active principle components of petroleum ether extract from leaves of Lawsoniainermis L.

 

B. Arirudran¹, V. Krishnan², Us Mahadeva Rao^3*

^1*Assistant Professor, P.G. Department of Biochemistry, SRM Arts and Science College, Kattankulathur,  Kanchipuram District, Tamil Nadu - 603203.

²Post Graduate Student, P.G. Department of Biochemistry, SRM Arts and Science College, Kattankulathur, Kanchipuram District, Tamil Nadu - 603203.

³Professor (VK7), School of Basic Medical Sciences, Faculty of Medicine, University of Sultan Zainal Abidin, 20400 Terengganu, Malaysia.

 

ABSTRACT:

Background: Lawsoniainermis L belongs to the Lythraceae family, generally known as Henna, cultivated for its leaves although stem, bark, roots, flowers and seeds. They were used in traditional medicine to treat a variety of ailments as rheumatoid arthritis, headache, ulcers, diarrhea, leprosy, fever, leucorrhoea, diabetes, cardiac disease. They were used for dye making. Aim: The present study focus on preliminary phytochemical analysis and determination of bioactive components from the leaf of Lawsoniainermis L. Methods: Preliminary phytochemical analysis and identification of active components by GC/MS were done. Result: The preliminary phytochemical analysis reveals that the aqueous and petroleum ether extracts from leaf of Lawsonia inermis Lconsists of alkaloids, anthraquinones, flavonoids, phenols, reducing sugars, saponins, steroids, tannins, and terpenoids.GC/MS analysis reveals that petroleum ether extracts consists of phthalic anhydride, 4-methylformanilide, acetamide, N-(2-methylphenyl)-, 3-hydroxycarbofuran, 2-hydroxy-1,4-naphthalenedione, dodecyl acrylate,3,7,11,15-tetramethyl-2-hexadecen-1-ol, trans-geranylgeraniol, n-hexadecanoic acids, 9,12,15-octadecatrienoic acids, (Z,Z,Z)-octadecanoic acids, phytol, acetate, cis-13-eicosenoic acid, cyclohexadecane-1,2-diethyl-, 4,8,12,16-tetramethylheptadecan-4-olide, eicosane, tetratriacontane, supraene, 1,6,10,14-hexadecatetraen-3-ol, 3,7,11,15-tetramethyl-, (E,E)-, stigmastan-3,5-diene, triacontane, and vitamin E. Conclusion: This study shows that Lawsoniainermis L. leaves contain bioactive phytochemicals that may serve as a biocompatible and eco-safe agent in formulation of drugs, natural dyes in colouration of textile materials and other purpose.

 

 

 

INTRODUCTION:

Currently, in the world of growing environmental consciousness, natural colourants have caught the attention of everyone. Natural dyes have been a part of human life since time of immemorial. The alchemy of colours started its use from an early time.^[1]

 

Natural organic dyes exhibit better biodegradability and generally have a better compatibility with the environment. Also they possess lower toxicity and allergic reactions than synthetic dyes.^[2] Lawsoniainermis L(henna) belongs to the Lythraceae family, generally known as Henna. It is tree species native to North Africa, now a dayit is used all over world. Leaves of henna plant are entirely, opposite, sub-sessile, oval-shaped and smooth.^[3] Leaves have a length of 2-3cm with 1-2cm width.^[4] Henna shrub is highly branched and has greyish-brown barks grown up to 25 feet.^[5] Leaves of henna are an ancient dye, evidence being the Egyptian mummies found in the tombs that had their nails dyed with henna. Currently, it is used in many countries for dyeing hair, eyebrows and fingernails during religious festivals and marriages etc. The use of henna for dyeing the palms and fingernails is an auspicious ritual mostly in Asian countries.^[6] The powdered leaves of this plant (aqueous paste) are used as a cosmetic for staining hands, palms, hairs and other body parts.^[7,8,9] Traditionally in India, Mehndi is applied to hands and feet. Its use became popular in India because of its cooling effect in the hot summer.^[10,11] The leaf have a bitter bad taste and used in vulnerary, diuretic, headache, hemicranias, lumbago, bronchitis, boils, ophthalmia, syphilitis, sores, amenorrhoea, scabies, and spleen diseases and favours the growth of the hair.^[12] The Ayurvedic Pharmacopoeia of India indicated the use of leaves in dysuria, bleeding disorder, prurigo and other obstinate skin diseases.^[13] In traditional medicine they were used to treat a variety of ailments as rheumatoid arthritis, headache, ulcers, diarrhea, leprosy, fever, leucorrhoea, diabetes, cardiac disease.^[10,11] In this present study we are mainly focused on, preliminary phytochemical analysis and GC-MS determination of bioactive components from petroleum ether extract of leaf from Lawsoniainermis L.

 

MATERIALS AND METHODS:

Collection of plant materials:

Fresh plant materials of Lawsoniainermis L. were collected from Kancheepuram District of Tamilnadu. The plant materials were identified and authenticated by the botanist of this institute using the Flora of the Presidency of Madras.^[14,15] Sample was authenticated based on organoleptic, macroscopic examination and certified, by department staffs.

 

Chemicals and reagents:

All the chemicals and reagents in this study were of Laboratory grade and implemented without further purification.

 

Preparation of aqueous and petroleum ether extract:

The extracts were prepared as described by the standard method.^[16] 500gm of dried clean Lawsoniainermis L. leaf were coarsely powdered and weighed. The dried powder 250gm was soaked with aqueous and petroleum ether separately for 72 hours with intermediate shaking separately in two beakers. At the end of the extraction, it was to be sent through Whatman No.1filter paper (Whatman Ltd., England). Then the filtrate was extracted with soxhlet apparatus, filtered and concentrated to dryness.^[17] The last traces of solvent were removed by transferring them into a china dish and allow heating the china dish using a sand bath at normal temperature, carefully in order to prevent charring and denaturation of compounds due to overheating. The yield of aqueous extracts (18.5gm) and petroleum ether (13gm) extract was noted. Dried crude extract is kept in sterile amber colored storage vials in refrigerator until used for the further studies.

 

Preliminary Phytochemical Analysis:

The stock solution was prepared from both the crude extracts (aqueous and petroleum ether). The obtained stock solutions were subjected to phytochemical analysis based on standard methods described.^{[18, 19, 20]}

 

Gas Chromatography–Mass Spectrometry (GC/MS) Analysis:

GC/MS-a combination of two different analytical techniques, Gas Chromatography (GC) and Mass Spectrometry (MS), is used to analyze complex organic and biochemical mixtures.^[21] GC/MS analysis of this extract was performed using a Perkin Elmer GC Claurus 500 systems and Gas Chromatograph interfaced to a Mass Spectrometer (GC/MS) equipped with arElite-1 fused silica capillary column (30m×0.25mm ID.×1 ìMdf, composed of 100% Dimethyl poly siloxane with 0.25μm film thickness), Agilent technologies 6890 N JEOL GC Mate II GC-MS model. For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium as carrier gas (99.999%) was used as the carrier gas at a constant flow rate of 1ml/min and an injection volume of 2 ìl was employed (split ratio of 10:1). Injector was operated at temperature 200 to 250°C, Ion-source temperature 250 to 280°C. The column oven temperature was programmed from 110°C (isothermal for 2min.), with an increase of 10°C/min to 200°C, then 5°C/min to 280°C, ending with a 9min. isothermal at 280°C. Mass spectra were taken at 70 Ev, a scan interval of 0.5 seconds and fragments from 45 to 450Da., interface temperature of 250°C, mass range of 50-600 mass units. Total GC running time was 36minutes. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was Turbo Mass Ver5.2.0.

 

Identification of components:

The database of the National Institute Standard and Technology (NIST) having more than 62,000 patterns was used for the interpretation on mass spectrum of GC-MS. The mass spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library.

 

RESULT AND DISCUSSION:

Preliminary Phytochemical Analysis :

The table1 shows that the result of preliminary phytochemical analysis of aqueous and petroleum ether extracts of leaf from Lawsonia inermis L. The aqueous extract consists of alkaloids, anthraquinones, flavonoids, phenols, reducing sugars, saponins, tannins and terpenoids. The petroleum ether extract consist of alkaloids, flavonoids, phenols, steroids, tannins and terpenoids.

 

Table:1 Preliminary phytochemical analysis of aqueous and petroleum ether extracts of leaf from Lawsonia inermis L.

Phytochemical analysed	Aqueous	Petroleum ether
Alkaloids	+	+
Anthraquinones	+	-
Flavonoids	+	+
Phenols	+	+
Reducing sugars	+	-
Saponins	+	-
Steroids	-	+
Tannins	+	+
Terpenoids	+	+

 

In my present study the result of preliminary phytochemical analysis from table 1 shows that both aqueous and petroleum ether extract comprise secondary metabolites compound like alkaloids, flavonoids, phenols, tannins and terpenoids etc, Early report said that the leaves of Lawsonia inermis L. contain soluble matter tannin, gallic acid, glucose, mannitol, fat, resin and mucilage.^[22] Bark of Lawsonia inermis L. also reported to contain napthoquinone, isoplumbagin, triterpenoids-Hennadiol, aliphatics (3-methylnonacosan-1-ol).^[23] The secondary metabolites mainly attribute the antimicrobial activity of plants.^[24]The bioactive compounds from the plant material are secondary metabolites and their derivatives like flavonoids, saponins, quinines, terpenoids, steroids, alkaloids, anthroquinones, and phenolic compounds etc, which are used as drugs,^[25]as curative agents for many diseases drugs.^[26]The result of this study shows that leaves of Lawsonia inermis L may possess antimicrobial activity and hence the leaf of Lawsonia inermis L. may be used as drugs as curative agents for many diseases drugs.Recent studies with similar secondary metabolites screening in different herbal samples were reported with pronounced therapeutic properties. ^{[27 – 34]}

 

 

Figure 1(a): Phthalic anhydride

Isolation and analysis bioactive principle components by GC/MS:

The petroleum ether extract of leaf from Lawsoniainermis L. were subjected to GC/MS analysis and the active principle compounds were identified based on active principles with their Retention time (RT), Molecular formula, Molecular weight (MW) and Concentration (%).

 

 

Figure 1(b):4-Methylformanilide

 

 

Figure 1(c): Acetamide, N-(2-methylphenyl)

 

 

Figure 1(d): 3-Hydroxycarbofuran

 

Figure 1(e): 2-hydroxy-1,4-Naphthalenedione

 

 

Figure 1(f): Dodecyl acrylate

 

 

Figure 1(g): 3,7,11,15-Tetramethyl-2-hexadecen-1-ol

 

 

Figure 1(h): trans-Geranylgeraniol

 

Figure 1(i):n-Hexadecanoic acids

 

 

Figure 1(j): 9,12,15-Octadecatrienoic acids

 

 

Figure 1(k): (Z,Z,Z)-Octadecanoic acids

 

 

Figure 1(l): Phytol, acetate

 

Figure 1(m): cis-13-Eicosenoic acid

 

 

Figure 1(n): 1,2-diethyl-Cyclohexadecane

 

 

Figure 1(o): 4,8,12,16-Tetramethylheptadecan-4-olide

 

 

Figure 1(p): Eicosane

 

Figure 1(q): Tetratriacontane

 

 

Figure 1(r):Supraene

 

 

Figure 1(s): 1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl-, (E, E)-

 

 

Figure 1(t): Stigmastan-3,5-diene

 

Figure 1(u): Triacontane

 

 

Figure 1(v): Vitamin E

 

Figure 1(w): Total ionic chromatogram.

Individual ionic chromatogram(a - v) and Total ionic chromatogram (w) GC/MS of petroleum ether extract from leaves of Lawsoniainermis L.

 

Table 2: Isolation of active principle components of petroleum ether extract from leaves of LawsoniainermisL. based on Retention time, Molecular formula,and Molecular weight by using GC/MS.

S No	Name of the compound	Molecular formula	Retention time	Molecular weight g/mol
1	Phthalic anhydride	C8H4O3	5.893	148.117
2	4-Methylformanilide	C8H9NO	6.695	135.166
3	Acetamide, N-(2-methylphenyl)-	C9H11NO	7.283	149.1897
4	3-Hydroxycarbofuran	C12H15NO4	8.491	237.255
5	2-hydroxy-1,4-Naphthalenedione,	C10H6O3	8.816	174.155
6	Dodecyl acrylate	C15H28O2	10.359	240.387
7	3,7,11,15-Tetramethyl-2-hexadecen-1-ol	C20H40O	12.013 - 12.521	296.539
8	trans-Geranylgeraniol	C20H34O	12.998	290.491
9	n-Hexadecanoic acids	C16H32O2	13.698	256.43
10	9,12,15-Octadecatrienoic acids,	C18H30O2	16.327	278.436
11	(Z,Z,Z)-Octadecanoic acids	C18H36O2	16.469	284.484
12	Phytol, acetate	C22H42O2	16.987	338.576
13	cis-13-Eicosenoic acid	C20H38O2	18.438	310.522
14	Cyclohexadecane-1,2-diethyl-,	C20H40	18.743	280.54
15	4,8,12,16-Tetra-methylheptadecan-4-olide	C21H40O2	18.966	324.549
16	Eicosane	C20H42	20.955	282.556
17	Tetratriacontane	C34H70	23.787	478.934
18	Supraene	C30H50	25.888	410.73
19	1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl-, (E, E)-	C20H34O	26.050	290.491
20	Stigmastan-3,5-diene	C29H48	27.898	396.703
21	Triacontane	C30H62	28.507	422.826
22	Vitamin E	C29H50O2	31.958	430.717

 

In this present study, we were observed and reported that about twenty-two active principle phytochemical compounds by GC/MS analysis of petroleum ether extracts from Lawsonia inermis leaf based on retention time, molecular formula, and Molecular weight. The figure 1 (a to w) shows the individual and total ionic chromatogram (GC/MS) of petroleum ether extract of leaf from Lawsoniainermis L obtained with 70eV using Elite-1 fused silica capillary column with He gas as the carrier. The prevailing and abundant compounds were listed as, Phthalic anhydride, 4-Methylformanilide, Acetamide, N-(2-methylphenyl)-, 3-Hydroxycarbofuran, 2-hydroxy-1,4-Naphthalenedione, Dodecyl acrylate, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, trans-Geranylgeraniol, n-Hexadecanoic acid, 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-Octadecanoic acid, Phytol, acetate, cis-13-Eicosenoic acid, Cyclohexadecane-1,2-diethyl-, 4,8,12,16-Tetramethylheptadecan-4-olide, Eicosane, Tetratriacontane, Supraene, 1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl-, (E,E)-, Stigmastan-3,5-diene, Triacontane, and Vitamin E. Each and every compound has a unique property and its uses and natures were collected from PubMed^[26] as curative agents for many diseases.^[25]

 

Early report said that, the main chemical constituents of henna are Lawsone (2-hydroxynaphthoquinone), mucilage, mannite, gallic acid and tannic acid.^[35] 2-Hydroxy-1, 4-napthoquinone (HNQ, Lawsone) is the principle natural dye contained at 1.0-1.4 % in the leaves of Henna.^[36] Other related compounds present in the leaves are 1, 4-dihydroxynaphthalene.1,4-naphthoquinone, 1,2-dihydroxy-glucoyloxynaphthalene and 2-hydroxy-1,4-diglucosyloxynaphthalene, Flavonoids (luteolins, apigenin, and their glycosides). Coumarins (esculetin, fraxetin, scopletin). Steroids (β-sitosterol).^[23] Hexadecanoic acid methyl ester, also known as Methyl palmitate, in the methanol fraction is an aliphatic acid ester reported to causing growth inhibition and apoptosis induction in human gastric cancer cells.^[37]

 

Previously it was reported that chloroform extract of Lawsoniainermis L. and a pure compound was isolated and identified, using chromatographic and spectroscopic techniques, as 2-hydroxy-1,4-naphthaquinone (lawsone). The isolated compound was found to possess significant anti-inflammatory, analgesic, and antipyretic activity.^[38] Phytol was also present in Lawsoniainermis L. which is a di-terpene alcohol which functions as a precursor for vitamins E and K1 and an antioxidant and a preventive agent against epoxide-induced breast cancer.^[39] It’s also an effective vaccine adjuvant with no adverse auto-immune effects.^[40] His plant has been described in Charaka Samhitaa for the treatment of epilepsy and jaundice, and for dyeing grey hair. In Sushruta Samhitaa it has been recommended as a remedy for malignant ulcers.^[23] Among the isolated compounds, Phthalic anhydride, 3-Hydroxycarbofuran, 2-hydroxy-1,4-Naphthalenedione, n-Hexadecanoic acid, Phytol acetate, Vitamin E were obtained through GC/MS of Lawsoniainermis L are well known for their medicinal properties may possess anti-inflammatory, analgesic, and antipyretic activity, antioxidant and a preventive agent against epoxide-induced breast cancer, growth inhibition and apoptosis induction in human gastric cancer cells. It may also use as a remedy for jaundice, and malignant ulcers.

 

Early report said that henna is known to be used as a cosmetic agent for dyeing hair, nails and skin.^[41] Henna powder is pasted with water and applied to the part to be dyed, used as a colouring material. For dyeing hair, it is applied as a pack; it acts as a substantive dye for keratin and imparts an orange red colour. It is harmless and causes no irritation of skin.^[42] Use of natural dyes in colouration of textile materials and other purpose is just one of the consequences of increased environmental awareness.^[43] This study reported that leaves of Henna are harmless and because no irritation of skin. It may be used as a cosmetic agent and natural dyes for colouring the hair, nails and skin.

 

CONCLUSION:

From this study it may be concluded that leaf of Lawsonia inermis L contains many important phytochemical compounds like phthalic anhydride, 3-hydroxycarbofuran, 2-hydroxy-1,4-naphthalenedione, n-hexadecanoic acid, phytol acetate, vitamin E, alkaloids, anthraquinones, flavonoids, phenols, saponins, tannins and terpenoids. They may prove to be a potent antimicrobial, anti-inflammatory, analgesic, and antipyretic activities. This result may provide scientific support and potentially useful for the development of new chemotherapeutic agent. Lawsonia inermis L also consists of dye with lower toxicity and allergic reactions than synthetic dyes. Safety of natural dyes needs to be proved if they are used more widely and in commercial process.

 

ACKNOWLEDGMENT:

The authors would like to acknowledge to Mrs. A. Thenmozhi, Head of the Department and other faculty members, P.G. Department of Biochemistry, SRM Arts and Science College, Kattankulathur, Kanchipuram, district, for providing me the laboratory facilities, encouragement and support during this work.

 

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Received on 18.03.2019           Modified on 14.04.2019

Accepted on 15.05.2019         © RJPT All right reserved