Identification of Secondary Metabolites from the Ethanol extract of the leaves of Bauhinia tomentosa by GC-MS Analysis

 

R. Balabhaskar¹*, K. Vijayalakshmi²

¹Research Scholar, Department of Biochemistry, Bharathiar University, Coimbatore - 641046.

²Associate Professor, Department of Biochemistry, Bharathi Womens’ College, Chennai - 600 108.

 

ABSTRACT:

Gas Chromatography-Mass Spectrometry (GC-MS) is normally used for direct analysis of chemical components existing in herbal medicines. The medicinal plants are having numerous bioactive components which are identified even at less than 1ng by using GC-MS or LC-MS analysis. The aim of this study is to identify the secondary metabolites present in the leaves of B. tomentosa using gas chromatography-mass spectrometry (GC-MS) analysis. In the present study the ethanol extract of the leaves of Bauhinia tomentosa has been subjected to GC-MS analysis, while the mass spectra of the compounds found in the extract was matched with the National Institute of Standards and Technology (NIST) library. GC-MS analysis revealed the presence of 14 secondary metabolites. These compounds were identified by comparing their retention times and peak areas with those from the literature and by interpretation of the mass spectra. The major secondary metabolites were DL-.alpha.-tocopherol (14.84%), 2-[(trimethylsilyl oxy]-, methyl ester, 1-alpha,2-alpha.-epoxy-1-beta-methylcholesta-4,6-dien-3-one (12.93%), pentacosenoic acid (12.71%), phytol (10.28%), Ethyl Isoallocholate (8.197%), Spirost-8-en-11-one-3-hydroxy-,(3-beta,5 alpha,14 beta,20 beta, 22 beta,25R)-(8.162%), Urs-12-en-28-ol (6.675%), 1-Octadecyne (5.702%) and Cholest-8-en-3-beta-ol,Acetate (5.426%). The compounds having area less that 5% were considered of no significance. These findings suggest that the presence of these secondary metabolites may be the cause for the properties exhibited by Bauhinia tomentosa. Thus, presence of various bioactive compounds justifies the use of the leaf for various ailments by traditional practitioners.

 

 

 

INTRODUCTION:

Plants have always played a central role in traditional systems of medicine for the prevention and treatment of disease worldwide¹. It is important to understand that for thousands of years the traditional medicine in all countries exclusively employed naturally occurring plant products because they have vast and diverse assortment of organic compounds that can produce a definite physiological action on the human body². In the last years, interest in the antioxidant activity of plant extracts, or isolated substances from plants, has grown, especially in countries possessing great biodiversity.

 

Bauhinia tomentosa commonly known as Yellow bell orchid tree belongs to Fabaceae family and is one of the best, versatile and most commonly used household remedy for many manifestations. Tomentosa is derived from tomentose, meaning with dense, interwoven hairs. It is commonly known as ‘Iruvatchi’ in Tamil and ‘Kachnar’ in Hindi³. The decoction of the plant extract is used for the treatment of liver ailments and abdominal problems. Fruit is used as a diuretic. Flowers, buds, and dried leaves are used for dysentery treatment. Root bark is used for inflammation of liver. Seeds are tonic and aphrodisiac. Infusion of stem bark is useful as an astringent gargle. Leaves have anti-diabetic potential⁴. It is used for snake bite and scorpion sting⁵.

 

The modern methods describing the identification and quantification of active constituents in plant material may be useful for proper standardization of herbal drug formulations. In the recent years, gas-chromatography mass-spectrometry has become a key technological platform for metabolite profiling in plant^6,7. Gas Chromatography-Mass Spectroscopy (GC-MS), a hyphenated system is a very compatible technique and the most commonly used technique for the identification and quantification purpose. Mass spectrometry coupled with chromatographic separations, such as gas chromatography, is normally used for the direct analysis of the com­ponents that exist in both traditional medicines and medicinal plants. The medicinal plants are having numerous bioactive components which are identified even at less than 1ng by using GC-MS or LC-MS analysis. The unknown organic compounds in a complex mixture can be determined by interpretation and also by matching the spectra with reference spectra⁸. In recent years, GC-MS studies have been increasingly applied for the analysis of medicinal plants as this technique has proved to be a valuable method for the analysis of non-polar components and volatile essential oils, fatty acids, lipids and alkaloids^9,10.

 

The aim of the present study is to identify the phyto components of B.tomentosa by subjecting the ethanol extract of the leaves to GC-MS analysis. This work will help to identify the compounds, which may have therapeutic value.

 

MATERIALS AND METHODS:

Plant materials:

The leaves of Bauhinia tomentosa Linn were collected from Villivakkam, Chennai and authenticated by Director of Plant and Anatomy Research Centre, West Tambaram, Chennai (PARC/2014/2294).

 

Preparation of extract:

The leaves of Bauhinia tomentosa were first washed, shade dried and then ground into coarse powder using a mechanical grinder. 50g of the powdered leaf material was extracted with ethanol by hot percolation method.1μl of the ethanolic extract of leaves of Bauhinia tomentosa L. was employed for GC-MS analysis¹¹.

 

GC-MS analysis:

Instruments and chromatographic conditions:

The Clarus 680 GC used for the analysis employed a fused silica column, packed with Elite-5MS (5% biphenyl 95% dimethyl poly siloxane, 30m × 0.25mm ID × 250μm df) and the components were separated using Helium as carrier gas at a constant flow of 1 ml/min. The injector temperature was set at 260°C during the chromatographic run. The 1μL of extract sample injected into the instrument and the oven temperature was as follows: 60°C (2 min); followed by 300°C at the rate of 10°C min⁻¹; and 300°C, where it was held for 6 min. The mass detector conditions were: transfer line temperature 240°C; ion source temperature 240°C; and ionization mode electron impact at 70 eV, a scan time 0.2 sec and scan interval of 0.1 sec. The fragments were from 40 to 600 Da. The spectrums of the components were compared with the database of spectrum of known components stored in the GC-MS NIST (2008) library.

 

Identification of components:

The relative percentage of each component was calculated by comparing its average peak area to the total area. Interpretation of mass spectrum of GC-MS was done using the database of National Institute of Standard and Technology (NIST) having more than 62,000 patterns. The mass spectrum of the unknown component was compared with the spectra of the known components stored in the NIST library. The name, molecular weight and structure of the constituents isolated were ascertained. The compound prediction is based on Dr. Duke’s Phytochemical and Ethnobotanical Databases by Dr. Jim Duke of the Agricultural Research Service/USDA.

 

RESULTS AND DISCUSSION:

Gas Chromatography-Mass spectrometry (GC-MS) chromatogram of the ethanol extract of the leaves of Bauhinia tomentosa (Figure 1) showed 14 peaks indicating the presence of fourteen secondary metabolites. On comparison of the mass spectra of the constituents with the NIST library the 14 phytoconstituents were characterized and identified (figure 2.1-2.14). The chemical name, retention time (RT), molecular formula, molecular weight (MW), peak area (%) and biological activity of various phytochemicals (Dr. Duke's Phytochemical and Ethnobotanical Databases) are presented in Table 1 and Table 2.

 

The GC-MS analysis of the ethanol extract of Bauhinia tomentosa leaves revealed the presence of 14 secondary metabolites. Each of the secondary metabolite had different retention times and area percentage. In the present study, the secondary metabolites whose percentages were less than 5% were considered to be insignificant. The secondary metabolites that had significant area percentage were DL-.alpha.-tocopherol (14.84%), 2-[(trimethylsilyloxy]-, methylester, 1-alpha,2-alpha.-epoxy-1-beta-methylcholesta-4,6-dien-3-one (12.93%), pentacosenoic acid (12.71%), phytol (10.28%), Ethyl iso-allocholate (8.197%), Spirost-8-en-11-one-3-hydroxy-,(3-beta,5 alpha,14 beta,20 beta, 22 beta,25R)- (8.162%), Urs-12-en-28-ol (6.675%), 1-Octadecyne (5.702%) and Cholest-8-en-3-beta-ol,Acetate (5.426%).

 

Phenol and polyphenols are considered to be major contributors to the antioxidant property of fruits, vegetables and mushrooms. Phenolic compounds are potent water soluble and free radical scavenger which prevent oxidative cell damage¹². A number of studies have been focused on biological activities of phenolic compounds as potential antioxidants and free radical scavengers.

 

The phytochemical analysis conducted on the extract of Bauhinia tomentosa revealed the presence of constituents that are known to exhibit medicinal as well as physiological activities¹³. Ethanol extract of Bauhinia tomentosa contains 14.841% of alpha tocopherol. Alpha.-tocopherol has anti­oxidant uses in humans¹⁴. Natural antioxidants, which mainly come from plants, are in the form of phenolic compounds, such as flavonoids, phenolic acids and tocopherols¹⁵. Tocopherols, which are known biological antioxidants have also been shown to scavenge singlet molecular oxygen¹⁶ and to intercept lipid peroxyl radicals and their by terminating lipid peroxidation chain reactions¹⁷. Moreover, vitamin E has been reported to inhibit biosynthesis of prostaglandins in chickens¹⁸. Therefore, tocopherols by virtue of their free radical scavenging property and suppression of prostaglandin biosynthesis could be expected to possess anti-inflammatory activity.

 

12.936% of 1-alpha, 2-alpha.-epoxy-1-beta-methylcholesta-4,6-dien-3-one is present in ethanol extract of Bauhinia tomentosa. 1-alpha, 2-alpha.-epoxy-1-beta-methylcholesta-4,6-dien-3-one is also called as Cholesta-4, 6-dien-3-one . Cholesta-4, 6-dien-3-one is a product of the oxidation of cholesterol. Oxygenated derivatives of cholesterol (oxysterols) are widely distributed in nature, being found in the blood and tissues of animals and man as well as in foodstuff. They exhibit many biological activities which are of potential physiological, pathological or pharmacological importance. Oxy sterols also inhibit cell replication and have cytotoxic properties which suggest that these sterols may participate in the regulation of cell proliferation and may be potentially useful as therapeutic agents for cancer¹⁹.

 

10.280% of Phytol is present in ethanol extract of Bauhinia tomentosa. It has been reported to possess antitumor activity and antioxidant activity. Phytol was able to reduce the production of free radicals, and this activity can be attributed to their structural feature, since phytol is a branched-chain unsaturated alcohol and its antioxidant properties may be related to the hydroxyl group (OH) present in its molecule. Probably, phytol, by reacting with a free radical, donates hydrogen atoms with an unpaired electron (H^∙), converting free radicals into less reactive species²⁰. This result suggests that phytol can exert an antioxidant activity that protects the lipid biomolecules²¹. The ability of a substance scavenging OH is directly related to its antioxidant activity. The OH is an extremely reactive species capable of causing damage to DNA, proteins, and lipids^22,23,24. Phytol could remove OH^∙, exhibiting antioxidant activity which may be capable of inhibiting cell damage caused by this radical.

 

Ethyl iso-allocholate has been reported for its anti-inflammatory, anticancer, antimicrobial, antiasthma and diuretic properties^25,26. The area of ethyl iso-allocholate was found to be 8.197%.

 

Pentacosenoic acid was found to have an area of 12.710%. They have been reported for diverse biological functions including antioxidant, acidifier and acidulant activities. Pentacosenoic acid, 2-[(trimethylsilyl)oxy]-, methyl ester are methylguanidines and they are hydroxyl radical scavengers. However, guanidine has no effect on hydroxyl radical generating system, suggesting that amino or methyl substitution of a guanidine molecule is necessary for the hydroxyl radical scavenging activity. Since aminoguanidine^27,28 as well as methylguanidine and guanidine²⁹ are shown to be NOS inhibitors and inhibition of nitric oxide formation will inhibit the peroxynitrite production.

 

Hexadecanoic acid was reported with following activities like flavour, antibacterial³⁰, cosmetic and perfumery³¹, hypercholesterolemic and lubricant³². 1.832 % of Hexadecanoic acid was found to be present in ethanol extract of Bauhinia tomentosa.

 

Urs-12-en-28-ol belongs to class of natural products found especially in plants. Urs-12-en-28-ol is also called as α-amyrin. This triterpene exhibits important biological and pharmacological activity, including anti-inflammatory, antimicrobial, antiviral, cytotoxic and cardiovascular effects³³. The area of α-amyrin was found to be 6.675%. The compounds α-amyrin and β-amyrin, commonly found in medicinal plants, have many bio-active properties. Some studies have demonstrated that the α/β amyrin triterpene mixture also has many biological functions, including analgesic, antimicrobial, anti-inflammatory properties³⁰. Spirost-8-en-11-one,3-hydroxy-,(3.beta.,5.alpha.,14.beta.,20.beta.,22.beta.,25r)-has a area of 8.162%. It has been found to possess anti-cancer activity³⁴.

 

 

Figure 1: GC-MS Chromatogram of ethanol extract of Bauhinia tomentosa

 

2.1. Phytol:

 

 

2.2.  3,7,11,15-tetramethyl-2-hexadecen-1-ol

 

 

2.3.  1-octadecyne

 

 

2.4. Hexadecanoic acid, ethyl ester

 

 

2.5. Pentacosenoic acid, 2-[(trimethylsilyl)oxy]-, methyl ester

 

 

2.6. 1,2-pentanediol,5-(6-bromodecahydro-2-hydroxy-2,5,5a,8a-tetramethyl-1-Naphthalenyl-3-Methylene

 

 

2.7. 1.alpha.,2.alpha.-epoxy-1.beta.-methylcholesta-4,6-dien-3-one

 

 

2.8. Ethyl iso-allocholate

 

 

2.9. Urs-12-en-28-ol

 

 

2.10. DL-.alpha.-tocopherol

 

 

2.11. Spirost-8-en-11-one,3-hydroxy-, (3.beta., 5.alpha.,14.beta., 20.beta.,22.beta., 25r)-

 

 

2.12. 22,23-dibromostigmasterol acetate

 

 

2.13. 2,4,4-trimethyl-3-hydroxymethyl-5a-(3-methyl-but-2-enyl)-cyclohexene

 

 

2.14. Cholest-8-en-3.beta.-ol, acetate:

 

Figure 2.1.to 2.14.: Mass Spectrum of all the bioactive components in Ethanol extract of Bauhinia tomentosa.

 

Table 1: Compounds present in ethanol extract of Bauhinia tomentosa leaves

S. No	Peak No.	RT	Area %	Name of Compound	M.Wt	Formula
1	2	16.944	10.280	Phytol	296	C20H40O
2	3	17.239	3.857	3,7,11,15-tetramethyl-2-hexadecen-1-ol	296	C20H40O
3	4	17.439	5.702	1-octadecyne	296	C20H40O
4	5	18.760	1.832	Hexadecanoic acid, ethyl ester	284	C18H36O2
5	6	24.792	12.710	Pentacosenoic acid, 2-[(trimethylsilyl)oxy]-, methyl ester	482	C29H58O3Si
6	7	25.212	2.619	1,2-pentanediol, 5-(6-bromodecahydro-2-hydroxy-2,5,5a,8a-tetramethyl-1-Naphthalenyl-3-Methylene	486	C24H39O5Br
7	8	25.903	12.936	1.alpha.,2.alpha.-epoxy-1.beta.-methylcholesta-4,6-dien-3-one	410	C28H42O2
8	9	26.188	8.197	Ethyl isoallocholate	436	C26H44O5
9	10	26.398	6.675	Urs-12-en-28-ol	426	C30H50O
10	11	26.823	14.841	Dl-.alpha.-tocopherol	430	C29H50O2
11	12	27.163	8.162	Spirost-8-en-11-one,3-hydroxy-, (3.beta.,5.alpha.,14.beta.,20.beta.,22.beta.,25r)-	428	C27H40O4
12	13	29.529	1.682	22,23-dibromostigmasterol acetate	612	C31H50O2Br2
13	15	30.644	2.986	2,4,4-trimethyl-3-hydroxymethyl-5a-(3-methyl-but-2-enyl)-cyclohexene	222	C15H26O
14	16	30.975	5.426	Cholest-8-en-3.beta.-ol, acetate	428	C29H48O2

 

Table 2: Biological activities of compounds in Bauhinia tomentosa

S. No.	RT	Area %	Name of Compound	Biological Activity
1	16.944	10.280	Phytol	Anti tumour
2	24.792	12.710	Pentacosenoic acid, 2-[(trimethylsilyl)oxy]-, methyl ester	Acidifier, Acidulant, Arachidonic acid inhibitor,
3	25.903	12.936	1.alpha.,2.alpha.-epoxy-1.beta.-methylcholesta-4,6-dien-3-one	Anti oxidant
4	26.188	8.197	Ethyl iso-allocholate	Anti tumour
5	26.823	14.841	DL-.alpha.-tocopherol	Antioxidant, Peroxide radical scavenger
6	27.163	8.162	Spirost-8-en-11-one,3-hydroxy-, (3.beta.,5.alpha.,14.beta.,20.beta.,22.beta.,25r)-	Anticancer
7	26.398	6.675	Urs-12-en-28-ol	Antiprotozoal, Antimicrobial, Anti inflammatory, Antitumor and chemopreventive properties
8	17.439	5.702	1-octadecyne	Anti inflammatory; cytotoxic activity; anti microbial
9	30.975	5.426	Cholest-8-en-3.beta.-ol, acetate	Inhibition of sodium fluorescein uptake

 

Thus, the results of GC-MS analysis of ethanol extract of Bauhinia tomentosa leaves demonstrate the presence of many secondary metabolites that possess a range of biological activities. The presence of these many compounds in the leaf extract of Bauhinia tomentosa suggests that the plant may play a significant role in the treatment of various ailments.

CONCLUSION:

In the present study fourteen secondary metabolites have been identified from ethanolic extract of the leaves of Bauhinia tomentosa by GC-MS analysis. The presence of various bioactive compounds justifies the use of the leaf for various ailments by traditional practitioners. However, isolation of individual secondary metabolites and subjecting it to elucidate their biological activity will be more beneficial. It could be concluded that the ethanol extract of the leaves of Bauhinia tomentosa contains various bioactive compounds. Hence, the plant can be considered to have therapeutic potential.

 

CONFLICT OF INTEREST:

The authors declare no conflict of Interest.

 

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Received on 15.05.2020           Modified on 24.06.2020

Accepted on 21.07.2020         © RJPT All right reserved