INTRODUCTION:

The importance of medicinal plant in drug development is known to us and humans have been used them for different diseases from the beginning of human history ^(1). Plants are now occupying important position in allopathic, Herbal, Homeopathy, Aroma therapy and also used for spices and food plants.

Plants are cheaper and accessible to most people especially in the developing countries than orthodox medicine, there is lower incidence of adverse effect after used. These reasons might account for their world-wide attention and use.

It is a liana that reaches a length of 6m (20Feet) and scrambles over the vegetation. Storms are covered in curved spines. Its 2 cm (0.79°in) grey seeds known as nicker nuts ⁽⁴⁾ are buoyant and durable enough to be dispersed by ocean currents. It is reported to have multiple therapeutic properties like adaptogenic, anti-diabetic ⁽⁵⁾ anti-tumor, anti-filarial and anti-inflammatory.

Despite its many uses for medicinal purpose, the seed of this plant as not yet been scientifically evaluated in India. In the last few years GC-MS as become firmly enhanced as a key technological platform for secondary metabolite profiling in both plant and non-plant species^{(6) (7)}.

Therefore, the present study was focused to explore the proximate, phytochemical constituents of Caesalpinia bonducella using GC-MS analysis.

MATERIALS AND METHODS:

Collection and preparation of plant material:

Healthy disease free seeds of Caesalpinia bonducella were procured from the natural habitants of Sivagangai District, Tamil Nadu, and India. Further taxonomic identification was authenticated by Director of the Rapinat Herbarium and Centre for molecular systematics, St. Josephs College, Trichy. The fresh seeds of Caesalpinia bonducella were shade dried separately and ground into fine powder. The powdered materials were stored in air tight polythene bags until use.

Proximate analysis:

The air dried seeds were ground into a fine powder about 10.0g of the grounded seeds are exhaustively processed for various parameters. The proximate analysis of carbohydrates, moisture, crude protein, crude fiber, ether extract, ash, gross energy) of the seeds were determined by using AOAC Methods^{(8) (9)}. The content of the seed weight difference, moisture and ash content were also determined. The fiber content was estimated from the loss in the weight of the crucible and its content on ignition. The carbohydrates was determined when the sum of the percentage of moisture, ash, crude protein were subtracted form100.The nitrogen value which is the precursor for protein of a substance was determined by microkjeldahl method which involving Digestion, Distillation and finally titration of the sample. The nitrogen value was converted to protein by multiplying with a factor of 6.25.

Preparation of extract for phytochemical analysis:

Briefly, 10gm powder of Caesalpinia bonducella was extracted with 100ml various solvents including methanol, ethanol, aqueous, petroleum ether and chloroform by using a soxhlet apparatus for 16 hours. The crude extract obtained was filtered through Whatman filter paper. The obtained extracts were collected and stored in a sterile glass container for further phytochemical evaluation.

Phytochemical analysis:

Test for steroids:

One ml of Caesalpinia bonducella seeds extract, 2ml of acetic acid anhydride,1ml of ethanolic extract and 2 ml of sulphuric acid were added. Dark green, violet and blue coloration of the solution indicated the presence of steroids. ⁽¹⁰⁾

Test for terpenoids:

One ml of the Caesalpinia bonducella seeds extract, 2ml of chloroform and 3ml of conc.H₂SO₄ were carefully added to form a layer. A reddish brown coloration of the interface indicated the presence of terpenoids.

Test for flavonoids:

Alkaline reagent test:

One ml of the Caesalpinia bonducella seeds extract, a few drops of dilute NH₃and concentrated HCL was added. Formation of intense yellow color in the extract indicated the presence of flavonoids.

Led acetate test:

One ml of the Caesalpinia bonducella seeds extract, led acetate solution was added. Formation of yellow precipitate in the extract indicated the presence of flavonoids.

Test for saponins:

The Caesalpinia bonducella seeds extract was diluted with 5ml of distilled water and it was agitated in a graduated cylinder for 15 minutes. The formation of 1cm layer of foam showed the presence of saponins.

Test for phenols:

One ml of Caesalpinia bonducella seeds extract and few drops of led acetate were added. The formation of white precipitate indicated the presence of phenols.

Test for tannins:

Led acetate test:

Few ml extract and few ml led acetate was added and then formation of white precipitate indicated the presence of tannins.

Ferric chloride test:

One ml of Caesalpinia bonducella seeds extract and 2 ml of ferric chloride solution were added. Blue, black and brownish green coloration of the solution indicated the presence of tannins.

Test for cardiac glucosides:

One ml of Caesalpinia bonducella seeds extract, few ml of glacial acetic acid, 1ml of ferric chloride and concentrated H₂SO₄was added. Appearance of the reddish brown layer and bluish green colour showed the presence of cardiac glycosides.

Test for amino acid:

Dissolved a small quantity of the Caesalpinia bonducella seeds extract in few ml of water and added 1ml of ninhydrin reagent. Purple or blue colour indicated the presence of amino acids.

Test for proteins:

Xanthoprotein test:

One ml of Caesalpinia bonducella seeds extract with 1ml of con. HNO₃ was added then formation of white precipitate. Then it was boiled with 20% NaOH or NH_3. Appearance of orange colour showed the presence of proteins.

Test for carbohydrates:

One ml of Caesalpinia bonducella seeds extract boiled with barford reagent. Appearance of reddish brown precipitate showed the presence of carbohydrates.

Test for reducing sugar:

Fehling test:

A few drops of fehlings solution A and B were added in equal volumes were diluted with Caesalpinia bonducella seeds extracts, heated for 30 minutes and observed for the formation of brick red colour precipitate.

Benedicts test:

Five ml of benedict reagent were added to 1ml of Caesalpinia bonducella seeds extract, heated for 30 minutes and observed for the formation of red precipitate.

Test for alkaloids:

One ml of Caesalpinia bonducella seeds extract and 1ml of Dragendrop’s reagent were added and then formation of orange red precipitate indicated the presence of alkaloids.

Seed sample extraction for GC-MS:

10 g of the powdered seed of Caesalpinia bonducella was equilibrated with 200d/m of ethanol for 24h. The volume of the supernatant was later reduced by carefully heated to 2 d/m. The concentrated ethanolic extracts of the seed of Caesalpinia bonducella were analyzed using the Clarus 500 GC-MS (Perkin Elmer) equipment at Trichy Research Institute of Biotechnology, Trichy, Tamil Nadu, India.

GC-MS analysis:

GC-MS analysis of these extracts was carried out by following the method ⁽¹¹⁾.GC-MS analysis were performed using a Perkin-Elmer GC clauses 500 system and Gas Chromatograph interfaced to a mass spectrometer (GC-MS) equipped with a Elite-1, fused silica capillary column (30 m × 0.25 mm ID × 1 µ df, composed of 100% Dimethyl poly siloxane). For GC/MS detection, an electron ionization system with ionizing energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at constant flow rate 1 ml/min and an injection volume of 2 µl was employed (Split ratio of 10:1) injector temperature 250°C; ion-source temperature 280°C. The oven temperature was programmed from 110°C (isothermal for 2 min) with an increase of 10°C/min to 2000°C, then 5°C/min to 280°C, ending with a 9 min isothermal at 280°C. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 45 to 450 Da.

Total GC running time was 36 minutes. The relative % 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 a Turbo mass. The components were identified on the basis of retention time (RT) for GC and interpretation of mass spectrum was done by comparing spectral fragmentation obtained, to the database provided by Wiley Library and National Institute Standard and Technology (NIST 14LIB).

RESULTS:

The proximate analysis revealed the presence of Moisture content is (9.20%), Crude protein (10.05%), Crude Fiber (9.85%), Ether extract is (2.86%), Total Ash (7.33%), Carbohydrate is (18.41 mg g^-1) and Total Gross energy (3660Kcal/kg) were measured in the seed of Caesalpinia bonducella. All the proximate values were reported in % (AOAC 1990: AOCS 2000) ^{(8) (9)}. (Table:1)

Table: 1 Determination of proximate content from Caesalpinia bonducella seeds

S. No	Proximate content	Quantity (%)
1.	Moisture	9.20
2.	Crude protein	10.05
3.	Crude Fiber	9.85
4.	Ether extract	2.86
5.	Total Ash	7.33
6.	Carbohydrate	18.41
7.	Gross Energy	3660Kcal/kg

The phytochemical constituents of Caesalpinia bonducella seeds extracted by different solvents were analyzed and identified as multiple medicinally active components. (Table: 2) The methanol extract of Caesalpinia bonducella revealed the presence of saponins, tannins, Cardiac glycosides strongly and presence of proteins, carbohydrates, reducing sugars and alkaloids moderately. Its ethanol extract revealed the presence of terpenoids, saponins, cardiac glycosides, reducing sugars strongly and presence of flavonoids, phenols, tannins, amino acids, protein, carbohydrates and alkaloids were measured moderately.

Its aqueous extract revealed the presence of saponins, phenols, tannins, amino acids, proteins, alkaloids and steroids, flavonoids, carbohydrates, reducing sugar moderately. Its petroleum ether revealed the presence of terpenoids, saponins strongly and phenols, tannins, proteins, carbohydrates moderately. Its chloroform extract revealed the presence of steroids, terpenoids, cardiac glycosides, proteins, reducing sugar strongly and saponins, carbohydrates were measured moderately.

Figure 1- GC-MS spectrum analysis of Caesalipinia bonducella seeds

Table:3 GC-MS analysis of phytoactive components from Caesalpinia bonducella seeds

Peak	Retention time	Start time	End Time	m/z	Area	Area %	Height	Height %	A/H	M A R K	Name of the molecules
1	5.051	5.03	5.07	TIC	31798	0.08	29306	0.24	1.09		Silicic Acid (H4sio4), Tetraethyl Ester
2	13.524	13.495	13.545		49326	0.13	35400	0.3	1.39		1,2-Benzenedicarboxylic Acid, Diethyl Ester
3	13.775	13.74	13.845		1373428	3.58	837404	6.98	1.64		Benzene, 1,2,4-Trimethoxy-5-(1-Propenyl)-, (Z)-
4	14.54	14.52	14.68		32515	0.08	25413	0.21	1.28		1-Undecanol
5	14.705	14.68	14.74		200084	0.52	136320	1.14	1.47	V	1-Tetradecanol, Acrylate
6	15.088	15.055	15.15		488931	1.27	286533	2.39	1.71		Benzene, Ethylphenoxy-
7	15.428	15.39	15.48		228299	0.59	129125	1.08	1.77		Tetradecanoic Acid
8	16.009	15.98	16.055		125918	0.33	76063	0.63	1.66		Phenol, 2-(1-Phenylethyl)-
9	16.492	16.465	16.52		54034	0.14	35266	0.29	1.53		Pentadecanoic Acid
10	17.322	17.28	17.4		501370	1.31	210111	1.75	2.39		2,4-Decadienamide, N-Isobutyl-, (E,E)-
11	17.559	17.48	17.905		7174679	18.7	2187404	18.24	3.28		N-Hexadecanoic Acid
12	17.727	17.705	17.77		121275	0.32	91561	0.76	1.32		Benzenecarbothioic Acid, 2,4,6-Triethyl-, S-(2-Phenylethyl) Ester
13	17.849	17.785	17.895		145319	0.38	85712	0.71	1.7		Hexadecanoic Acid, Ethyl Ester
14	18.495	18.465	18.53		56703	0.15	34737	0.29	1.63		Heptadecanoic Acid
15	18.74	18.705	18.765		79924	0.21	46688	0.39	1.71		N-Nonadecanol-1
16	18.82	18.765	18.845		49130	0.13	24580	0.2	2	V	9,12-Octadecadien-1-Ol
17	19.211	19.13	19.23		2884557	7.52	895972	7.47	3.22		9,12-Octadecadienoic Acid (Z,Z)-
18	19.27	19.23	19.405		5070364	13.21	1418350	11.83	3.57	V	9-Octadecenoic Acid, (E)-
19	19.434	19.405	19.47		971828	2.53	325672	2.72	2.98	V	Linoleic Acid Ethyl Ester
20	19.486	19.47	19.525		469587	1.22	290432	2.42	1.62	V	Ethyl Oleate
21	19.849	19.825	19.89		57279	0.15	37242	0.31	1.54		Eicosyl Acetate
22	21.06	21.025	21.1		388269	1.01	262207	2.19	1.48		Retrofractamide-A
23	21.557	21.525	21.6		106348	0.28	42770	0.36	2.49		Tetrapentacontane, 1,54-Dibromo-
24	21.639	21.6	21.68		400682	1.04	239850	2	1.67	V	Phenol, 2,4-Bis(1-Phenylethyl)-
25	21.769	21.72	21.805		430676	1.12	240115	2	1.79		Phenol, 2,4-Bis(1-Phenylethyl)-
26	22.012	21.97	22.06		455164	1.19	242429	2.02	1.88		2-Hydroxy-4-Methoxy-7-Methyl-7,8,9,10,11,12,13,14-Octahydro-6-Oxabenzocyclododecen-5-One
27	22.223	22.185	22.28		886719	2.31	466878	3.89	1.9		Phenol, 2,4-Bis(1-Phenylethyl)-
28	22.402	22.365	22.44		198304	0.52	83385	0.7	2.38		Hexadecanoic Acid, 2-Hydroxy-1-(Hydroxymethyl)Ethyl Ester
29	22.488	22.44	22.535		568403	1.48	150626	1.26	3.77	V	Stigmasterol
30	22.604	22.535	22.69		33447177	8.72	1064205	8.87	3.14	V	2-T-Butyl -3-[ 2',7'-Di-T-Butylfluoren-4'-Yl] Propanoc Acid
31	22.934	22.835	23.02		3163196	8.24	824984	6.88	3.83		2-T-Butyl -3-[ 2',7'-Di-T-Butylfluoren-4'-Yl] Propanoc Acid
32	23.356	23.3	23.4		164799	0.43	53693	0.45	3.07		Carpesterol
33	23.945	23.91	24.055		140177	0.37	25945	0.22	5.4		Pregn-4-En-17(Alpha),20(Alpha)-Diol-3-One
34	24.09	24.055	24.11		148021	0.39	56347	0.47	2.63	V	Octadecanoic Acid, 2,3-Dihydroxypropyl Ester
35	24.231	24.11	24.325		2689211	7.01	387366	3.23	6.94	V	Stigmast-5-En-3-Ol, (3.Beta.,24s)-
36	24.405	24.325	24.45		729280	1.9	110077	0.92	6.63	V	N'-(2,2,6,6-Tetramethyl-4-Piperidinylidene) Acetohydrazide, Tbdms Derivative
37	24.49	24.45	24.585		1466560	3.82	183993	1.53	7.97	V	Piperine
38	24.674	24.585	24.86		2928992	7.63	320196	2.67	9.15	V	Fucosterol

1,2-Benzenedicarboxylic Acid, Diethyl Ester , Benzene, 1,2,4-Trimethoxy-5-(1-Propenyl)-, (Z)-, 1-Undecanol, 1-Tetradecanol, Acrylate, Benzene, Ethylphenoxy-, Tetradecanoic Acid, Phenol, 2-(1-Phenylethyl)-, Pentadecanoic Acid, 2,4-Decadienamide, N-Isobutyl-, (E,E)-, N-Hexadecanoic Acid, Benzenecarbothioic Acid, 2,4,6-Triethyl-, S-(2-Phenylethyl) Ester, Hexadecanoic Acid, Ethyl Ester, Heptadecanoic Acid, N-Nonadecanol-1, 9,12-Octadecadien-1-Ol, 9,12-Octadecadienoic Acid (Z,Z)-, 9-Octadecenoic Acid, (E)-, Linoleic Acid Ethyl Ester, Ethyl Oleate, Eicosyl Acetate, Retrofractamide-A, Tetrapentacontane, 1,54-Dibromo-, Phenol, 2,4-Bis(1-Phenylethyl)-, Phenol, 2,4-Bis(1-Phenylethyl)-, 2-Hydroxy-4-Methoxy-7-Methyl-7,8,9,10,11,12,13,14-Octahydro-6-Oxabenzocyclododecen-5-One, Phenol, 2,4-Bis(1-Phenylethyl)-, Hexadecanoic Acid, 2-Hydroxy-1-(Hydroxymethyl)Ethyl Ester, Stigmasterol, 2-T-Butyl -3-[ 2',7'-Di-T-Butylfluoren-4'-Yl] Propanoc Acid, Carpesterol, Pr, Egn-4-En-17(Alpha),20(Alpha)-Diol-3-One, Octadecanoic Acid, 2,3-Dihydroxypropyl Ester,Stigmast-5-En-3-Ol, (3.Beta.,24s)-, N'-(2,2,6,6-Tetramethyl Piperidinylidene) Acetohydrazide, Tbdms Derivative, Piperine, Fucosterol. The pharmacological activities recorded are based on Dr. Dukes phytochemical and ethno botanical database by Dr. Jim Duke of the Agricultural Research Service / USDA. (Table:4)

Table: 4 Compound Nature and Biological activity of Caesalpinia bonducella seeds in Ethanol extract

Compound Nature	Biological Activity
Silicic Acid (H4sio4), Tetraethyl Ester	Anti-diabetic activity
1,2-Benzenedicarboxylic Acid, Diethyl Ester	Antimicrobial and antifouling activity
Benzene, 1,2,4-Trimethoxy-5-(1-Propenyl)-, (Z)-	Antimicrobial activity
1-Undecanol	Flavoring agent
1-Tetradecanol, Acrylate	Antimicrobial activity
Benzene, Ethylphenoxy-	Antimicrobial activity
Tetradecanoic Acid	Larvicidal and repellent activity
Phenol, 2-(1-Phenylethyl)-	Antimicrobial activity
Pentadecanoic Acid	Antibacterial and antifungal activity
2,4-Decadienamide, N-Isobutyl-, (E,E)-	Hepatoprotective activity
N-Hexadecanoic Acid	Antimicrobial, anti-inflammatory, antioxidant, anti-diabetic, analgesic, hemolytic activity
Benzenecarbothioic Acid, 2,4,6-Triethyl-, S-(2-Phenylethyl) Ester	Antibacterial activity
Hexadecanoic Acid, Ethyl Ester	Hypercholesterdemic activity, lubricant, flavoring, cosmetics.
Heptadecanoic Acid	Antimicrobial activity
N-Nonadecanol-1	Antibacterial activity, anti-tubercular, cytotoxicity.
9,12-Octadecadienoic Acid (Z,Z)-	Antioxidant, hepatoprotective, Nematicide
9-Octadecenoic Acid, (E)-	Antimicrobial, anti-inflammatory, Hypercholesterdemic activity.
Phenol, 2,4-Bis(1-Phenylethyl)-	Antifungal activity
2-Hydroxy-4-Methoxy-7-Methyl-7,8,9,10,11,12,13,14-Octahydro-6-Oxabenzocyclododecen-5-One	Anti-inflammatory activity, Tissue building mechanism.
Phenol, 2,4-Bis(1-Phenylethyl)-	Hemolytic, pesticide, antioxidant, flavoring agent.
Pregn-4-En-17(Alpha),20(Alpha)-Diol-3-One	Co-solvent, oil carrier, anti-acne, anti-oxidant activity
Piperine	Anti-tumor, anti-oxidant, Antibacterial, anti-inflammatory, Insecticidal activity.
Fucosterol	Anti-diabetic, cytotoxic, antioxidant activity.

DISCUSSION:

The result of proximate analysis showed variant properties of biochemical and other contents. The result will serve as standard reference for identification and distinguishing characteristics of seed powder of Caesalpinia bonducella., its substituents and adulterants.

The phytochemical screening reveals the presence of Steroids, Terpenoids, Flavonoids, Saponins, Phenols, Tannins, Glycosides, Amino acid, proteins, Carbohydrates, reducing sugars and alkaloids.

Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties ⁽¹²⁾. Different phytochemicals have been found to possess a wide range of activities. The phytochemicals are known to have antimicrobial activity ⁽¹³⁾. Tannin has been found to possess astringent properties hasten the healing of wounds and inflamed mucous membranes ⁽¹⁴⁾. Tannin and flavonoid are thought to be responsible for antidiarrheal activity ⁽¹⁵⁾.

Phytochemicals such as terpenoids, flavonoid, tannin, steroid, and alkaloid have anti-inflammatory effects ⁽¹⁶⁾.Glycoside, flavonoid, tannin and alkaloid have hypoglycemic activities ⁽¹⁷⁾.Alkaloids are heterocyclic indole compounds which have proved to be having pharmacological properties such as hypotensive activity⁽¹⁸⁾ anticonvulsant activity ⁽¹⁹⁾.antiprotozoal, antimicrobial and antimalarial activities ⁽²⁰⁾.

Flavonoids are antioxidants and free radical scavengers which prevent oxidative cell damage, have strong anticancer activity and protect the cell against all stages of carcinogenesis ⁽¹⁴⁾.Flavanoids show anti allergic, anti-inflammatory, anti-microbial and anti-cancer activity ⁽²¹⁾.In the present report showed evidence that solvent extract of Caesalpinia bonducella contains medicinally important bioactive compounds and this justifies the use of Caesalpinia bonducella as traditional medicine for treatment of various diseases.

The prediction of the biological activities by applying the Duke’s databases was confirmed with previous observations and supplemented the traditional usage of the Caesalpinia bonducella. Among the identified phytochemicals, n-Hexadecanoic acid, Hexadecanoic acid, ethyl ester, Palmitic acid have the property of antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant activities and hemolytic 5-alpha is a reductase inhibitors ⁽²²⁾.

Hexadecanoic acid and ethyl ester acts as antifungal, antitumour, anti-bacterial. Hexadecanoic acid, 2-hydroxy-1- (hydroxymethyl) ethyl ester found to act as Hemolytic, pesticide, flavor and antioxidant.⁽²³⁾. Linolenic acid, ethyl ester found to act as Hypocholesterolemic, Nematicide, Antiarthritic, Hepatoprotective Antiandrogenic, Hypocholesterolemic, 5- Alpha reductaseinhibitor, Antihistaminic, Anticoronary, Insectifuge, Antieczemic, and Antiacne⁽²⁴⁾

Silicic acid have the property of anti- diabetic ⁽²⁵⁾, Tetradecanoic acid act as Antioxidant, antimicrobial, anticancer, hyper cholesteredemic and Cosmetic ^{( 26)(27)} 2-Hydroxy-4-Methoxy-7-Methyl-7,8,9,10,11,12,13,14-Octahydro-6-Oxabenzocyclododecen-5-One act as anti inflammatory activity⁽²⁸⁾. Phenol, 2,4-Bis(1-Phenylethyl)- acted as Anti inflammatory and tissue building mechanism^(29).

The identified compounds found in the ethanol extract of Caesalpinia bonducella seeds are being used for the pharmacological work. Thus this type of GC-MS analysis is the first step towards understanding the nature of active principles in the medicinal plants. This also upgrades several known and unknown bioactive compounds. Further exploration may lead to the development of drug formulation.

CONCLUSION:

Proximate, Phytochemical screening and GC-MS analysis of ethanol extract of Caesalpinia bonducella seeds revealed the presence of secondary metabolites of antidiabetic, anticancerous, antimicrobial, antioxidant, anti-inflammatory, antiproliferative, cytotoxic, Hypercholesterdemic activities and provides a potential source of industrial application. The present study concluded that the biological values of Caesalpinia bonducella seeds contain pharmacological active compounds that may enhance its used as a traditional drug.

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Received on 25.03.2019 Modified on 11.05.2019

Research J. Pharm. and Tech. 2019; 12(10):4628-4634.

DOI: 10.5958/0974-360X.2019.00796.0

Phytochemicals	Methanol	Ethanol	Aqueous	Pet ether	Chloroform
Steroids	-	-	++	-	+++
Terpenoids	-	+++	-	+++	+++
Flavanoids	-	++	++	-	-
Saponins	+++	+++	+++	+++	++
Phenols	-	++	+++	++	-
Tannins	+++	++	+++	++	-
Cardiac Glycoside	+++	+++	-	-	+++
Amino acids	-	++	+++	-	-
Proteins	++	+	+++	+	+++
Carbohydrates	++	++	+	+	+
Reducing sugars	++	+++	++	-	+++
Alkaloids	++	++	+++	-	-