Quantitative Determination of Alkaloid, Allicin, Glycoside and Saponin Constituents of the Leaves of Sansevieria senegambica Baker by Gas Chromatography.

Ikewuchi Catherine C., Ikewuchi Jude C.*, Ayalogu Edward O. and Onyeike Eugene N.

Department of Biochemistry, Faculty of Science, University of Port Harcourt, P.M.B. 5323, Choba, Nigeria.

ABSTRACT:

The alkaloid, allicin, glycoside and saponin levels of the leaves of Sansevieria senegambica were determined by gas liquid chromatography. The leaves are rich in alkaloids (93.76mg/kg wet weight and 293.91 mg/kg dry weight), with low allicin (1.2115 mg/kg wet weight and 3.7979 mg/kg dry weight) and saponin (0.94978 mg/kg wet weight and 2.97737 mg/kg dry weight), and very low glycoside (0.02547 mg/kg wet weight and 0.07985 mg/kg dry weight) contents. Twelve alkaloids were detected, consisting mainly of ambelline (about 24.39%), 6-hydoxybuphanidrine (18.10%) and crinamidine (17.73%). Of the three allicins detected, diallylthiosulphinate (about 52.70%) was the most abundant, while the most abundant saponin was avenacins B-1 (about 37.75%). These results show that the leaves are rich in alkaloids, lending credence to their use for medicinal purposes.

KEYWORDS: Alkaloids, allicins, glycosides, medicinal plants, phytochemical, Sansevieria senegambica, saponins.

INTRODUCTION:

Sansevieria senegambica (family Agavaceae or Ruscaceae) is one of the sixty (60) species of the genus Sansevieria¹, whose common names include mother-in-laws tongue, devils tongue and snake plant². It is native to tropical and subtropical regions of the world. It is grown as an ornamental plant². In southern Nigeria, it is used in traditional medical practice, for curing bronchitis, inflammation, cough and boils. It is also used in arresting snake bites, as well as in compounding solutions used as hair tonic.

The role of medicinal plants in disease prevention or control has been attributed to bioactive properties of their constituents, usually associated to a wide range of molecules, including alkaloids, allicins, glycosides and saponins. These compounds are commonly found in, and often responsible for actions of most medicinal plants. Alkaloids are compounds that contain nitrogen at a negative oxidation level³; allicins are sulfur-containing, while saponins are a large family of structurally diverse compounds containing a steroidal or triterpenoid aglycone linked to one or more oligosaccharide moieties. The pharmacological properties of alkaloids include antitumor^4,5,6, antidiabetic⁷, inflammatory⁸, antimalarial, antimicrobial and antituberculosis activities⁶. Those of allicin (diallylthiosulfinate) are insecticidal, hypolipidemic, antimicrobial, antioxidant, anti-thrombotic and anti-inflammatory activities^9,10. Saponins have detergent, piscicide, molluscicide¹¹, spermicidal, anti-inflammatory¹², hypocholesterolemic, diuretic, anti-diabetic, anti-ulcer⁸, hemolytic, immunostimulatory, antitumorigenic^4,8, chemoprotective¹³, hypoglycemic¹⁴ and cognition enhancing activities¹⁵.

A search of the literature showed that nothing is known about the chemical composition of Sansevieria senegambica. So, in the present study, we evaluated the alkaloid, allicin and saponin contents of the leaves of Sansevieria senegambica.

MATERIALS AND METHOD:

Collection of Plant Samples: Samples of fresh Sansevieria senegambica were procured from a horticulturist by Air Force Gate, Aba Road, Port Harcourt, Nigeria and from within Alikor Estate, Choba, Rivers Sate, Nigeria. After due identification at the University of Port Harcourt Herbarium, Port Harcourt, Nigeria, their leaves were collected, rid of dirt and stored for subsequent use. All reagents used were GC-grade purity.

Calibration: Standard solutions were prepared in methanol for alkaloids and allicins, ethanol for saponins. The linearity of the dependence of response on concentration was verified by regression analysis. The result of the calibration of the GC system is shown in Table 1.

Determination of Alkaloid Composition: The method reported by Tram et al.¹⁶ was adopted. To 30 g of the ground sample was added 250 mL of boiling deionized water and allowed to soak for 30minutes, before filtration. The filtrate was acidified to pH 4 with acetic acid, before extracting with 30 mL of petroleum spirit and chloroform. The acidic aqueous phase was made alkaline (pH 9), with 25% aqueous ammonia, and then extracted three times with 30 mL of chloroform. The chloroform extract was concentrated to 1.0 mL with the aid of rotary evaporator. The chloroform extract was then analyzed with gas chromatography. Chromatographic analyses were carried out on an HP 6890 (Hewlett Packard, Wilmington, DE, USA), GC apparatus, fitted with a flame ionization detector (FID) (range scanned: 220–500 nm), and powered with HP Chemstation Rev A 09.01 (1206) software, to quantify and identify compounds. The column was a capillary DB-5MS (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320°C. Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier gas. The hydrogen and compressed air pressures were 28 psi and 38 psi. The oven was programmed as follows: initial temperature at 60°C for 5 minutes. First ramping at 10°C/min for 20 min, followed by a second ramping at 15°C/min for 4 min. Identification was performed by comparisons of retention times with those of standard samples. Quantification was performed by establishing calibration curves for each compound determined, using the standards.

Determination of Allicin Composition: To 5.0 g of the pulverized sample in a pre-cleaned 500 mL borosilicate beaker was added 50 mL of 98% ethanol, and covered tightly with a Petri dish. The mixture was allowed to stand for 48 hours before filtering with a Whatman No. 1 filter paper, into a clean borosilicate container. The extract was concentrated in a rotary evaporator, and the resultant residue was dissolved in methanol for gas chromatographic analysis.

GC analysis was performed with a Hewlett–Packard (HP 6890) Series system, with a flame ionization detector (FID), and powered by HP Chemstation Rev A 09.01 (1206) software. The column was DB-5MS–capillary (30 m × 0.32 mm × 0.25 μm film thickness). Injector and detector temperatures were set at 220ºC and 250ºC, respectively. Split injection was adopted with a split ratio of 20:1. Helium was used as the carrier gas, at a flow rate of 1.0mL/min. The hydrogen and compressed air pressures were 22 psi and 28 psi. The column was held initially at 110°C for 2 min and then increased by 5°C per min up to 280°C. Identification was based on comparison of retention times and spectral data with standards. Quantification was performed by establishing calibration curves for each compound determined, using the standards.

Determination of Glycosides: 1.0 g of the pulverized sample was weighed into a pre-cleaned borosilicate beaker, and extracted by pouring 10 mL of ethanol/water (7:3) mixture on it, and allowing to stand for 2 hours. The mixture was filtered with Whatman No. 1 filter paper. The extract was purified by washing with lead acetate. The purified extract was further purified by adding sodium hydrogen phosphate. The extract was concentrated to 1 mL, for gas chromatographic analysis.

Chromatographic analyses were carried out on an HP 6890 (Hewlett Packard, Wilmington, DE, USA), GC apparatus, fitted with a flame ionization detector (FID), and powered with HP Chemstation Rev. A 09.01 [1206] software, to quantify and identify compounds. The column was a capillary DB-225MS Column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320°C. Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier gas. The hydrogen and compressed air pressures were 28 psi and 40 psi. The oven was programmed as follows: initial temperature at 60°C for 5 minutes. First ramping at 12°C/min for 18 min, followed by a second ramping at 15°C/min for 5 min. Identification was based on comparison of retention times and spectral data with standards. Quantification was performed by establishing calibration curves for each compound determined, using the standards.

Determination of the Saponin Composition: The method of Hanafy and Lobna¹² was adopted. The pulverized sampled was defatted with petroleum ether at 40⁰C for 3 hours. After filtering the petroleum ether, the sample was extracted with methanol for 3 hours, with mild heating.

Table 1: Calibration data of the HPLC system

Compound

Correlation constant

Relative resolution (%)

Equation

a. Alkaloids

Ø 9-octadecenamide

Ø Dihydro-oxo-demethoxyhaemanthamine

Ø Augustamine

Ø Oxoassoamine

Ø Crinane-3α-ol

Ø Buphanidrine

Ø Powelline

Ø Undulatine

Ø Ambelline

Ø 6-Hydroxybuphanidrine

Ø 6-Hydroxypowelline

Ø Crinamidine

Ø 6-Hydroxyundulatine

Ø 1β,2β-Epoxyambelline

Ø Epoxy-3,7-dimethoxycrinane-11-one

Ø 6-Hydroxycrinamidine

Ø Mitraphylin

0.99827

0.99970

0.99992

0.99969

0.99956

0.99955

0.99968

0.99912

0.99987

0.99949

0.99987

0.99974

0.99982

0.99983

0.99733

0.99997

0.99971

-11.764

-4.868

-2.530

-4.987

-5.909

-6.018

-5.075

-8.416

-3.226

-6.373

-3.226

-4.603

-3.846

-3.670

-14.634

-1.544

-4.828

Area=13.604448*Amt+0

Area=7.08082178*Amt+0

Area=5.56479993*Amt+0

Area=6.4172004*Amt+0

Area=5.76675781*Amt+0

Area=4.9754859*Amt+0

Area=63.21632*Amt+0

Area=3.0988*Amt+0

Area=0.496*Amt+0

Area=0.34648*Amt+0

Area=0.992*Amt+0

Area=0.26416*Amt+0

Area=0.832*Amt+0

Area=8.72*Amt+0

Area=0.984*Amt+0

Area=20.72*Amt+0

Area=7.21937244*Amt+0

b. Allicins

Ø Diallylthiosulphinate

Ø Methylallylthiosulphinate

Ø Allyl methylthiosulphinate

0.99761

0.99936

0.99965

-8.333

-4.211

-3.106

Area=7000*Amt-14000

Area=23600*Amt-24333.333

Area=170000*Amt-130000

c. Glycosides

Ouabain

Digitoxin

Digoxin

Salicin

Amygdalin

Arbutin

0.99957

0.99986

0.99983

0.99987

0.99980

0.99990

-5.858

-3.329

-3.723

-3.177

-3.972

-2.801

Area=76.381*Amt+0

Area=74.381*Amt+0

Area=64.4006*Amt+0

Area=74.409*Amt+0

Area=74.2626*Amt+0

Area=84.409*Amt+0

d. Saponins

Ø Avenacin A-1

Ø Avenacin B-1

Ø Avenacin A-2

Ø Avenacin B-2

0.99963

0.99930

0.99999

0.99981

-3.185

-4.418

-0.5571

-2.247

Area=4.55E13*Amt-3.5667E13

Area=620*Amt-670

Area=480*Amt-66.666667

Area=600*Amt-333.33333

Amt = amount.

Table 2: The alkaloid composition of the leaves of Sansevieria senegambica

Compounds

R. time (min)

Composition (mg/kg)

/Wet weight

/Dry weight

Total alkaloid content

Ø 9-Octadecenamide

Ø Dihydro-oxo-demethoxyhaemanthamine

Ø Augustamine

Ø Oxoassoamine

Ø Crinane-3α-ol

Ø Buphanidrine

Ø Powelline

Ø Undulatine

Ø Ambelline

Ø 6-Hydroxybuphanidrine

Ø 6-Hydroxypowelline

Ø Crinamidine

Ø 6-Hydroxyundulatine

Ø 1β,2β-Epoxyambelline

Ø Epoxy-3,7-dimethoxycrinane-11-one

Ø 6-Hydroxycrinamidine

Ø Mitraphylin

Ø Unidentified component

10.650

12.250

13.896

15.077

16.561

17.345

18.257

19.016

20.341

20.681

22.451

23.471

23.727

24.740

25.564

26.619

27.180

2.594

93.76

0.00

2.16

0.68

0.99

3.81

0.13

9.27

22.87

16.97

5.41

16.62

4.18

0.00

7.70

0.00

3.88

293.91

0.00

6.76

2.14

3.09

11.93

0.40

29.06

71.69

53.21

14.11

52.10

13.12

0.00

24.14

0.00

12.17

R. time = retention time.

The methanol extract was concentrated and re-extracted with methanol/acetone (1:5) mixture. The precipitate obtained was dried in vacuo, until it turned to a whitish amorphous powder, on complete drying. It was eluted on a silica gel (230-400 mesh) column, with chloroform/methanol/water (7:3:1). The first fraction collected was air dried at room temperature and the residue obtained was treated as pure saponins. The residue was dissolved in methanol for gas chromatographic analysis.

Chromatographic analyses were carried out on an HP 6890 (Hewlett Packard, Wilmington, DE, USA), GC apparatus, fitted with a flame ionization detector (FID), and powered with HP Chemstation Rev. A 09.01 [1206] software, to quantify and identify compounds. The column was a capillary DB-225MS Column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320°C. Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier gas. The hydrogen and compressed air pressures were 28 psi and 40 psi. The oven was programmed as follows: initial temperature at 60°C for 5 minutes. First ramping at 12°C/min for 18min, followed by a second ramping at 15°C/min for 5 min. The compounds appearing in chromatograms were identified on retention times and spectral data by comparison with standards. Quantification was performed by establishing calibration curves for each compound determined, using the standards.

Data Analysis: Comparisons were based on simple percentages.

RESULTS AND DISCUSSION

The alkaloids composition of the leaves of Sansevieria senegambica is shown in Table 2. They have high alkaloid content, which is mainly made up of ambelline (about 24.39%)^a, 6-hydoxybuphanidrine (18.10%) and crinamidine (17.73%), with moderate levels of undulatine (9.89%), epoxy-3,7-dimethoxycrinane-11-one (8.21%), 6-hydoxypowelline (4.80%) and 6-hydroxyundulatine (4.46%). Augustamine (2.30%), oxoassoamine (0.73%), crinane-3α-ol (1.05%), buphanidrine (4.06%), powelline (0.14%) and unidentified component were present in low levels, while 9-octadecenamide, dihydro-oxo-demethoxyhaemanthamine, 1β,2β-epoxyambelline, 6-hydroxycrinamidine and mitraphylin were not detected. Ambelline in a 1:1 combination with epoxyambelline produces pronounced activation of the spleen lymphocytes¹⁷.

The allicins composition of the leaves of Sansevieria senegambica is given in Table 3. The leaves have moderate levels of allicin content. It consisted mainly of diallylthiosulphinate (about 52.70%), with moderately lower levels of allyl methylthiosulphinate (20.15%) and methylallylthiosulphinate (27.15%). Diallylthiosulphinate (mainly called allicin) is reported to have antimicrobial, insecticidal, hypolipidemic, antihypertensive, anti-thrombotic, anti-inflammatory, antioxidant and anti-ulcer activities^9,10.

Table 3: Allicins composition of the leaves of Sansevieria senegambica

Compounds

R. time (min)

Composition (mg/kg)

/Wet weight

/Dry weight

Total allicins

Ø Diallylthiosulphinate

Ø Methyl allylthiosulphinate

Ø Allyl methylthiosulphinate

16.563

16.950

18.079

1.212

0.639

0.329

0.244

3.798

2.002

1.031

0.765

R. time = retention time.

The glycoside composition of Sansevieria liberica is given in Table 4. The leaves have very low glycosides content. The main glycosides were salicin (about 26.96%) and amygdalin (21.76%); others detected include ouabain (7.48%), digitoxin (12.78%), digoxin (15.69%) and arbutin (15.35%). Ouabain is a cardiotonic steroid¹⁸.

Table 4: Glycosides composition of the leaves of Sansevieria senegambica

Compounds

R. time (min)

Composition (x 10^-3 mg/kg)

/Wet weight

/Dry weight

Total glycosides

Ø Cardiac glycosides

· Ouabain

· Digitoxin

· Digoxin

Ø Salicin

Ø Amygdalin

Ø Arbutin

20.591

21.427

23.261

18.846

19.466

17.484

25.47

1.91

3.25

4.00

6.87

5.54

3.91

79.85

5.97

10.21

12.53

21.53

17.37

12.25

R. time = retention time.

Low saponin levels were recorded in the leaves (Table 5). This consisted mainly of avenacins B-1 (about 37.75%), with a moderately lower level of avenacin A-1 (26.33%) and B-2 (25.67%), and a low level of A-2 (10.25%). Saponins are reported to have broad range of pharmacological properties⁸. Avenacins have antimicrobial properties^19,20. Avenacin A-1 has antifungal activity^19,21.

Table 5: Saponins composition of the leaves of Sansevieria senegambica

Compounds

R. time (min)

Composition (x10^-1 mg/kg)

/Wet weight

/Dry weight

Total saponins

Ø Avenacin A-1

Ø Avenacin B-1

Ø Avenacin A-2

Ø Avenacin B-2

7.688

9.850

11.117

11.478

9.498

2.501

3.586

0.973

2.439

29.774

7.839

11.240

3.051

7.644

R. time = retention time.

In conclusion, this study showed that Sansevieria senegambica leaves are rich in alkaloids and moderate in allicins. This finding supports their medicinal uses.

Footnote:

Percentages are based on the weight of the compounds per its total extract, whether alkaloid, allicin, glycoside or saponin.

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Received on 25.01.2011

Modified on 14.02.2011

Accepted on 28.02.2011

Research J. Science and Tech. 3(6): Nov.-Dec. 2011: 308-312