Isolation, structural characterization and anti – inflammatory screening of three compounds from Strychnos colubrina Linn extract

Preethimol Francis, Suseem SR*

Department of Chemistry, School of Advanced Sciences, VIT, Vellore - 632014, Tamil Nadu, India.

*Corresponding Author E-mail: srsuseem@vit.ac.in

ABSTRACT:

Plants are the natural wealth of any nation. India is a country rich in medicinal plants. Most of the herbal medicines are the only available therapy for certain diseases; for which there are no alternative synthetic therapies are available, eg; chloroquinine for malaria, digoxin for CHF etc. The side effects of medicinal plants are less compared to synthetic medicines. The plant Strychnos colubrina Linn was widely used by the tribes for the treatment of many diseases like inflammations, chicken pox, tumor etc. The dried powdered plant parts were subjected to successive extractions using, pet. ether, chloroform, methanol and water. The extracts of the plant S.colubrina Linn was subjected to preliminary qualitative and quantitative analysis. Then based on the GC – MS analysis, the chloroform extract was subjected to isolation. Three compounds were identified (two isoflavone: daidzein and 5, 7-dihydroxy- 3- ( 3- hydroxy-4- 3,4,5 trihydroxy-6-(hydroxymethyl) tetrahydro pyran-2-yloxy) phenyl) chromen-4-one) and a phenolic compound (isovanillic acid)  The extract was subjected to in vitro anti – inflammatory screening and the isolated compounds were subjected to in vivo anti-inflammatory screening. All the three compounds were showed significant anti – inflammatory activity.

KEYWORDS: Medicinal plants, Strychnos, Anti – inflammatory, Daidzein, Isovanillic acid.

INTRODUCTION:

Plants form the basis of all life as it is lived upon the earth, because they alone have the power of making new supplies called organic substances, which are the only substances on which animals can feed out. The plant resources are providing us essential raw material for food, shelter, climatic balance and medicine. The existence of life on earth cannot be imagined without plants¹. The medicinal value of plant kingdom is only partially exploited. The various parts of the plants such as leaves, fruits, barks, roots, rhizomes or even flowers are used as medicines. All these plant parts are packed with active biomolecules like flavonoids, sterols, terpenes, nucleic acids, saponins, glycosides, alkaloids etc. These natural compounds formed the foundations of modern prescription drugs as we know today^2,3,4.

People turn to traditional medicine for complementary care is the increasing cases of chronic and debilitating diseases for which there is no cure. Scientific studies of several traditional medicine therapies show that their use is effective, e.g. for HIV / AIDS and cancer patients⁵. The advantages of traditional medicine include its diversity and flexibility; its availability and affordability in many parts of the world; its widespread acceptance in low - and middle - income countries; it’s comparatively low cost; and the relatively low level of technological input required. As a result, traditional medicine therapies have the potential to contribute to a better health care system in many countries^6,7.

The genus Strychnos is the largest genus of the family Loganiaceae. The plant Strychnos colubrina Linn was distributed in West coast tropical evergreen and semi evergreen forests. It extended over West deccan peninsula, Konkan to cochin. It was a large climbing shrub with thickened bifid tendrils. The root is used in snake bite, dyspepsia, malaria, intermittent fevers, swellings in  chicken pox and rhematism,  joint    pain, diarrhoea, anthelmint. The fresh leaves are used in tumor, rheumatism, inflammation. The bark is used in febrifuge, intermittent fever, dyspepsia, malarial cachexia, cutaneous eruptions and the fruit in mania⁸.

MATERIAL AND METHODS:

The plant Strychnos colubrina Linn was collected from Idukki district of Kerala in the month of October 2014. The plant was identified and authentified  from NISCAIR, Delhi. (NISCAIR / RHMD / Consult / 2016 / 2997 – 24). The collected whole plant was chopped into small pieces and the various parts were shade dried for a month. After sufficient drying, the bark and arieal parts were taken and ground into fine powder. The powdered sample was weighed and preserved in dry place, away from moisture. About 200 g of powdered plant material was weighed and defatted with petroleum ether (1 L) using soxhlet apparatus at 40°C for 48 hrs. Then the residue was extracted with 750 ml of chloroform, methanol and water successively. The extracts were concentrated in a rotary evaporator to yield a dark brown mass.

GC –MS Analysis

The GC - MS analysis of the S. colubrina Linn was performed using a Clarus 680 Perkin Elmer gas chromatography equipped with an Elite - 5 capillary column (5 % diphenyl, 95 % dimethyl polysiloxane) (30.0 m × 0.25 mm ID× 250 m) and mass detector turbo mass of the company which was operated in EI mode. Helium was the carries gas used at a flow rate of 1 ml / min. The injector was operated at 200°C and the oven temperature was programmed as follows: 60°C for 2 min and 10°C / min until 300 ° C. Interpretation of GC - MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight, and structure of the components of the test materials were ascertained^{9,10, 11}.

Isolation and structural characterization of compounds from chloroform extract

The chloroform extract of the plant was subjected to column chromatographic separation using normal phase silica gel column. The dark green solid plant extract was adsorbed on silica gel (20 g) and transferred to a column of silica gel (200 g equilibrated with pet.ether). Elution was performed with different solvents. Fractions of 100 ml were collected every time, distilled off the solvent and the homogeneity of the resulting residues was examined on TLC by using different solvent systems and similar fractions, identified by their TLC behaviour, were mixed together. The isolated compounds were subjected to NMR, IR, UV and LCMS. After isolation, the spectral analysis and interpretation of compounds were carried out^12-15.

In vivo anti - inflammatory screening by Carrageenan induced paw oedema method

Albino Wistar rats (120 – 180 g) were procured from in - house animal facility of J.S.S College of Pharmacy, Ootacamund. Animals were housed under standard laboratory conditions, (22 ± 3^° C) and relative humidity (30–70 %) with a 12:12 light: dark cycle] and maintained in a group of six, were housed in a standard polypropylene cage The animals were fed with standard pellet diet (M / s. Amrit feeds Ltd, Banglore, India) and water ad libitum. The experiment was conducted with prior approval of Institutional Animal Ethical Committee of JSS College of Pharmacy, Ooty, India (Approval No. JSSCP / IAEC / Ph.D / Ph.cology / 08 / 2016 -17).

The animals were grouped into five groups. Each group was consisted of six animals. Group – 1 was treated with distilled water and it was control group. Group – 2 was the standard group, animals treated with 10 mg / kg of indomethacin. Group -3, 4 and 5 were treated with 100 mg / kg of compound -1, compound – 2 and compound – 3 respectively.

About 60 min after, control, standard and test samples administration acute inflammation was induced in each group of animals. The inflammation was induced by the administration of 0.1 ml of 1 % (10 mg in 10 ml of normal saline) carrageenan solution to sub plantar region of right hind paw. A mark was made at the malleolus of leg to ensure uniform paw oedema measurement. The paw volume was measured at 0, 1, 2, 3, 4 and 5 hrs after induction of inflammation. The paw volume was measured by dipping the paw (only till the malleolus marking) in mercury in the plethysmograph. The initial and final volumes of mercury level during paw dipping were noted. The increase in the volume was calculated.^16-19

           (Paw volume of standard / sample treated group) 

%  Inhibition  =   1 -  ----------------------------------  x 100

                              Paw volume of control group 

RESULTS AND DISCUSSION:

The GC – MS analysis revealed the presence of more compounds in the chloroform and methanolic extracts. The spectras obtained for pet. ether, chloroform, methanol and water were shown in fig. 2, 3, 4 and fig . 5.  

Isolation of chloroform extract and structural interpretation of isolated compounds

Three compounds were isolated from the chloroform extract. The following was the ratio of solvent system and R_f value on TLC plate for the compounds.

Table. 1 Isolation of compounds from chloroform extract

Compound	Solvent system	Ratio	Rf value
Compound - 1	Chloroform: Methanol	7.5: 2.5	0.33
Compound - 2	Chloroform: Methanol	8.0: 2.0	0.65
Compound - 3	Chloroform: Methanol	6.5: 3.5	0.43

Structural characterization of isolated compounds

Compound-1

Colour                    :  Yellow, amorphous

Rf value                :  0.78 (solvent system BAW: 4 : 1 : 5)

Melting point       :  316 ° C

Solubility               :  Soluble in polar solvents like water, methanol

Qualitative test     :  Positive for flavonoids

Absorption maxima by UV Spectra :  374 nm, 256 nm

Figure. 1 UV, IR, LC - MS spectra, Proton NMR and ¹³C NMR of compound - 1

The ESI - MS (positive mode) spectra showed a pseudo molecular ion peak at m / z 255 [M + H]⁺ ion indicating a molecular formulae of C₁₅H₁₀O₄.The UV spectra showed an absorption peak with λ _max of 375 nm indicating  the presence of an α, β- unsaturated carbonyl system.The IR spectra showed absorption bands at 3000 cm^-1 for a hydroxyl group (OH), 1700 cm^-1 for a carbonyl group (C = O), 1,600 cm ^-1 for  C = C group  and at 1050 cm^-1 for a C-O group.In the ¹H - NMR spectra it showed signals at δ 6.58 (m, 2 H), 6.73 (m, 2 H), 7.2 (m, 2 H) and 7.91 (m, 2 H) suggesting the presence of eight aromatic / unsaturated protons and two hydroxyl protons at δ 8.1(m, 2 H) in the molecule. Further signals were observed in the aliphatic region at δ 0.34, 1.46 and 2.92 which were not integrated (it means they were from the solvent molecules). Further in the ¹³ C - NMR spectra signals were observed only in the aromatic region at δ 98.52, 103.42, 119.90, 125.3, 149.6, 152.1, 168.9 and 170.0 suggesting the presence of eight carbon atoms in the aromatic region of the molecule. Thus the compound was identified as Daidzein

Figure. 2 (7 – hydroxyl – 3 - (4 - hydroxyphenyl) – 4 H – 1 – benzopyran – 4 - one)

Compound - 2

Colour                :  Ivory, amorphous

Rf value             :  0.90 (solvent system BAW: 4 : 1 : 5)

Melting point    :  272 – 275 ° C

Solubility           :  Soluble in polar solvents like water, methanol

Qualitative test :  Positive for Phenolic compounds

Absorption maxima by UV Spectra :  280 nm

Figure. 3 UV, IR, LC - MS spectra, ¹³C NMR and Proton NMR of compound - 2

The ESI - MS (positive mode) spectra showed a pseudo molecular ion peak at m / z 157 [M + H] ⁺ ion indicating a molecular formulae of C₇H₁₀O₄. The UV spectra showed an absorption peak with λ _max of 280 nm indicating the presence of conjugated unsaturated system. The IR spectra showed a broad absorption band between 2500 and 3400 cm ^-1 showing the presence of a COOH group,    1650 cm ^-1 for a carbonyl group (C = O), 1600 cm ^-1 for  C = C group  and at 1150 cm ^-1 for a C - O group. In the ¹ H - NMR spectra it showed signals at δ 6.95 (m, 1 H), 7.71 (m, 2 H) suggesting the presence of only two aromatic protons and signals at δ 8.3 (m, 1 H) and 10.89 for phenolic and carboxylic protons in the molecule. Further signal observed at δ 3.93 may be due to the presence of a methoxy group.  In the ¹³ C - NMR spectra signals were observed at δ 23.4 for methoxy carbon, and in the aromatic region at δ 107.70, 115.51, 117.97, 130.59, 157.90, 160.02 and a carbonyl carbon at δ 166.1. Thus the compound was identified as Isovanillic acid.

Figure. 4 Isovanillic acid (3 - Hydroxy, 4 methoxybenzoic acid)

Compound - 3

Colour    :  white crystalline

Rf value :  0.92 (solvent system BAW: 4 : 1 : 5)

Melting point       :  185 – 190 ° C

Solubility               :  Not Soluble in water but soluble in methanol

Qualitative test     :  Positive for flavonoids

Absorption maxima by UV Spectra :  323 and 215 nm

Figure. 5 UV, IR,LCMS spectra, Proton NMR and ¹³ CNMR of compound - 3

The UV spectra showed an absorption peak with λ _max of 323 nm indicating the presence of an α, β - unsaturated carbonyl system. The IR spectra showed absorption bands at 3200 cm ^-1 for a hydroxyl group (OH), 1700 cm ^-1 for a carbonyl group (C = O), 1600 cm ^-1 for  C = C group  and at 1050 cm ^-1 for a C - O group. In the ¹ H - NMR spectra it showed signals at δ 6.14 (m, 1 H), 6.28 (m, 1 H), 6.9 (m,  1H) ,  7.0 (m, 1 H) , 7.15(m, 1 H) and 7.8 (m, 1 H) suggesting the presence of  six  aromatic  protons and four phenolic / hydroxy protons at δ 8.36 (m,1 H), 8.55 (1 H), 8.83 (1 H) and 8.96 (1H)  in the molecule. Further signals were observed in the aliphatic region at δ 2.78, 3.12, 3.66, and 4.24 for hydroxyl methane protons and the signal at δ 4.84 for an anomeric carbon atom. The compound was 5, 7 – dihydroxy – 3 - (3 hydroxy – 4 - (3,4,5 trihydroxy – 6 - (hydroxymethyl) tetrahydro pyran – 2 – yl oxy) phenyl) chromen – 4 – one.

Figure. 6 ( 5, 7 – dihydroxy – 3 - (3 hydroxy – 4 - (3, 4, 5 trihydroxy – 6 - (hydroxymethyl) tetrahydro pyran – 2 – yl oxy) phenyl) chromen – 4 - one

In vivo anti - inflammatory screening by Carrageenan induced paw oedema method

The inflammation induced by carrageenan is due to activation of inflammatory pathway through the release of various chemical mediators. The NSAID indomethacin was taken as the standard. The result of anti - -inflammatory screening was depicted in Table 2. The results of the study showed that compound -1 and compound - 3 were significant anti - inflammatory agents with a comparable potency with the standard drug.

Table 2 In vivo anti - inflammatory activity

Sl. No	Treatment (p.o)	1 hr	2 hr	3 hr	4 hr	5 hr
1.	Control (distilled water)	0.26 ± 0.28	0.62 ± 1.28	0.82 ± 1.27	1.02 ± 1.81	0.94 ± 0.64
2.	Indomethacin (10 mg / kg)	0.24  ± 1.28	0.28 ± 1.37 *	0.33 ± 1.72*	0.26 ± 1.64*	0.18 ± 1.39*
3.	PS - I (100 mg / kg)	0.27  ± 0.58	0.35 ± 1.11	0.46 ± 1.42*	0.42 ± 1.78*	0.24 ± 1.31*
4.	PS - I (200 mg / kg)	0.25  ± 0.31	0.29 ± 1.17 *	0.38 ± 1.78*	0.29 ± 1.84*	0.18 ± 1.13*
5.	PS - II (100 mg / kg)	0.24  ± 0.58	0.41 ± 1.36	0.48 ± 1.67	0.60 ± 1.29*	0.35 ± 1.18*
6.	PS - II (200 mg / kg)	0.27  ± 0.11	0. 36 ±1.08	0.42 ± 1.78*	0.55 ± 1.38*	0.30 ± 1.18*
7.	PS - III (100 mg / kg)	0.26  ± 0.51	0.37 ± 1.36	0.47 ± 1.12*	0.44 ± 1.18*	0.29 ± 1.31*
8.	PS - III (200 mg / kg)	0.25  ± 0.71	0.29 ± 1.58 *	0.39 ± 1.58*	0.27 ± 1.34*	0.19 ± 1.04*

Values were Mean ± SD, n = 6, * p < 0.05 when compared to control group. ANOVA followed by turkey’s test.

CONCLUSION:

The plant Strychnos colubrina Linn was a widely used plant by the tribes. There were only a few number of scientific studies on this plant. Thus the plant was selected for the study and it was collected from Idukki district of Kerala. The dried plant parts were dried, extracted and GC –MS analysis was carried out. The isolation on the chloroform extract yielded two isoflavones and a phenolic compound. The anti – inflammatory activity on this plant showed significant efficacy for the isolated compounds.

ACKNOWLEDGEMENT: 

The authors would like to thank VIT University for providing a platform for the study.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 21.11.2020            Modified on 19.06.2021

Accepted on 11.12.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(3):1235-1240.

DOI: 10.52711/0974-360X.2022.00206