Therapeutic properties of a poisonous plant Cleistanthus collinus

A. Amala Lourthuraj^1*, M. Masilamani Selvam¹, Bharathi Ravikrishnan², Murugan R²

¹Department of Biotechnology, Sathyabama University, Rajiv Gandhi Salai, Chennai, India.

²Department of Biotechnology, Hindustan College of Arts and Science, Chennai, India.

*Corresponding Author E-mail: a.amalraaj@gmail.com

ABSTRACT:

Cleistanthus collinus (Roxb.) Benth. ex Hook. f. is a familiar plant which is available in many parts of India. It is commonly known as Oduvanthalai in Tamil. Though, this plant is widely used for suicidal purposes, it is now increasingly investigated for its therapeutic properties in the area of phytochemical research. The present study is designed to check the antimicrobial properties of Cleistanthus collinus leaf extract against the pathogens like Methicillin Resistant Staphylococcus aureus (MRSA), Enterococcus and Candida spp.. Mostly these pathogens are the causative agents for skin infections and urinary tract infections. Cleistanthus collinus leaves were collected and the crude extract was prepared using various organic solvents and water. Thin layer chromatography with suitable stationary phase and mobile phase was employed to check the presence of active principles of the plant leaves. One of the chief active principles, Cleistanthin A was isolated and partially characterized using chromatography methods and spectroscopy analysis. Antioxidant assay was also performed with the plant extract. The results reveal positive medicinal properties of the plant leaves.

KEYWORDS: Cleistanthus collinus, Skin infection, Cleistanthin A, Antimicrobial assay, Antioxidant assay.

INTRODUCTION:

Among various needs and desires, living a healthy life always remains on top of everyone’s desire, since nothing could worth better than health. Hence, the ultimate intention for most of the research is the wellbeing of all forms of life on earth. In all age of human life, we relied on plants for most of our basic needs (David J. Newman, 200). Recent advancement in drug development technology significantly increased the interest on natural products (Frank E. Koehn, 2005). Currently, More than 100 numbers of anticancer and anti-infective drugs of plant origins are in clinical drug development stages (Alan, 2008).

Cleistanthus collinus (Benth and Hook F. 1887) is an interesting type of plant, which is usually known as Oduvanthalai in Tamil. It’s a kind of shrub grows on hot hill side area in most parts of rural India. The active principles of the plant C. collinus were isolated and reported by various groups worldwide.

The chief chemical constituents of the plant leave Collinusin, Diphyllin, and Cleistanthin was reported (Govindachari et al. 1969). Later, Cleistanthin was renamed as Cleistanthin A, after another similar type of compound from the same plant was found and lately isolated compound was named as Cleistanthin B (Laxmi et al., 1970).

The most interesting part of this plant relies on its dual performance. One part of report highlights their toxicological profiles. Especially, the leaves are widely used for suicidal purposes in rural area. The consumption of leaves results in renal tubular dysfunction (dRTA) (Delinda et al., 2010), acute respiratory distress syndrome (ARDS), hypokalemia, cardiac abnormalities, renal failures, metabolic acidosis etc (Benjamin et al., 2006). However, the other research groups pointed out the therapeutic potentials like insecticidal (Bharti Ahiwar., 2011), larvicidal (Ramar et al., 2014), antimicrobial (Kalaivanan et al., 2014), antifungal and antiseptic (Maji et al., 2010), diuretic (Parasuraman et al. 2012) and anticancer properties (Pradheep kumar CP, 1999) of the same plant.

The versatile actions of such plant are mainly possible because of the existence of diverse group of phytochemicals and other definite active principles of the plant. Advancements in the field of bioseparation and characterization technologies support the researchers to work on their determined point of view. In this current research, we report the effect of one of the purified active principles of the plant against the organisms causing skin infections. We also report the antioxidant properties of the crude extract and the purified fraction Cleistanthin A.

MATERIAL AND METHODS:

Sample collection: The fresh plant leaves were collected from a hill side near Vellore, Tamil Nadu state in India.

Extract Preparation:

The fresh leaves were cleansed with distilled water, shade dried, semi crushed and stored in glass bottles. 25grams of semi-crushed leaves were soaked in 100 ml of Acetone of AR grade in a 250 ml conical flask. The flask was covered with thick folds of aluminium foil and kept in hot plate at mild temperature not exceeding 30° C for 6 hours. The flask was taken out from the hot plate at every hour, shaken well then kept back. After 6 hours, the flask was taken out from the hot plate and kept in room temperature overnight. The next morning, the extract was filtered using a piece of cotton and funnel. The filtrate was concentrated using rotary evaporator and final volume was brought around 20 ml, poured into a Petri plate and kept in oven at 40° C overnight. The next day, the dried powder was scrapped out from the plate using a sterile blade, stored in eppendorf tubes and used for further analysis.

TLC Profiling:

Silica gel – G was used as stationary phase and Hexane: Chloroform: Ethanol in the ratio of 1:1:0.1 were used as mobile phase (Annapoorani KS et al., 1984). The dried form of acetone extract was taken in a small quantity, mixed with acetone and applied to the activated TLC plate, which was kept in a closed TLC chamber and left undisturbed. When the solvent front reached the top, the plate was taken and visualized under UV light illuminator chamber.

Compound Isolation:

Two important active principles were isolated using liquid-liquid extraction method. Preparatory TLC was employed to isolate and purify the compounds. (The detailed methodology is obtained and followed from a well known medical institution Christian Medical College, Vellore. (unpublished).

Characterization:

UV-Vis and FR-IR analysis were done to characterize the purified compound. Analytical TLC was done to compare the isolated compound with the authenticated samples.

Clinical Isolates Description:

The three clinical isolates used in this research are MRSA strain, Enterococcus sp. and Candida sp. These cultures were obtained from the Pathology Lab of Rajiv Gandhi Government Hospital, Chennai. MRSA strain was maintained in Mannitol salt agar slant, Enterococcus sp. and Candida sp. were maintained in Nutrient agar. All these cultures were obtained from the patient with specific infections.

Antimicrobial assay:

This assay was performed as disc diffusion methodology (Kirby Bauer’s method, 1959). 10µl of standard drugs are used as control antibiotics. For MRSA and Enterococcus bacteria Bactroban was used as standard drug, for Candida sp., itch guard was used. The anti bacterial activity of the plant sample was detected by disc diffusion method, in which the plant samples were suspended in DMSO (as the solvent) and discs were prepared by adding 50µl of each sample.

Antioxidant Activity:

The antioxidant activity of the samples were analyzed using Vitamin C as standard and DPPH as the substrate. The antioxidant activity of DPPH activity was performed by Brand-Williams methodology, 1995. The antioxidant activity was calculated by the below mentioned formula:

% of inhibition = (Abs control- Abs sample) x 100

Abs control

Where, Abs of control- Vitamin C in distilled water and Abs of plant sample- DSMO

RESULTS AND DISCUSSION:

The conventional method of bioseparation was very handful in the preparation of crude extract as well as the isolation and purification of active principles present in the plant leaves. The below figures depicts the steps involved in isolation (Fig. 1, 2 and 3).

Fig.3. Dried acetone extract

TLC profiling (Annapoorani KS., 1984) was done with the crude extract, purified fraction along with the authenticated sample. The authenticated sample used here was obtained from the Dept. of Physiology, Christian Medical College, Vellore. The image was also taken at the same place. The first lane from left was acetone extract, which shows most of the active principles. The second lane represents the purified Cleistanthin A and the third lane represents authenticated Cleistanthin A. The Rf value of the purified compound matched with the crude extract and the authenticated sample. The TLC profile shows the presence of single fluorescent spot in lane 2 (Purified compound) and lane 3 (authenticated sample).

Fig.4. Lane 1 – Acetone extract, Lane 2 – Purified Cleistanthin A, Lane 3 – Authenticated Cleistanthin A

UV –Vis Spectrophotometer was done with one of the purified active principle. There is a major peak present at 263 nm. Since, cleistanthin A is a glycoside, the major peak at 263 represents our compound.

Graph 1. UV Data of Cleistanthin A

The FT –IR Analysis of Cleistanthin A (Graph 2) is given below. The IUPAC name of our compound is 9-(1,3-benzodioxol-5-yl)-4-[(3R,4R,5R)-3-hydroxy-4,5-dimethoxyoxan-2-yl]oxy-6,7-dimethoxy-3Hbenzo[f][2] benzofuran-1-one. The FT-IR data represents the presence of all functional groups.

Graph 2. FT – IR data of Cleistanthin A

The antimicrobial property of our purified compound and crude acetone extract were compared with the commercially available drugs. Cleistanthin A showed significant activity with all the three pathogens. Acetone extract showed moderate inhibitory action with Enterococcus and Candida spp.

Antioxidant assay:

Antioxidant properties of the samples were checked using DPPH assay (Brand-Williams., 1995). The OD values of the standard samples of different concentrations, acetone extract and Cleistanthin A were taken using UV- Vis spectrophotometer. Based on the value and the specific formula, the percentage of inhibition was calculated.

Table 2: Antioxidant Activity

Sl. No.	Name of the Sample	Absorbance of Sample	% of Inhibition
1	S1	0.10	-
2	S2	0.12	-
3	S3	0.15	-
4	S4	0.18	-
5	S5	0.22	-
6	Clei A	0.19	-87.55
7	AE	0.26	-58.61

Calculation:

Percentage of inhibition (Cleistanthin A)

=0.18-0.19÷0.18×100 = -87.55%

Percentage of inhibition (Acetone Extract)

=0.22-0.26÷0.22×100 = -58.61%

CONCLUSION:

There are many reports which confirm the toxic effect of Cleistanthus collinus leaf extract. From this current study the biopotentials of Cleistanthus collinus leaf is evident. The extracted compound from Cleistanthus collinus leaf is Cleistanthin A which exhibited antimicrobial activity against MRSA, Candida sp. and Enterococcus sp. Even though the zone of inhibition exhibited by test microbes are lesser than the standard compounds, the natural compound when used for in vivo purpose, side effects can be prevented. The antibacterial activity of Cleistanthin A may be due to its antioxidant property. The antioxidant property of Cleistanthin A was confirmed by the rate of inhibition of DPPH activity. The antioxidant property exhibited by Cleistanthin A is greater than the standard compound Vitamin C. Further research must be generated to confirm the same effect of natural occurring compound Cleistanthin A in in vivo conditions also.

ACKNOWLEDGEMENT:

Authors sincerely thank Dr. Sathya Subramani, Professor and Head, Department of Physiology, Christian Medical College, Vellore for her valuable guidance and support to carry out the isolation work.

REFERENCES:

1. Alan L. Harvey. Natural products in drug discovery. Drug Discovery Today, 2008. Volume 13, Issue 19, 894-901.

2. Bauer, A. W., D. M. Perry, and W. M. M. Kirby. 1959. Single disc antibiotic sensitivity testing of Staphylococci. A.M.A. Arch. Intern. Med. 104:208–216.

3. Benth. ex Hook.f. Cleistanthus collinus (Roxb.) Fl. Brit. India 5: 274. 1887;

4. Benjamin, M Edwin Fernando, J Jerene Jayanth, B Preetha. Cleistanthus Collinus Poisoning. JAPI. 2000; (54): 742-746

5. Bharti Ahirwar , Dheeraj Ahirwar and Sanjay Lanjhiayana. Insecticidal activity of Cleistanthus collinus Roxb. Der Pharma Chemica, 2011; 3 (1):49-52.

6. Brand-Williams W, Cuvelier ME, Berset C. Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie. 1995;28:25-30.

7. Delinda Maneksh, Anitha S, Pragalathan K, , Amala Lourthuraj A, Anup R, Sathya S. Cleistanthus collinus induces type I distal renal tubular acidosis and type II respiratory failure in rats. Indian J of Pharmacol. 2010; (42), 3: 178-184.

8. Govindachari T.R, Sathe S, Viswanathan N, Pai B.R, Srinivasan M. Chemical constituents of Cleistanthus collinus (Roxb.). Tetrahedran. 1969; 25 (14), : 2815-2821.

9. Kalaivanan, M. Chandrasekaran and V. Venkatesalu. Evaluation of antibacterial efficacy of leaves of Cleistanthus collinus and Strychnos nux-vomica. International Journal of Natural Products Research 2014; 4(2): 65-71

10. Kavitha Priya Kettimuthu, A. Amala Lourthuraj, A. Soosai Manickam, Sathya Subramani, and Anup Ramachandran. Mechanisms of toxicity of Cleistanthus collinus: Vacuolar ATPases are a putative target. Clinical Toxicology (2011), 49, 457–463

11. Lakshmi TG, Srimannarayana G and Subbha Rav NV. A new glycoside from Cleistanthus collinus. Curr. Sci. 1970;(39): 395-396 .

12. Maji S, Dandapat P, Ojha T, Maity C, Halder SK, Das Mohapatra PK , Pathak TK, Pati BR, Samanta A and Mondal KC. In vitro antimicrobial potentialities of different solvent extracts of ethnomedicinal plants against clinically isolated human pathogens. Journal of Phytology. 2010, 2(4): 57–64

13. Pradeepkumar, C,P; Shanmugam,G. Anticancer potential of cleistanthin A isolated from tropical plant Cleistanthus collinus. Oncol. Res. 1999; 11(5): 225-32.

14. Pasrasuraman S and Raveendran R. Diuretic Effects of Cleistanthin A and Cleistanthin B from the Leaves of Cleistanthus collinus in Wistar Rats. J Young Pharm. 2012 Apr-Jun; 4(2): 73–77.C.

15. Ramar G, Suman T, Elangomathavan R, Jeyasankar A. Larvicidal activity of biologically synthesised silver nanoparticles against dengue vector Aedes Aegypti (Culicidae). Discovery. 2014, 9(23), 65-68.

Received on 16.03.2016 Modified on 04.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 365-368

DOI: 10.5958/0974-360X.2016.00065.2

Sl. No.	Pathogens	Zone of inhibition in mm
Sl. No.	Pathogens	Acetone Extract	Cleistanthin A	Standard
1	MRSA	13	16	25
2	Candida non albicans	09	08	20
3	Enterococcus sp.	10	14	20