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Phytochemical Investigation of Irritant Constituents of Cuscuta reflexa.

Byline: Syed Saeed ul Hassan Shahid Rasool Muhammad Khalil-ur-Rehman Saiqa Ishtiaq Shahid ul Hassan Imran Waheed and M. Asif Saeed

Abstract

Dodder (Cuscuta reflexa Roxb.) is found locally as parasitic weed on host plants. This plant often causes irritation on the hands. The main objective of the present investigation was to evaluate its irritation potential on animal skin. Skins irritating chemical constituents were separated in the form of various fractions. To do this solvents with various polarities were used to extract least-polar compounds (petroleum ether extract) constituents of intermediate polarities [Chloroform (CHCl3) extract] and polar constituents [methanol (MeOH) extract] from pulverized biomass of Cuscuta reflexa. Ten fractions were collected from methanol extract by Liquid Column Chromatography and purified by Thin Layer Chromatography. The irritation potential of these purified fractions was evaluated on rabbit's skin. Fractions eluted by CHCI3 / MeOH (90:10) CHCI3 / MeOH (40:60) and CHCI3 / MeOH (20:80) showed more irritant potential. These biologically active purified fractions were characterized by Ultra Violet (UV) and

Fourier Transform Infrared (FTIR) spectroscopy. The presence of OH COOH or ketonic group and a double bond in these fractions were liable to be reacted with the cell membrane and cellular contents of both superficial and deeper layers of epidermis causing irritancy. It was concluded that Cuscuta reflexa contained skin irritant compounds. Copyright 2014 Friends Science Publishers

Keywords: Cuscuta reflexa; Solvent extraction; Liquid Column Chromatography; Irritating compounds; UV and FTIR spectroscopy

Introduction

Dermatitis caused by plants is commonly encountered in the practice of dermatology. Irritant properties of the plants have long been discovered by Indians Chinese and Arab physicians (Bah et al. 2012). Different plants produce different reactions on coming in contact with the skin depending upon nature of plant the type of skin and other varying factors (Behl et al. 2004)

Cuscuta reflexa commonly known as Dodder or Akash belongs to family Convolvulaceae. It is tropical and subtropical herb found as parasite weed on host plants. There are about 15 species of genus Cuscuta (Kirtikar 1985; Battacharjee 2001). It contains coumarin flavonoids aamarin AYamarin oleonolic acid stigmasterol and AYsitisterol which were detected from roots of the plant (Ramachandran et al. 1992; Anis et al. 1999). The presence of high molecular weight carboxymethylcellulose was also found in this plant (Chatterjee et al. 1997). Soluble phenolic constituents mainly caffeic acid were extracted from C. Reflexa (Leoffler et al. 1995). Seedlings of this parasitic plant synthesize ascorbic acid (Tommasi and Gara 1990).

Cuscuta has ability to take up and accumulate alkaloids of host plant (Czygan et al. 1988). The major glycoside Isohamentin 3-O-neohesperidoside was isolated along with flavonol glycoside (Dandapani et al. 1989). In addition to these compounds aromandendrin and taxi-folin were also isolated from this plant which has anti-HIV activity (Mahmood and khan 1989). C. reflexa is purgative expectorant tonic diaphoretic diuretic and aphrodisiac (Kirtikar 1985). This and other related plants have antiviral activity against HIV (Mahmood and khan 1989; Salehan et al. 2013). This plant shows inhibitory effect towards Bacillus subtilus Pseudomonas aeuroginosa and Shigella dysenteriae (Anjum and Khan 2003). It also has antifungal activity against Colletotrichum capsici (Sinha et al. 2004). Methanolic extract of C. reflexa delayed maturation in mice due to suppressed ovarian steroidogenesis (Mazumdera and Bhattacharya 2003).

It was observed in practice that C. reflexa often cause irritation on the hands of those who collect it. As our efforts to explore the flora of Pakistan with respect to irritancy (Hassan et al. 2012; Hassan et al. 2013) the main objective of the present investigation was to evaluate its irritant potential on animal's skin.

Materials and Methods

Plant Material

Fresh wines of Cuscuta reflexa were collected from the villages of tehsil Phalia district Mandi Bahawal Din Punjab Pakistan. Plant was authenticated by Dr. Zaheer ud DinDepartmentofBotanyGovernmentCollege

University Lahore (Voucher specimen No. 651). Plant was spread on the laboratory tables and dried under the shade at room temperature for 7 days. Dried plant material was pulverized by using an electric mill.

Chemicals

Petroleum ether (40-60C) Chloroform Methanol Distilled water and Acetone were of analytical grade (BDH Company England). Silica gel 60 (70-230 mesh ASTM) was used for Column Chromatography (E. Merck Germany). Prepared TLC plates (20 x 20 cm) coated with silica gel on aluminium foils were used (E. Merck Germany). A boro-silicate glass column (Pyrex) of 50 x 2.5 cm size was used for column chromatography.

Instruments

Rotary vacuum evaporator (Tokyo Rikakikai Co. Ltd Japan) UV spectrophotometer (Hitachi-270-30) FTIR (Pye-Unicam SP-8-400).

Animals

Healthy adult male/female albino rabbits were purchased from the local market and identified as Caprolagus hispidus. These animals (1.0-1.5 kg body weight) were acclimatized in the animal house University College of Pharmacy University of the Punjab Lahore for three days and were ad libitum fed carrots fresh green fodder (clover) and tap water.

Solvent Extraction

Dried pulverized plant material (1 kg) was extracted successively in petroleum ether (40-60oC) chloroform and methanol using 2.5 L of each solvent for soaking. Maceration was carried out in each solvent for four days at room temperature (252.5oC). Solvent of each extracted material was removed with rotary evaporator under reduced pressure and the extracts were weighed as shown in Fig. 1 (Brain and Turner 1975).

Column Chromatography

Column was packed uniformly with 250 g of silica gel (which was already activated by heating at 120C in an oven for 3 h) by slurry method. Chloroform was used for packing the column. A 20 g of methanol extract was adsorbed on 20 g of silica gel using chloroform. Chloroform was completely evaporated and the dried silica gel adsorbed material after pulverization was put on top of the column. The column was first run with a mixture of chloroform and methanol then the polarity of the system was changed by increasing the quantity of methanol in chloroform.

Table 1: Grading of irritant reactions

Reaction###Explanation

Grades

_###No reaction

+###Doubtful reaction diffused inflammation with no clear visible

###symptoms.

+###Slight reddening of the main vessels without reddening the

###area in between.

++###Marked Reddening of the main vessels with reddening of the

###area in between.

+++###Intense reddening of the entire ear often accompanied with

###macroscopic visible hyperplasia.

++++###Visible exudative lesion with marked epidermal damage.

Ten fractions were collected in glass test tubes. These fractions were purified by PTLC using different solvent systems.

Irritancy Test

The biological assay for irritancy was adopted from Evans and Schmidt method (Evans and Schmidt 1980; Schmidt and Moult 1983). A 2050 L solution from different dilutions was applied to the inner surface of rabbit's ear. Acetone was used as solvent as well as control. The ears were examined for redness after 15 min. of application and then after 30 min. intervals until two examinations indicated that further redness would not occur. Time for maximum erythema was noted. The animals were also examined after 24 and 48 h to ascertain the chronic inflammatory dose. Evaluation of irritant response of the extracts / fractions was done as given in Table 1. The dose causing an ear redness to the degree ++ was defined as irritant unit (IU) and expressed in g/mL per ear (Hecker 1971; Evans and Schmidt 1980; Schmidt and Moult 1983).

Characterization of Active Purified Fractions

Biologically active purified fractions were characterized by UV and FTIR spectroscopy. Ultraviolet Spectra were recorded on Hitachi-270-30 spectrophotometer using ethanol as a solvent. Infrared Spectra were measured on Pye-Unicam SP-8-400 spectrophotometer using thin film on sodium chloride disc.

Results

Solvent Extraction and %age Yield of Extracts

Out of the three types of extracted materials the polar components (11.20%) which were extracted in methanol were yielded grater than other extracts. The components with intermediate polarity (9.33%) which were extracted by chloroform were next in the yield. Conversely non-polar fractions (7.24%) which were extracted in petroleum ether were the lowest in yield. Thus the powdered plant of C. reflexa contained greater fraction of polar compounds and those with intermediate polarities as compared to non-polar components.

Separation of Fractions

The methanol extract of C. reflexa which was present in sufficient amount was used for column chromatography to obtain the fractions. Ten pooled fractions were collected and purified by PTLC using different mobile phases (Table 2).

Irritancy Assay

Results of preliminary irritation responses of crude extracts on rabbit's ear are given in Figs. 24. Methanol extract causes more irritancy as compared to chloroform and petroleum ether extracts. Results of irritant reactions of purified column fractions of methanol extract on rabbit's ear have been shown in Fig. 5. The result indicated that fractions eluted by CHCI3/MeOH (90:10) CHCI3/MeOH (40:60) and CHCI3/MeOH (20:80) showed more irritant potential.

Characterization of Active Purified Fractions

Purified fractions 4 6 7 9 and 10 showed more irritant potential. These biologically active purified fractions were characterized by UV and FTIR spectroscopy as shown in Table 3 and Fig. 6 and 7. UV spectra of these purified fractions showed absorption at max (nm) 225 245270280 and 255 respectively which was probably due to n p transition which suggested the presence of some double bonds in their molecules.

The available FTIR spectral evidence showed that the Fractions 4 9 and 10 probably contained methyl/aryl/ketonic/carboxylic acid/acid anhydride/amine/nitrile or some secondary amide group along with some OH groups due to some alcohol or phenol. Fractions 6 and 7 contained methyl/aryl/ketonic/carboxylic acid/acid anhydride/amine/ nitrile or some secondary amide group without OH group.

Table 2: PTLC Analysis of pooled column fractions of methanolic extract of C. reflexa

Pooled Eluting###PTLC solvent###No. of hRf###Detecting Agents

Fraction Solvent###systems###Spots values

###UV###Iodine Liebr

###light###Bur

###Reagent

1###CHCI3(100%) CHCI3 / MeOH###1###89###l-pink l yel l- bro

###(40:60)

2###CHCI3 /###CHCI3 / MeOH###1###76###d-###d yel d- bro

###MeOH (95:5) (30:70)###pink

3###CHCI3 /###CHCI3 / MeOH###1###85###l blu Yel###Bro

###MeOH(90:10) (20:80)

4###CHCI3 /###CHCI3 / MeOH###1###83###l blu l yel l- bro

###MeOH(90:10) (10:90)

5###CHCI3 /###CHCI3/MeOH/Ace.A 1###45###d pur d- yel l- bro

###MeOH(80:20) (90:10:1)

6###CHCI3 /###CHCI3/MeOH/Ace.A 1###41###d###d yel l- bro

###MeOH(70:30) (80:20:2)###pink

7###CHCI3 /###CHCI3/MeOH/Ace.A 1###50###l - blu l- yel d- bro

###MeOH(60:40) (90:10:5)

8###CHCI3 /###CHCI3/MeOH/Ace.A 1###62###l pur Yel###l- bro

###MeOH(50:50) (80:20:5)

9###CHCI3 /###CHCI3 / MeOH/HCl 1###89###l###l yel d- bro

###MeOH(40:60) (80:20:2)###pink

10###CHCI3 /###CHCI3 / MeOH/HCl 1###33###d-###d- yel l- bro

###MeOH(20:80) (80:20:5)###pink

Table 3: UV and FTIR spectra characterization of active fractions

###Active###UV###FTIR (cm)

fractions###max (nm)###OH###C=O###CH3###NH2

4###225###3358###1622###1399 2410###2930

6###245###-###700 864###1012 1413###2962

7###270###-###1730###1463 1272###2950

9###280###3388###1500 1462 1462 1074###2920

10###255###3600###1733###1378 1174###2914

Discussion

The results indicated that two solvent extracts except methanol extract exhibited either no or doubtful irritant responses when the low doses of 20 30 and 40 L were used but at 50 L dose level redness of + ++ and +++ intensity was observed on rabbit's ears (Table 2). Methanol extract seemed to be more irritant than other two extracts at this dose level. Chloroform extract showed little irritation response. Petroleum ether extract seemed to be nearly inert in its irritation reaction with all the four doses used. Thus to conclude polar constituents of C. reflexa were responsible for such adverse reaction on the animal's skin.

The fractions No. 4 6 7 9 and 10 seemed to be most active fractions (Fig. 5). They exhibited a strong to moderate irritant responses on rabbit's skin. Maximum irritant response was demonstrated by fractions 4 9 and 10 when the dose of 20 L was applied on rabbit's ears. The irritant responses of ++ intensity by these fractions were observed after 2 h of their application which continued to increase with time and gained +++ intensity

levels in about 4 h. These reactions lasted for about 48 h then faded away. On the other hand two other fractions 6 and 7 demonstrated moderate irritant response (Fig. 5). It was further postulated from UV and FTIR spectra (Table 5; Fig. 6 and 7) that the fractions 4 9 and 10 probably penetrated through the skin of rabbit's ear with much ease. The presence of OH COOH or ketonic group and a double bond in these compounds were liable to be reacted with the cell membrane and cellular content of both the superficial and deeper layers of epidermis. As a result inflammation of superficial as well as the deeper layers occurred which probably causes damage to epidermis. The mechanism of action of these compounds was probably like the other strong to moderately irritating previously (Mitchell

and Dupuis 1971; Evens and Schmidt 1980; Fregret 1981; Benazra et al. 1985). Fractions 6 and 7 displayed a moderate to weak reactions (++ to + intensity) which might be due to two possible reasons. 1) The compounds themselves entered the skin with some difficulty and were not completely accessible for the skin and 2) the nature of their molecules was not much strong to severely damage to epidermis of skin.

In conclusion phytochemical investigation indicated that C. reflexa contains several constituents that can be isolated by column chromatography. Five of the fractions were biologically active and caused skin irritation. The level of activity varies from slight to severe irritation. In our best of knowledge irritant activity of crude extracts and fractions of C. reflexa is first time reported. Furthermore the mechanism of irritancy can be investigated and different drugs can be formulated for the treatment of this irritancy.

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Author:Saeed, M. Asif; Hassan, Syed Saeed ul; Rasool, Shahid; Khalil-ur-Rehman, Muhammad; Ishtiaq, Saiqa; H
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2014
Words:2936
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