INTRODUCTION:

Herbal drugs derived from plant extracts are being increasingly used for prevention and treatment of a wide range of diseases, though relatively little knowledge about their mode of action is available. There is a growing interest in the pharmacological evaluation of various plants used in Indian traditional systems of medicine. Thus, the present investigation was carried out to evaluate the Anti-inflammatory, Antioxidant and Antipyretic potential of C.occidentalis in experimental animal models.

Cassia occidentalis Linn has been used as indigenous and folklore medicine systems to treat several illnesses. Its roots, leaves, flowers, and seeds have been employed in herbal medicine around the world. In Unani medicine its used as an antidote of poisons, blood purifier, expectorant, anti-inflammatory agent and a remedy for the treatment of liver diseases¹. It is also an important ingredient of several polyherbal formulations marketed for liver diseases. C.ocidentalis is used in herbal medicine for a variety of purposes such as laxative, expectorant, analgesic, anti-malarial², hepatoprotective³, relaxant⁴, anti-inflammatory⁵ and wound for healing⁶.

This study was aimed at investigating the antipyretic and anti inflammatory activity of the COMF and chrysophanol in Albino Wistar rats.

MATERIALS AND METHODS:

Extraction, separation and purification of the compound:

Fresh leaves of C. occidentalis L. were collected from the premises of Loyola College Chennai, and it was authenticated by Dr. M. Ayyanar, Taxonomist, Entomology Research Institute, Chennai and a voucher specimen was preserved. The leaves were shade dried, coarsely powdered and was used for extraction. The dried leaf material (3kg) was extracted with methanol (w/v 1:3) three times at room temperature for a week. The methanol crude extract was combined and concentrated to yield a residue (140 g) which was subjected to successive solvent partitioning to give chloroform (26g), methanol (114g) soluble fractions. Thus, the methanol extract (100g) was chromatographed on a silica gel column (100-200 mesh) using a gradient solvent system of chloroform: methanol (100: 0, 95:5, 90:10, 85: 150: 100) to give 25 fractions. The fractions were combined based on the TLC profile. Finally 7 fractions were obtained. The fractions were screened for inflammatory activity and antipyretic activity. Fourth fraction showed good activity and its yield was 10.6 g. It was further purified by column chromatography using chloroform and methanol solvent system. Fraction 2 showed single (yellow colour) spot on TLC over silica gel with CHCL₃: methanol (9:1) as the developing solvent system (Fig. 1) and the yield was 4.2 g. The spot turned in pink color on exposure to ammonia vapour or spraying with 5% alcoholic sodium hydroxide. It indicated the presence of anthraquinones. The pure compound was subjected to IR ¹H and ¹³C NMR and MASS spectrum analyses. Solvents used for NMR spectra are; Tetra Methyl Saline (TMS), which shows chemical shift value at zero on the δ scale. ¹H and ¹³C NMR spectra were recorded with a JEOL 500 MHz FT NMR spectrometer (H¹), 500 MHz (C¹³) and chemical shifts were recorded in ppm. The solvent used for IR spectra was CHCl₃- Ccl₄. Liquid samples were taken in KBr crystals and solid samples were grinded with KBr or Nujomull. IR spectra were recorded in Shimadszu by KBr pellet method. IR spectra were taken on a Perkin Elmer FT-IR spectrophotometer. EI-MS were taken on a JEOL-GC mate spectrum in Indian Institute of Technology, Chennai.

Fig.1: TLC of purified fraction of C. occidentalis and Purified compound Chrisophanol

Drugs and chemicals:

The drugs and fine chemicals were purchased from Sigma Aldrich (USA) and Ranbaxy (India). All other chemicals and solvents were obtained from local firms and were of analytical grade

Phytochemical screening:

The COLMF was screened for the presence of various phytoconstituents (Anthraquinones, Sitosterols,Tannins, Xanthorin,Tannin, Saponin, and Flavonoids) using wellestablished methods reported in literature^7,8.

Acute Toxicity Studies:

A separate experiment was conduct to know whether any toxic effect was produced by COLMF and Chrysophanol over a period of 72 h by the method of Lorke⁹. Behavioral changes (irritation, restlessness, respiratory distress, abnormal locomotion and catalepsy) were observed over a period of 72 h for sign of acute toxicity. Rats fasted for 12 h were randomly divided in to drug treated ‘test’ groups and vehicle treated ‘control’ group, totally making up five groups of six rats. COLMF 150, 300, 600, 900 and 1200 mg/kg bw and chrysophanol 50, 150,300, 600, and 100 mg/kg b.w was separately administered orally to the rats in each of the test groups, respectively. Control group was treated with vehicle alone (CMC 0.1%; 1 ml/kg bw). The behavioral changes were observed over a period of 72 hours for sign of acute toxicity. The number of mortality caused by the compound/COLMF within this period of time was observed.

Animals:

Male Albino Wistar rats weighing approximately 110-150 g were obtained from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India. They were acclimatized to animal house conditions,and they were fed with pellet chow (Hindustan Lever Ltd., Bangalore, India) and had free access to water. All the animal experiments were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee guidelines.

Animal treatment:

Carrageenan induced paw edema:

Animals were randomly divided into 4 groups (I-IV), each having 6 animals. COLMF (200mg/kg b.w) dissolved in 1% carboxymethylcellulose and chrysophanol was suspended in 0.4% sodium carboxymethylcellulose and administered orally using an intragastric tube.

Group I Carrageenan-treated control group receiving vehicle, orally.

Group II Carrageenan + Indomethacin (10 mg/kg bw)

Group III Carrageenan +COLMF (200mg/kg bw)

Group IV Carrageenan +Chrysophanol (30 mg/kg bw)

Paw edema was induced by injecting 0.1ml of 1% carrageenan in normal saline into the sub plantar tissues of the left hind paw of test group rats. Each test group of animals was pretreated with oral administration of COLMF and chrysophanol. One hour later all the animals received carrageenan. The paw volume was measured at 1h, 2h, 3h and 4h according to the method of Bhatt et al.¹⁰.

The percentage inhibition of increase in the volume of paw edema was calculated for each animal by the following formula:

V_c - V_t

Paw edema inhibition = -------------- X 100

V_c

Where V_c= mean increase of paw volume in control animals; V_t = mean increase of paw volume in treated animals.

Cotton pellet induced granuloma:

Cotton pellet induced granuloma was assessed by the method of D’Arcy et al¹¹. Male Albino Wistar rats weighing 110-150g were divided into four groups of 6 rats each and were given the following treatments.

Group I Control animals treated with vehicle (CMC), orally for 7 days.

Group II Indomethacin (10 mg/kg bw) orally for 7 days.

Group III COLMF (200mg/kg bw) orally for 7 days.

Group IV Chrysophanol (30 mg/kg bw) orally for 7 days.

30 mg of cotton pellets were surgically inserted into the groin of animals for 7 days along with the administration of COLMF and chrysophanol, for 7 days. On the eighth day animals were sacrificed under light ether anesthesia, the cotton pellets with the attached granuloma were dissected out, dried and theweights of the dried granuloma were determined.

Antipyretic activity:

Male Albino Wistar rats weighing 110-150g were divided into five groups of 6 rats each and were given the following treatmen ts.

Group I	Brewer’s yeast -treated control group received, vehicle orally
Group II	Brewer’s yeast + Paracetamol 200mg/kg bw, orally
Group III	Brewer’s yeast+ COLMF (200 mg/kg bw)
Group IV	Brewer’s yeast + Chrysophanol (30mg/kg bw)

Hyperpyrexia was induced in rats by subcutaneous injection of 10 ml/kg of a 15% aqueous suspension of dried Brewer’s yeast in the subcutaneous route of hind limbs of the rats¹². The animals were then fasted for the duration of the experiment, water being made available ad libitum. Temperature was recorded 24 h after the yeast injection. Temperatures were recorded 1 h prior to drug administration in fevered animals served as the pre drug control. COLMF and chrysophanol in doses of 200 and 30 mg/kg bw. were given orally 24 h after yeast injection. Paracetamol (200 mg/kg bw, orally) served as the reference drug. The rectal temperature was recorded hour by hour using a flexible thermister probe coated with lubricant.

RESULTS:

Preliminary qualitative chemical analysis of COLMF (10.6g) revealed the presence anthraquinones, sitosterols,tannins, xanthorin, tannin, saponin, and flavonoids.

Toxicity studies:

The results of oral acute toxicity studies revealed the non-toxic nature of the chrysophanol and COLMF of C.occidentalis. No mortality was observed in the treated rats and behavior also appeared normal. Therefore the LD₅₀ value was fixed as above 1200 mg/kg b.w in COLMF and 1000 mg/kg in chrysophanol.

IR:

The IR spectrum (Fig. 2) showed the presence of chelated hydroxyl (3435 cm^-1), chelated carbonyl (1627 cm^-1), unchelated carbonyl (1676 cm^-1), and aromatic system (3055,1606, 1568,1475 and 1453, 903,868, 839,815 and 753 cm^-1): corresponding to the reported of Choi et al ¹³.

Fig 2 . IR spectrum of isolated (Chrysophanol

¹H NMR:

The¹H NMR spectrum (Fig. 3) showed two metacoupled protons at δ 7.05 and 7.59 corresponding to H-2 and H-4. The three protons of A ring forming an ABC system appeared at δ 7.77 (H-5), 7.63 (H-6) and 7.25 (H-7). The aromatic methyl at c-3 appeared as a three proton singlet at δ 2.44 (Table 1).

Fig 3. ¹H NMR spectrum of Chrysophanol

Fig 3a. ¹H NMR spectrum of Chrysophanol

1H NMR spectrum Cδ Found: H-2- 7.05 (1H,brs), H-4-7.59 (1H,brs),H-5-7.77 (1H,brdJ=7.5 Hz),H-6-7.63 (1H.t, J=7.6 Hz),H-7-7.25 (1H,br d J= 8.5 Hz),CH3-2.44(3H, S),2-X-OH, 11.5 (1H,br s),

The 13^C NMR spectrum Cδ Found: 1-162.65, 2-124.32, 3-149.32,4-121.31, 4a-133.56, 5-119.89, 6-136.91, 7-124.51, 8-162.36, 8a-115.80, -192.42, 9a-113.66, 10-181.85, 10a-133.19, CH3-22.24

¹³C NMR:

The 13C NMR spectrum (Fig.4) also confirmed that isolated compound has to be chrysophanol (Table 1).

Fig 4.¹³C NMR spectrum of Chrysophanol

Fig 4a. ¹³C NMR spectrum of Chrysophanol

Fig 5. MASS spectrum of Chrysophanol

MASS spectrum:

The MASS spectrum (Fig. 5) showed highest peak at EI-MS m/z 253 [M+1]⁺: 234,223, 194, 177, 166, 149, 136, 124, 113, 102 were corresponded to Chrysophanol. Its melting point was 196, corresponded to molecular formula C₁₅H₁₀O₄

The above information and other spectral data in the IR,¹H NMR, ¹³C NMR and MASS determined the structure of the compound as Chrysophanol (Fig.6). Chrysophanol has been previously reported by Lee and Sohn¹⁴.

These spectral data suggested that the compound was an anthraquinone derivative. The structure of the compound was determined to be 1,8- dihydroxy-3-methyl anthraquinone (chrysophanol). The NMR spectral and physical data of compound were in good agreement with those reported in a previous paper Kim et al.¹⁵.

Fig.6: Chrysophanol isolated from C. occidentalis

Carrageenan induced paw edema:

Significant increase in volume of paw edema was observed in control group of animals, further inflammation was observed in early phase (1 h) and late phase (1-4 h). The early phase was associated significantly with severe inflammation i.e., 1.23 ± 0.08 in control group, 1.18 ± 0.08 in indomethycine group, 1.22 ± 0.12 in COLMF treated group and 1.19 ± 0.09 in chrysophanol treated group. After 4 h, there was an automatic regression of inflammation and the reduction percentage of inflammation, which was compared between the groups (Table 2). After the lapse of 4 h chrysophanol showed 33.89 % paw edema inhibition and COLMF showed 32.30 %. This result ravels the efficacy of chrysophanol and COLMF wahich can be comparable to the reference drug indomethacin.

Cotton pellet granuloma model:

In cotton pellet granuloma model Chrysophanol and COLMF showed significant reduction in the weight of the cotton pellet granuloma. and the percentage of inhibition was 51.34% in Chrysophanol group and 49.16% in COLMF treated group (Table 3).This was almost equal to Indomethacin treated group (59.04%).

Table: 2. Anti-inflammatory activity of COLMF and Chrysophanol on carrageenan induced paw edema in experimental rats. (Volume in ml)

Groups	1 h	2 h	3 h	4 h	Reduction of paw edema
I	1.23 ± 0.08	1.26 ± 0.05	0.87 ± 0.12	0.65±0.14	-
II	1.18 ± 0.08	0.87 ± 0.10	0.65 ± 0.14	0.41±0.06	36.92
III	1.22 ± 0.12	0.88 ± 0.17	0.69 ± 0.13	0.44 ± 0.05*	32.30
IV	1.19 ± 0.09	0.87 ± 0.17	0.62 ± 0.19	0.43 ± 0.07*	33.89*

Values are expressed as mean ± S.D (n=6). Statistically significant alterations are expressed as *p<0.05. Group III and IV are compared with group I.

Table 3: Anti-inflammatory activity COLMF and Chrysophanol on Cotton pellet granuloma in experimental rats

Groups	Granuloma weight (g)	Percentage of inhibition
I	70.39 ± 2.29	-
II	32.31 ± 2.10	54.09
IV	35.78 ± 0.45	49.16*
V	34.25 ± 1.56	51.34*

Values are expressed as mean ± S.D (n=6). Statistically significant alterations are expressed as *p<0.05. Group III and IV are compared with group I.

Table 4: Effect of COLMF and Chrysophanol on Yeast Induced Pyrexia

Groups	Temperature	After 18 h yeast treatment
	Before yeast treatment	After 18 h yeast treatment
	Before yeast treatment	0 h	1 h	2 h	3 h	4h
I	35.86±0.33	38.11±0.91	38.15±0.13	37.98±0.80	37.95±0.97	37.27±0.11
II	35.78±0.93	38.15±1.19	36.87±0.10	36.64±0.32	35.68±0.42	35.56±0.15
III	35.98±0.98	38.22±0.76	37.15±0.56	37.12±1.07	37.01±0.16	36.61±1.17
IV	35.91±0.65	38.04±1.50	36.70±1.19	36.52±1.04	36.33±1.01*	35.98±0.62*

Values are expressed as mean ± S.D (n=6). Statistically significant alterations are expressed as *p<0.05. Group III and IV are compared with group II.

Antipyretic activity:

The effect of COLMF and its pure compound Chrysophanol on body temperature in rats is given in Table (4) the experimental rats showed a mean increase of about 38^oC in rectal temperature, 24 h after Brewer’s yeast injection. chrysophanol and COLMF at 200 and 30 mg/kg bw produced significant (P<0.05) antipyretic activity at 1 h after drug administration. Control groupof animals did not show any decrease in body temperature. The maximum lowering of body temperature was noticed by chrysophanol treated group, as the mean temperature of 38.04°C was reduced to 36.33°C at 3h and 35.98^oC at 4 h. Interestingly the COLMF, also showed maximum reduction in temperature, from 38.22°C to 37.01°C at 3h and 36.61°C at 4h. The temperature lowering activity of chrysophanol (P>0.05) was similar to Paracetamol treated group.

DISCUSSION:

Carrageenin induced hind paw edema and cotton pellet induced granulomas are the two standard experimental models of acute and subacute inflammation respectively. The results of the present investigation suggest that chrysophanol and COLMF have significant anti-inflammatory effect against carrageenan induced paw edema and in cotton pellet induced granuloma in rats.

In cotton pellet granuloma study, the fluid absorbed by the pellets greatly influences the weight of granuloma. The dry weight of the granuloma increased the alkaline phosphatase activity and decreased albumin/globulin ratio of plasma in rats¹⁶. Treatment with Chrysophanol and COLMF significantly inhibited the inflammation. This result was similar to commercial drug indomethacin.

Oral administration of COLMF and chrysophanol showed significant anti-inflammatory effect in carrageenan induced paw edema. This effect was due to the inhibition of mediators of inflammation; such as histamine, serotonin and prostaglandin¹⁷. These results are in agreement with that of a previous study of Bhakta et al.¹⁸, who confirmed that the methanolic extract of Cassia tora showed maximum inflammatory effect. Moriyama et al.¹⁹ reported that methanol extract of Cassia alata leaves exhibited stronger inhibitory effects in carregeenan induced model. In conclusion COLMF and chrysophanol showed inhibitory effect on carrageenan-induced paw edema and cotton pellet-induced granuloma formation, thus exhibiting antiinflammatory effect against acute and subacute phases of inflammation.

Since the anti pyretic activity of C.occidentalis has not been evaluated, hence the present study was under taken to determine the antipyretic effectiveness of chrysophanol and COLMF. It is well established that the yeast induced pyrexia is due to production of prostaglandins, which set thermoregulatory centre at a higher temperature²⁰. The temperature was brought back to normal after 3 hrs of post administration of COLMF and chrysophanol. The elevated body temperature proves the efficacy of the COLMF and chrysophanol and this effect was comparable with paracetamol. The antipyretic activity of COLMF and chrysophanol is probably due to inhibition of prostaglandin synthesis in hypothalamus. Purnima et al.²¹ observed significant antipyretic activity of Centratherum anthelminticum in yeast induced pyrexia. They suggested that the mechanism of antipyretic action of plant extract was similar to that of other Non Steroidal Anti-inflammatory Drugs. Salawu et al.²² reported that the extract of Crossopteryx febrifuga produced a significant reduction in yeast induced pyrexia in rats in a dose-dependent manner. Based on these results, it can be conclude that COLMF and chrysophanol possesses significant antipyretic effect. Hence, proves the traditional use by traditional medicine practitioners as antipyretic and anti-inflammatory remedies.

REFERENCES:

1. Nadkarni AK. Indian Materia Medica. Bombay Popular Book Dept 1954; 3:181.

2. Tona LK. Mesia NP, Ngimbi B, Chrimwami, Okond'ahoka K, Cimanga T, De Bruyne, Apers S, Hermans N, Totte J, Pieters L, Vlietinck AJ. In-vivo antimalarial activity of Cassia occidentalis, Morinda morindoides and Phyllanthus niruri. Annals of Tropical Medicine and Parasitology 2001; 95: 47-57.

3. Usha K, Kasturi GM, Hemalata P.Hepatoprotective effect of Hygrophila spinosa and Cassia occidentalis on carbon tetrachloride induced liver damage in experimental rats. Indian J. Clin Biochem 2007; 22(2):132-135.

4. Ajagbonna OP, Mojiminiyi FBO, Sofola OA. Relaxant effects of the aqueous leaf extract of Cassia occidentailis on rat aortic rings, African J Bio Research 20001; 4:127.

5. Sadique J, Chandra T, Thenmozhi V, Elango V. Biochemical modes of action of Cassia occidentalis and Cardiospermum halicacabum in inflammation. J Ethnopharmacol 1987; 19:201-212.

6. Sheeba M, Emmanuel S, Revathi K, Ignacimuthu s. Wound healing activity of Cassia occidentalis L. in Albino Wistar rats. International J. Inte Biol 2009; 2:1-6.

7. Peach K, Tracey MV. Modern Methods of Plant Analysis, vol. I, Springler Verlag, Berlin 1955 pp. 367.

8. Wallis, TE. Textbook of Pharmacognosy, 3rd ed., J.A. Churchill Ltd., London, 1985, pp. 361.

9. Lorke, D.1983. Anewapproach to practical acute toxicity testing. Arch Toxicol 54: 275–287.

10. Bhatt KR, Mehta RK, Shrivastava PN. A simple method for recording anti-inflammatory effects on rat paw oedema. Indian J. Physiol Pharmacol 1977; 21:399-400.

11. D'Arcy PF, Howard EM, Muggleton PW, Townsend SB. The anti-inflammatory action of griseofulvin in experimental animals. J.Pharmmacol 1960;12: 659-665

12. Smith MK. Surgical Treatment of Toxic Goiter. Annal Surg 1935; 101:1358-1366.

13. Choi SZ, Lee SO, Jang KU, Chung SH, Park SH, Kang HC, Yang EY, Cho HJ, Lee KR. Antidiabetic stilbene and anthraquinone derivatives from Rheum undulatum. Arch. Pharm. Res 2005; 28: 1027-1030.

14. Lee MS, Sohn CB. Antidiabetic Properties of Chrysophanol and Its Glucoside from Rhubarb Rhizome. Biol. Pharm. Bull 2008; 31: 2154-2157.

15. Kim DK, Chi SU, RYU SY. Gcytotoxic constutents of Rumex japonicas, Akhak Hoej 1998; 42: 233-237.

16. Shah Biren N, Nayak BS, Bhatt SP, Jalalpure SS, Seth AK. The anti-inflammatory activity of the leaves of Colocasia esculenta. Saudi Pharma J 2007; 15:3-4

17. Asres K, Gibbons S, Hana E, Bucar F. Anti-inflammatory activity of extracts and a saponin isolated from Melilotus elegans. Pharmazie 2005; 60:310–312

18. Bhakta T, Mukherjee PK, Mukherjee K, Banerjee S, Mandal SC, Maity TK, Pal M, Saha BP. Evaluation of hepatoprotective activity of Cassia fistula leaf extract. J. Ethnopharmacol 1999; 66:277-282.

19. Moriyama H, Iizuka T, Nagai M, Miyataka H, Satoh T. Anti-inflammatory activity of heat-treated Cassia alata leaf extract and its flavonoid glycoside. Yakugaku Zasshi 2003; 123: 607-611.

20. Howard M. Neuroscience Behavioural Review. 1993; 17: 237-269.

21. Purnima A, Koti BC, Tikare VP, Viswanathaswamy AHM, Thippeswamy, Dabadi P. Evaluation of Analgesic and Antipyretic Activities of Centratherum anthelminticum (L) Kuntze Seed. Indian J. Pharm. Sci 2009; 71: 461-464.

22. Salawu A, Chindo BA, Tijani AY, Adzu B. Analgesic, antiinflammatory, antipyretic and antiplasmodial effects of the methanolic extract of Crossopteryx febrifuga. J. Med Plants Research 2008; 28: 213-218.

Received on 27.02.2010 Modified on 05.03.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 888-893