An in vitro study of the effects of Cassia podocarpa fruit on the intestinal motility of rats.

Summary

Folkloric evidence and scientific reports indicate the use of C. podocarpa fruit as a purgative recipe. This study attempts to find the in vitro effects of its aqueous infusion (ACPF) and methanolic extract (MCPF) on the motility of the intestine of albino rats of Wistar strain and to compare their effect with those of C. acutifolia fruit (ACAF and MCAF). MCPF relaxed both the ileum and colon dose dependently. Its effect was blocked by tolazoline ([10.sup.-9] M) and propranolol ([10.sup.-9] M). ACPF had no effect on the ileum, but contracted the colon dose-dependently. Its effect was blocked by nifedipine (2.8 X [10.sup.-10] M) and drastically reduced by atropine (3.4 X [10.sup.-6] M). MCAF has the same effect as ACPF on both ileum and colon and its effect was similarly affected by atropine (3.4 X [10.sup.-6] M) and nifedipine (2.8 X [10.sup.-8] M). ACAF relaxed the ileum, its effect was blocked by tolazoline (5.1 X [10.sup.-7] M). MCAF was more potent than ACPF in contracting the colon, Hexamethonium (2.8 X [10.sup.-8] M), chlorpheniramine (3.8 X [10.sup.-8] M) and promethazine (3.2 X [10.sup.-10] M) potentiated the effect of ACPF on the colon. The results suggest that both ACAF and MCPF have anti-diarrhoeal effect. MCPF acts via both [alpha] and [beta] adrenergic receptor stimulation, while ACAF stimulates [alpha]-receptor. ACPF and MCAF engage both the cholinergic system and calcium channel activation in causing purgation in the colon. The potentiation of the effect of ACPF by some blockers could be due to allosteric enhancement of the receptors involved in its action.

Key words: motility, anthraquinone, allosteric activation, Cassia podocarpa fruit, Cassia acutifolia fruit, purgation, anti-diarrhoea

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Introduction

Cassia podocarpa Guill, and Perr. (Caesalpinoidae) is a glabrous shrub found in the South Western part of Nigeria (Dalziel, 1937; Watts and Breyer-Brandwijk, 1962).

The infusion or decoction of the leaves is given as a mild laxative. In large doses it acts as a purgative (Dalziel, 1937). The decoction of the leaves, root and flowers is given for the treatment of veneral diseases in women (Watts and Breyer-Brandwjk, 1962). The fresh leaves are gound and applied as poultices to the swellings and wounds and are used both internally and externally for skin diseases and yaws. For headache, they are rubbed on the forehead and temple and a lotion is made from them for opthalmia (Dalziel, 1937).

Many scientific reports have been published validating the use of Cassia species in the disorders of gastrointestinal tract particularly constipation and gastrointestinal inertia (Elujoba et al. 1989, 1999).

The chemical constituents of Cassia species responsible for this important therapeutic activity has been identified as anthraquinone derivatives (Elujoba et al. 1989). The % total glycosidic and aglycone contents (w/w) of Cassia podocarpa has been reported as 0.25% and 0.05% respectively (Abo et al. 1999). However there is little evidence in literature about the effects of extracts of Cassia species on the motility of the intestine, the mechanisms of action of these extracts and the effects of solvent used in extraction on the activity of the extracts. This work was designed mainly to investigate and examine the physiological effects of extracts of Cassia podocarpa fruit (prepared by using methanol and water) on intestinal motility of rats, with a view to further promoting the understanding of their mechanisms of action as vegetable laxatives, using isolated ileum and colon of albino rats.

Experimental

Plant material

Ripe C. podocarpa fruits were collected from the campus of the Obafemi Awolowo University, Ile-Ife in November 1998 and authenticated by Professor A. A. Elujoba, Department of Pharmacognosy, Obafemi Awolowo University, Ile-Ife. Voucher specimen are preserved in the Herbarmium of the Department of Pharmacognosy, Obafemi Awolowo University, Ile-Ife.

The fruits were, oven-dried at 60[degrees]C for about ten hours immediately after collection to prevent the enzymic hydrolysis of the anthraquinone glycosides which usually occurs a few hours after collection (1). The dried fruits were powdered with a GEC Grinding Machine (Christy and Norris Model) and were kept in black polythene bags at room temperature until ready for use.

Extraction

* Methanolic extract. The coarsely powdered dried fruits (200 g) were moistened with methanol and allowed to stand overnight and then soxhlet extracted with 95% methanol. The extract was concentrated under vacuum at 40[degrees]C by using a rotary evaporator. The syrup residue was weighed (10.2% yield) and then kept at 0[degrees]C until ready for use Standard solutions of the extract were prepared by using distilled water as diluent.

* Hot Aqueous Infusion. 50 ml of boiling distilled water was poured unto 5 g of the powdered fruit of C. podocarpa in a beaker. The mixture was allowed to stand for thirty minutes before it was filtered with a Whatman's Number 1 filter paper. An equivalent of 100 mg dried plant per ml aqueous infusion was thus obtained. Each infusion was always freshly prepared in order to prevent hydrolysis of the anthraquinone component of the plant during storage.

* Reference drug

Methanolic extract: Weighed tablets of Senokot-Cassia acutifolia. (Reckitt and Colman Product Limited. Hull England), were soaked in methanol in a well-corked conical flask overnight. The mixture was filtered into a weighed evaporating basin and allowed to stand in the laboratory until all the methanol had evaporated. This was confirmed by repeated weighing of the basin and its content until a constant weight was obtained (22.6% yield). Standard solutions of the extracts were prepared by using distilled water as diluent.

-- Hot Aqueous Infusion: 22.4 ml of boiling distilled water was poured over 2.24 g of Senokot tablet in a conical flask which was corked. The mixture was allowed to stand for thirty minutes. It was filtered wit Whatman's Number 1 filter paper. An equivalent of 100 mg of the tablet per ml of aqueous infusion was thus obtained.

Animals

Albino rats of Wistar strain (200-250 g) obtained from the Animal House of the Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, were used. All the animals were kept under standardized environmental condition and received standard diet (Pfizer Feed Plc. Lagos), and water ad libitum.

Effect of extracts and infusions on rat ileum and colon

Animals of either sex were starved overnight but had free access to water. A segment of the ileum or colon, (2-cm-long) was suspended in a 20 ml organ bath containing Tyrode's solution, gassed with air and maintained at 37[degrees]C. The tissue was allowed to equilibrate for one hour during which the Tyrode's solution was replaced at a ten minute interval. Graded doses of the methanolic extracts or aqueous infusions were applied to the tissues and their responses were recorded by microdynamometer (Ugo Basile Model) using isotonic transducer. Contact time was between 30 and 45 seconds. Experiments were done in quadriplicate (n = 4).

Effects of antagonists on tissue responses to the extracts or infusions

The dose of the extract or infusion which produced the highest response was selected as the working dose for that particular extract or infusion. The response of the tissue to the working dose was determined 3 minutes after pre treating it with graded doses of the antagonists. The following antagonists were used when the tissue responded by contracting: Atropine (3.4 X [10.sup.-6] M), Chlorpheniramine (3.8 X [10.sup.-10] M), Promethazine (3.2 X [10.sup.-10] M), Hexamethonium (2.8 X [10.sup.-8] M) and Nifedipine (2.8 X [10.sup.-10] M). Tolazoline (1.0 X [10.sup.-9] M) and Propanolol (1.0 X [10.sup.-9] M) were used in cases of relaxation.

Drugs

Atropine (British Drug House Ltd. England), Chlorpheniramine (Alison's Drug Company), Promethazine (Shanghai Med Corp. China) Hexamethonium (BDH), Nifedipine (Alison's Drug Company), Tolazoline (Sigma) Propanol (Sigma).

Statistical analysis

Values obtained in this study are expressed as mean [+ or -] SEM of 'n'-number of observations. Differences between the tested and control groups were tested for statistical significance by student t-test, taken to be significant when P < 0.05.

Results and Discussion

Methanolic extract of C. podocarpa fruit (MCPF) relaxed the ileum dose dependently (Table 1). Its effect was blocked by tolazoline (1.0 X [10.sup.-9] M) and propranolol (1.0 X [10.sup.-9] M). On the colon, this extract elicited the same effect dose dependently and its effect was blocked by the same antagonists, tolazoline (5.1 X [10.sup.-9] M) and propranolol (3.4 X [10.sup.-9] M) (Table 2). The sensitivity of the extract induced relaxation to low concentration of tolazoline and propranolol tends to suggest that the effect was mediated via [alpha] and [beta] adrenergic receptor stimulation.

Aqueous infusion of C. podocarpa fruit (ACPF) had no appreciable effect on the ileum. However, it contracted the colon in a dose related manner (Table 3) Atropine (3.4 X [10.sup.-6] M) reduced the contractile effect drastically (to 20.0% of the maximum response), while nifedipine (2.8 X [10.sup.-10] M) abolished it, indicating a specific effect by both the cholinergic and L-type-calcium channel activation mechanisms.

Methanolic extract of C. acutifolia fruit "Senokot" (MCAF) produced no observable effect on the ileum. It contracted the colon dose dependently like in the case of ACPF. Atropine (3.4 X [10.sup.-10] M) reduced the contraction elicited by MCAF drastically (to 18.1% of the maximum response), while nifedipine (2.8 X [10.sup.-10] M) blocked it (Table 4). This is also an indication that MCAF and ACPF mediated their effects via the cholinergic and L-type calcium channel activation mechanisms.

Aqueous infusion of "Senokot" (ACAF) relaxed the ileum dose-dependently (Table 5). The effect was blocked by tolazoline (5.1 X [10.sup.-7] M) while propranolol (3.4 X [10.sup.-8] M) reduced it by 50.0%. This indicates that both [alpha] and [beta] adrenergic receptor stimulation were involved in this effect, but more of [alpha]-adrenergic stimulation than [beta]. On the colon, the infusion also caused dose related relaxation (Table 6) which was drastically reduced to 23.8% of the maximum response by propranolol (3.4 X [10.sup.-8] M) and was also abolished by tolazoline (5.1 X [10.sup.-9] M). The result also indicated that the relaxant effect was mediated via both [alpha] and [beta] the adrenergic mechanisms.

Hexamethonium, a ganglion blocker, promethazine and chlorpheniramine, both of which are [H.sub.1]-receptor blockers, potentiated or enhanced the effect of aqueous infusion of C. podocarpa fruits on the colon.

ACAF was more potent than MCPF in relaxing the ileum (Table 7) probably due to a difference in the chemical structure or concentration of the relaxant constituents of the two drugs. While the effect of MCPF on the ileum was completely blocked by tolazoline and propranolol, that of ACAF was blocked by tolazoline alone, (Table 1 and 5). However, a phytochemical study is essential to clarify the above assertion.

The physio-pharmacological effects of the two extracts of both Cassia podocarpa and Cassia acutifolia fruits on the colon clearly showed opposing actions. There is an indication that the constituents extracted by water in Cassia acutifolia (ACAF) and that extracted by methanol in Cassia podocarpa (MCPF) are similar, since both extracts produced relaxations of the colon and were blocked by tolazoline and propranolol. On the contrary, the constitutents extracted by methanol in Cassia acutifolia (MCAF) and that extracted by water in Cassia podocarpa (ACPF) also seem similar, since both extracts produced contraction of the colon which were blocked by atropine and nifedipine.

The solvent used in extraction could have an effect on free radical formation by dimeric glycosides, whose rate is determined by the substitution patterns of the bianthrones, and this is of much importance in their biological activity (Lemli, 1986). Generally, methanol is a better solvent than water for the extraction of anthraquinone glycosides (Elujoba et al. 1989).

Anthraquinones and hydroxyanthraquinones are the major constituents of Cassia (Senna) plants. Senna (C. acutifolia) pods had been used as laxative for centuries (Ben, 1972). The biological effect observed in this study could be due to an adverse alteration in the structure of the anthraquinone glycosides in both Cassia species by the solvents used for extraction. Also, the differences in activities among the Cassia plants could be attributed to the varying levels of anthraquinone glycosides in the fruits. It has been established that the total amount of glycosides and aglycone of anthraquinones in C. acutifolia is more than that in any other Cassia species (Elujoba et al. 1989; Abo et al. 1999). This could be the reason for C. acutifolia being more potent than C. podocarpa as observed in this study.

Cassia species have been shown to exhibit laxative activities (Elujoba et al. 1989; Elujoba et al. 1999; Abo et al. 1999), and also antidiarrhoea properties (Manandhar, 1989). This work revealed that, depending on the solvent used, C. podocarpa fruit extracts can serve as both antidiarrhoea and laxative recipe. The site of anthraquinone action in producing laxation is the colon, while anti-diarrhoea activity of Cassia species is observed in the ileum (Bowman and Rands, 1980; Curry, 1986). The pods of C. acutifolia are specially suitable for habitual constipation, the action being principally noted on the lower bowel (colon) as a result of long lasting contractile activity (Elujoba et al. 1999). Therefore, the effects of C. podocarpa fruit in this study is consistent in producing relaxation and contraction of the ileum and colon respectively. This confirms its potential usefulness as antidiarrhoea and laxative drug. The difference between the effect of the water and methanol extracts of Cassia podocarpa fruit could be caused by a different composition and amount of anthraquinones in both extracts. Tannins are known for their anti-diarrhoeal effects (Bowman and Rands 1980). However, it remains to be determined if there is any difference between the tannin contents of the two extracts.

The relaxant effects of ACAF, MCPF, MCAF were shown to be mediated by [alpha] and [beta] adrenergic receptors in the ileum and colon, since tolazoline and propranolol blocked the relaxations. However, the contractile effects of ACPF, and MCAF were mediated by stimulation of muscarinic, nicotinic receptors and activation Ca channel, since atropine, hexamethonium and nifedipine were able to antagonize the effects appreciably.

Most modulators diminish the affinity of the receptor for their agonists and antagonists, but some modulators have been discovered to enhance the affinity for agonists and antagonists (Tucek and Dolezal, 1998; Birdsall et al. 1997). The enhancement of the effects of ACPF by hexamethonium, promethazine and chlorpheniramine in this study could be due to allosteric activation of the cholinergic receptors through which ACPF manifests its effect on the colon.

Table 1. Response of the ileum to graded doses of MCPF and the effect of
some antagonists on its working dose (n = 4).

Dose (mg/ml)             Mean Response [+ or -]   Percentage  Response
                         S.E.M. (mm)              of maximum
                                                  relaxation

  2.5                     1.8 [+ or -] 0.53         18.4      Relaxation
  5.0                     5.5 [+ or -] 0.29         56.1      Relaxation
 10.0                     8.8 [+ or -] 0.75         89.8      Relaxation
*20.0                     9.8 [+ or -] 0.25        100.0      Relaxation
Effect of antagonists
20.0 + Tola
  (1.0 X [10.sup.-9] M)   --                        --        Blocked
20.0 + Prop.
  (1.0 X [10.sup.-9] M)   --                        --        Blocked

* working dose
This extract relaxed the ileum in a dose dependent manner. Its effect
was completely blocked by both tolazoline (1.0 X [10.sup.-9] M) and
propranolol (1.0 X [10.sup.-9] M).

Table 2. Response of the colon to graded doses of MCPF and the effect of
some antagonists on its working dose (n = 4).

Dose (mg/ml)              Mean Response [+ or -]  Percentage  Response
                          S.E.M. (mm)             of maximum
                                                  relaxation

  2.5                      1.5 [+ or -] 0.40         9.2      Relaxation
  5.0                      7.8 [+ or -] 0.25        47.9      Relaxation
 10.0                     12.3 [+ or -] 0.93        75.5      Relaxation
*20.0                     16.3 [+ or -] 0.48       100.0      Relaxation
Effect of antagonists
20.0 + Tola
  (5.1 X [10.sup.-9] M)   --                        --        Blocked
20.0 + Prop.
  (3.4 X [10.sup.-9] M)   --                        --        Blocked

* working dose
This extract relaxed the colon in a dose dependent manner. Its was
completely blocked by tolazoline (5.1 X [10.sup.-9] M) and propranolol
(3.4 X [10.sup.-5] M).

Table 3. Response of the colon to graded doses of ACPF and the effect of
some antagonists on its working dose (n = 4).

Dose (mg/ml)             Mean Response [+ or -]  Percentage  Response
                         S.E.M. (mm)             of maximum
                                                 relaxation

  0.5                     2.8 [+ or -] 0.25       14.0       Contraction
  1.0                     3.3 [+ or -] 0.25       16.5       Contraction
 *2.0                    20.0 [+ or -] 0.71      100.0       Contraction
  4.0                    18.3 [+ or -] 1.32       91.5       Contraction
Effect of antagonists
2.0 + Atr
 (3.4 X [10.sup.-6] M)    4.0 [+ or -] 0.71       20.0       Reduced
2.0 + Hex.
 (2.8 X [10.sup.-10] M)  40.0 [+ or -] 0.82      200.0       Increased
2.0 + Prom.
 (3.2 X [10.sup.-10] M)  24.0 [+ or -] 0.41      120.0       Increased
2.0 + Chl.
 (3.8 X [10.sup.-10] M)  27.0 [+ or -] 0.41      135.0       Increased
2.0 + Nif
 (2.8 X [10.sup.-10] M)  --                       --         Blocked

* working dose
This infusion contracted the colon dose dependently. Its effect was
completely blocked by nifedipine (2.8 X [10.sup.-10] M) and drastically
reduced by atropine (3.4 X [10.sup.-6] M). Hexamethonium, promethazine
and chlorpheniramine increased the response of the colon to this
infusion.

Table 4. Response of the colon to graded doses of MCAF and the effect of
some antagonists on its working dose (n = 4).

Dose (mg/ml)             Mean Response         Percentage   Response
                         [+ or -] S.E.M. (mm)  of maximum
                                               relaxation

0.4                       8.3 [+ or -] 0.48      39.5       Contraction
0.8*                     21.0 [+ or -] 0.71     100.0       Contraction
1.6                      12.8 [+ or -] 0.75      61.0       Contraction

Effect of antagonists
0.8 + Hex.
  (2.8 X [10.sup.-8]M)    8.0 [+ or -] 0.41      38.1       Reduced
0.8 + Atr.
  (3.4 X [10.sup.-10]M)   3.8 [+ or -] 0.63      18.1       Blocked
0.8 + Prom.
  (3.2 X [10.sup.-8]M)   14.5 [+ or -] 0.29      69.0       Reduced
0.8 + Nifl.
  (2.8 X [10.sup.-8]M)   --                      --         Reduced

* working dose
This extract contracted the colon dose dependently. Its effect was
completely blocked by nifedipine (2.8 X [10.sup.-8]M).

Table 5. Response of the ileum to graded doses of ACAF and the effect of
some antagonists on its working dose (n = 4).

Dose (mg/ml)             Mean Response         Percentage     Response
                         [+ or -] S.E.M. (mm)  of maximum
                                               relaxation

  1.0                    2.8 [+ or -] 0.25      20.0          Relaxation
  2.0                    3.8 [+ or -] 0.25      27.1          Relaxation
  4.0                    5.3 [+ or -] 1.11      37.9          Relaxation
  8.0                   13.8 [+ or -] 0.58      98.6          Relaxation
*16.0                   14.0 [+ or -] 0.71     100.0          Relaxation
Effect of antagonists
 16.0 + Prop.
  (3.4 X [10.sup.-8]M)   7.0 [+ or -] 0.41     50.0           Reduced
 16.0 + Tola.
  (5.1 X [10.sup.-7]M)  --                     --             Blocked

* working dose
This infusion relaxed the ileum in dose dependent manner. Its was
completely blocked by tolazoline (5.1 X [10.sup.-7]M).

Table 6. Response of the colon to graded doses of ACAF and the effect of
some antagonists on its working dose.

Dose (mg/ml)            Mean Response         Percentage  Response
                        [+ or -] S.E.M. (mm)  of maximum
                                              relaxation

 1.0                     2.8 [+ or -] 0.25     13.3       Relaxation
 2.0                     5.3 [+ or -] 0.48     25.2       Relaxation
 4.0                    12.3 [+ or -] 0.63     58.6       Relaxation
*8.0                    21.0 [+ or -] 0.58    100.0       Relaxation
Effect of antagonists
 8.0 + Tola.
  (5.1 X [10.sup.-9]M)  --                     --         Blocked
 8.0 + Prop.
  (3.4 X [10.sup.-8]M)   5.0 [+ or -] 0.41     23.8       Reduced

* working dose
This infusion relaxed the colon dose dependently. Its effect was totally
blocked by tolazoline (5.1 X [10.sup.-9]M).

Table 7. A Summary of the comparative effects of the extracts and
infusions with the standard/reference drugs.

S/N  Plant          Solvent     Intestinal  Response of
     name           for         segment     the tissue to
                    extraction  studied     the extract

A    C. podocarpa   Water       Colon       Contraction

A'*  C. acutifolia  Methanol    Colon       Contraction
B    C. podocarpa   Methanol    Colon       Relaxation

B'*  C. acutifolia  Water       Colon       Relaxation
C    C. podocarpa   Methanol    Ileum       Relaxation
#

C'*  C. acutifolia  Water       Ileum       Relaxation

S/N  DOSE RANGE             Comment on potency of extract
     Least       Most
     effective   effective
     dose        dose
     (mg/ml)     (mg/ml)

A    0.5          2.0       Methanolic extract of C. acutifolia fruit.
                            "Senokot, is more potent than aqueous
                            infusion of C. podocarpa fruit whose
                            methanolic extract relaxed the colon.

A'*  0.4          0.8
B    2.5         20.0       Aqueous infusion of C. acutifolia fruit.
                            "Senokot" is more potent than methanolic
                            extract of C. podocarpa fruit (Both of them
                            elicit colonic relaxation)

B'*  1.0          8.0
C    2.5         20.0       Aqueous infusion of C. acutifolia fruit.
#                           Senokot, is more potent than methanolic
                            extract of C. podocarpa fruit.

C'*  1.0         16.0

* reference drug

References

Abo KA, Lasaki SW, Adeyemi AA (1999) Laxative and Antimicrobial Properties of Cassia Species Growing in Ibadan. Nig J Nat Prod Med 3: 47-53

Ben CH (1972) "The Complete Herbal" 1st Edition, Published by Barre. p 165

Bowman WC, Rand MJ (1980) Textbook of Pharmacology, 2nd Edition, Blackwell Scientific Publications, Oxford. London. p 25.32-25.36

Birdsall NJM, Farries T, Gharagozloo P, Kobayashi S, Knonen D, Lazareno ST, Popham A, Sugimoto M (1997) Selective Allosteric enhancemnt of the Binding and Actions of Acetylcholine at Muscarinic Receptor Subtypes. Life Sciences 60: 1047-1052

Curry CE (1986) Laxative products. In: Handbook of Nonprescription Drugs. 8th Edition. Arcata Graphic Company, published by American Pharmaceutical Association, Washington D.C. p 75-97

Dalziel JM (1937) The Useful Plants of West Tropical Africa. Crown Agents Publishers, London. p 612

Elujoba AA, Abere AJ, Adelusi SA. (1999) Laxative Activities of Cassia Pods Sourced from Nigeria Nig J Nat Prod Med 3: 51-53

Elujoba AA, Ajulo OO, Iweibo GO (1989) Chemical and Biological Analysis of Nigeria Cassia species for Laxative Activity. J Pharmaceu Biomed Analy 7: 1453-1457

Lemli J (1986) the Chemistry of Senna. Fitoterap LVII: 33-40

Manandhar NP (1989) Medicinal Plants Used by Chepany Tribes of Makawanpur District, Nepal. Fitoterap LX: 61-68

Tucek S, Dolezal V (1998) Muscarinic Acetylcholine Receptors: Basic Properties and Allosteric Regulation. J Physiol 25: 511

Watt JM, Breyer-Brandwijk MG (1962) The Medicinal and Poisonous Plants of Southern and Eastern Africa 2nd edition. E & S. Livingstone. London 1: 102-132

R. O. Akomolafe (1), I. O. Adeoshun (1), A. O. Ayoka (1), A. A. Elujoba (2), and E. O. Iwalewa (3)

(1) Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria

(2) Department of Pharmacognosy, Obafemi Awolowo University, Ile-Ife, Nigeria

(3) Department of Pharmacology, Obafemi Awolowo University, Ile-Ife, Nigeria

Address

R. O. Akomolafe, Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria

e-mail: [e-mail]