Analgesic, anti-inflammatory and antimicrobial activities of Crinum augustum Rox. and Crinum asiaticum L.

John Refaat¹*, Mohamed S. Kamel¹, Mahmoud A. Ramadan² and Ahmed A. Ali²

¹Pharmacognosy Department, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt.

²Pharmacognosy Department, Faculty of Pharmacy, Assiut University, 71515 Assiut, Egypt.

ABSTRACT:

Many Crinum species are traditionally used in different parts of the world for various local pains, inflammatory processes and microbial infections. In the present study, the total ethanolic extracts of C. augustum Rox. bulbs and C. asiaticum L. leaves were fractionated separately into five fractions each. The resulting fractions (400 mg/Kg, orally) of the total extract of C. augustum Rox. bulbs were evaluated for their analgesic and anti-inflammatory effects in mice using the hot plate and carrageenan-induced paw oedema tests versus acetyl salicylic acid (ASA) (100 mg/Kg, orally) and indomethacin (15 mg/Kg, orally), respectively. Fractions II, III and ASA showed the highest analgesic effects, whereas; II, III, IV and indomethacin were the highest anti-inflammatory ones at that tested doses. On the other hand, a comparative study of the antimicrobial activities of the total extracts of both plants together with their fractions (at 5, 10 and 50 mg/ml) showed inhibitory effects on S. aureus and E. coli, especially at 50 mg/ml. In addition, the per oral LD₅₀ of the total extract of C. augustum Rox. bulbs were determined to be 1.6 g/Kg in mice.

KEYWORDS: Acute toxicity, Analgesic, Anti-inflammatory, Antimicrobial, Crinum.

INTRODUCTION:

Crinums (Amaryllidaceae) are perennial and bulbous herbaceous plants. They occur naturally throughout the tropics, subtropics and warm temperate regions of the world.¹ Many of Crinums' extracts and alkaloids were reported to possess analgesic and anti-inflammatory effects^2-8 that accounted for their use in traditional medicines of different parts of the world in rheumatism, earache, lumbago, headache, backache, oedema, swelling, haemorrhoids and various inflammatory processes.^9-12 They also found a wide use in treatment of skin diseases, kidney infections, tonsillitis, laryngitis, otitis, prostatitis and intestinal diseases such as diarrhea and dysentery.^12,13-17 Poultices and decoctions of Crinums are also prepared for abscesses and suppurating sores.^12,17 That's why; we devoted this study to evaluate the safety, analgesic, anti-inflammatory properties of C. augustum Rox., and finally, comparing its antimicrobial effects with those of C. asiaticum L.

MATERIALS AND METHODS:

Plants Collection

Bulbs of C. augustum Rox. were collected from the Experimental Station of Medicinal Plants, Faculty of Pharmacy, Assiut University, Assiut, Egypt, while C. asiaticum L. leaves were collected from the farm of Ornamental, Aromatic and Medicinal Plants, Faculty of Agriculture, Minia University, Minia, Egypt. Botanical authentication of C. augustum Rox. and C. asiaticum L. were confirmed by Prof. Abdel-Aziz Fayed, Department of Botany, Faculty of Science, Assiut University, Assiut, and Prof. Mahmoud Abdel-Hadi, Department of Horticulture, Faculty of Agriculture, Minia University, Minia, Egypt, respectively. Voucher samples were deposited in the Department of Pharmacognosy, Faculty of Pharmacy, Minia University.

Preparation of the extracts:

The air-dried powdered bulbs of C. augustum Rox. (4 Kg) and leaves of C. asiaticum L. (300 g) were macerated separately in ethanol (95%). The alcoholic extracts were then concentrated independently under reduced pressure to a syrupy consistency (795 g) and (75 g), respectively.

Fractionation of the total extracts:

The solvent-free residues of both plants were subjected to several fractionation procedures according to schemes shown in Figures 1 and 2 to yield five fractions I-V. Appropriate concentrations of plant fractions were dissolved or suspended in warm distilled water (for analgesic and anti-inflammatory studies) or in 20% methanol (for antimicrobial study). The remaining amounts of C. augustum Rox. fractions were reserved for further phytochemical investigation. All solutions or suspensions of drugs and plant fractions were freshly prepared prior to each biological study.

Experimental animals:

The present study was conducted on healthy adult male Swiss albino mice (obtained from Osman Animal House, Giza, Egypt) weighing 25-35 g, each according to the Institutional Animal Ethical Regulations. The animals were kept in mesh-bottomed stainless steel cages (six per cage) with free access to food and water. Mice were fed a standard diet and tap water, and left to acclimatize to the environment for at least one week prior to inclusion in the experiments. The animals were handled only at the time of experiments and during cages cleaning. All conditions were made to minimize animal suffering.

Acute toxicity (LD₅₀) study:

The acute toxicity of the total ethanolic extract of C. augustum Rox. bulbs was performed by observing the lethal dose for 50% of mice by oral route (LD₅₀) through 24 hours. Different dose levels (0.5, 1, 2, 4 g/Kg, p.o) of the total extract (suspended in 0.5% carboxymethylcellulose (CMC) solution (obtained from El-Nasr Company for Pharmaceuticals and Chemicals, A.R.E.)) were administered to different groups of mice, containing six mice each. The control group received the total extract vehicle; 0.5% CMC solution (10 ml/Kg, p.o).¹⁸

Analgesic activity (Hot plate test):

Different fractions obtained from fractionation procedure of the total extract of C. augustum Rox. bulbs were evaluated for their analgesic activity using the hot plate assay.¹⁹ Mice were randomly divided into seven groups (six mice per group). Groups 3-7 were administered 400 mg/Kg of fractions I–V, respectively, suspended in 0.5% CMC solution orally. Animals of group 2 (positive control) were administered ASA (100 mg/Kg), as the reference drug^20,21 (obtained from The Arab Drug Company, Cairo, A.R.E.), in 0.5% CMC solution orally, while those of group 1 (negative control) were administered the vehicle of plant fractions (0.5% CMC solution) orally. Mice were subjected to the hot plate test the third hour before treatment and then at 30 min intervals for three hours after treatment. A cut off time of 20 seconds were used to avoid tissue damage. The time taken to lick either hind paw or to jump up (reaction time) when placed on the hot plate maintained at 50 ºC was recorded. After each testing, the hot plate was cleaned with wet paper towels to remove urine and feces.

Figure 1. Fractionation procedure of the total extract of C. augustum Rox. Bulbs

Anti-inflammatory activity (Carrageenan-induced paw edema method)

Different fractions obtained from fractionation procedure of the total extract of C. augustum Rox. bulbs were evaluated for their anti-inflammatory activity using the carrageenan-induced paw oedema method.²² Mice were randomly divided into seven groups (six mice per group). Groups 3-7 were administered 400 mg/Kg of fractions I–V, respectively, suspended in 0.5% CMC solution orally. Animals of group 2 (positive control) were administered indomethacin (15 mg/Kg), as the reference drug⁸ (obtained from El-Nile Company for Pharmaceutical and Chemical Industries, Cairo, A.R.E.), in 0.5% CMC solution orally, while those of group 1 (negative control) were administered the vehicle of plant fractions (0.5% CMC solution) orally. The tested fractions and indomethacin were administered orally one hour before carrageenan (Sigma, U.S.A.) injection (0.02 ml, 1% w/v in normal saline, S.C) into the sub-plantar tissue of the right hind paw of mice. The linear paw circumference was measured using a micrometer before injection of the phlogistic agent at time (t₀) and at 30 min intervals for three hours after administration of the tested fractions and indomethacin. The anti-inflammatory activity to inhibit the swelling of the hind paw was calculated from the following formula:

% Inflammation = (A – B) / B ×100

Where A = measurement of paw thickness (swelling) at time (t) after carrageenan-induced oedema, B = initial measurement of paw thickness (swelling) at time (t₀) before carrageenan-induced oedema.

Figure 2: Fractionation procedure of the total extract of C. asiaticum L. leaves

Antimicrobial activity:

The total extracts and different fractions of both plants were tested for their antimicrobial activity using the Well technique²³ against Staphylococcus aureus (Gram +ve bacteria) and Escherichia coli (Gram –ve bacteria) (obtained from Microbiology department, Faculty of Pharmacy, Minia University).

Preparation of samples:

Weighed amounts of different fractions (I-V) together with the total extract of both plants were separately dissolved in 20% methanol (which has no inhibitory effect on the tested organisms) to obtain concentrations of 5, 10 and 50 mg/ml.

Preparation of the test organisms and agar plates:

Test organisms were grown in Muller-Hinton agar for 48 hours at 37 ºC.²⁴ A suspension of microorganisms matching 0.5 Mcfarland turbidity (St. colony forming unit) used to inoculate Muller-Hinton agar at 50 ºC.²⁵ Equal portions of the seeded agar were transferred into sterile glass Petri dishes under laminar flow to prepare plates of equal thickness. After solidification of them, circular cups were formed using a sterile Wassermann tube.

In vitro determination of the antibacterial activity:

For each concentration of the samples under investigation, a 50 µl were applied to each cup then the plates were incubated overnight at 37 ºC. The zones of inhibition of the growth around the cups were measured in mm. Ampicillin discs in a concentration of 500 ppm (obtained from El-Nile Company for Pharmaceutical and Chemical Industries, Cairo, A.R.E.) was used as the control for the antimicrobial activity test.

Statistical analysis:

Results in both the analgesic and anti-inflammatory tests were expressed as means ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett’s test which was used when groups were compared only to the control group; p values less than 0.05 and 0.01 were considered significant. Graph Pad Prism was used for statistical calculations (version 3.02 for Windows, Graph pad Software, San Diego California, U.S.A.).

RESULTS:

Acute Toxicity (LD₅₀):

The p.o. LD₅₀ of the total ethanolic extract of C. augustum Rox. bulbs was calculated to be 1.6 g/Kg in mice.

Analgesic activity (Hot plate test):

The results of the hot plate test are depicted in Table 1 and Figure 3. Fractions II and III caused significant prolongation of the reaction time (P < 0.05) versus control at 60 to 150 min and 60 to 180 min, respectively, that was comparable to ASA (100 mg/Kg) which showed its analgesic effect evidently up to 180 min and was significant (P < 0.05) at 150 and 180 min, and (P < 0.01) at 60, 90 and 120 min. The maximum effect of ASA was obvious after 60 min On the other hand; fractions IV and V significantly prolonged the hot plate reaction time only at 60 and 150 min, respectively. A higher increase in the reaction times was observed for fraction IV compared to V, which was evident from 30 to 120 min with the peak effect was seen after 60 min, while at 150 and 180 min, a relatively larger increase in reaction times of fraction V was observed. The overall effect of both fractions was less than that of ASA. On the contrary, fraction I failed to exhibit any significant increase in the hot plate reaction time.

Table 1. Effect of oral administration of different fractions of C. augustum Rox. bulbs on the hot plate reaction time in mice

Group No.	Treatment	Hot plate reaction time in (s) (Mean ± S.E.M.)
		Pretreatment	30 min	60 min	90 min	120 min	150 min	180 min
1	Control (vehicle)	6.53 ± 0.09	6.60 ± 0.04	6.58 ± 0.17	6.13 ± 0.21	6.47 ± 0.32	5.25 ± 0.30	5.23 ± 0.25
2	ASA (100 mg /Kg)	6.35 ± 0.06	6.60 ± 0.20	7.81 ± 0.34^**	7.75 ± 0.43^**	7.43 ± 0.15^**	6.60 ± 0.38^*	6.35 ± 0.06^*
3	Fraction I (400 mg /Kg)	6.45 ± 0.10	6.46 ± 0.06	6.70 ± 0.04	6.55 ± 0.05	6.51 ± 0.04	6.00 ± 0.22	5.87 ± 0.26
4	Fraction II (400 mg /Kg)	6.51 ± 0.09	6.70 ± 0.07	7.43 ± 0.18^*	7.40 ± 0.04^*	7.32 ± 0.06^*	6.42 ± 0.22^*	5.73 ± 0.06
5	Fraction III (400 mg /Kg)	6.40 ± 0.14	6.63 ± 0.08	7.50 ± 0.27^*	7.58 ± 0.09^*	7.35 ± 0.23^*	6.50 ± 0.29^*	6.30 ± 0.36^*
6	Fraction IV (400 mg /Kg)	6.44 ± 0.04	6.50 ± 0.01	7.40 ± 0.15^*	7.05 ± 0.17	6.83 ± 0.19	6.03 ± 0.23	5.80 ± 0.31
7	Fraction V (400 mg /Kg)	6.42 ± 0.40	6.40 ± 0.26	6.82 ± 0.17	6.85 ± 0.67	6.53 ± 0.16	6.35 ± 0.19^*	6.03 ± 0.39

* Values are significant at P < 0.05 versus control; ** Values are significant at P < 0.01 versus control

Figure 3. Effect of different fractions of C. augustum Rox. bulbs on the hot plate reaction time of mice

Table 2. Effect of oral administration of different fractions of C. augustum Rox. bulbs on the paw swelling in mice

Group No.	Treatment	Paw swelling (thickness) in mm. (Mean ± S.E.M.)
		Pretreatment	30 min	60 min	90 min	120 min	150 min	180 min
1	Control (vehicle)	1.90 ± 0.17	3.75 ± 0.19	3.70 ± 0.12	3.50 ± 0.20	3.38 ± 0.13	3.00 ±0.21	2.40 ± 0.13
2	Indomethacin (15 mg /Kg)	1.93 ± 0.13	2.75 ±0.14 ^**	2.55 ±0.17^*	2.17 ± 0.17 ^*	2.13 ±0.24^*	2.00 ±0.20^*	1.98 ± 0.17
3	Fraction I (400 mg /Kg)	1.80 ± 0.12	3.00 ± 0.20	2.75 ± 0.14	2.73 ± 0.16	2.60 ±0.14	2.20 ± 0.17	1.95 ±0.05
4	Fraction II (400 mg /Kg)	1.85 ± 0.12	3.25 ± 0.14	2.56 ± 0.13 ^*	2.39 ± 0.14^*	2.23 ±0.23 ^*	2.13 ± 0.13	1.90 ± 0.14
5	Fraction III (400 mg /Kg)	1.80 ± 0.20	3.20 ± 0.12	2.50 ± 0.29 ^*	2.25 ± 0.43 ^*	2.18 ± 0.35 ^*	2.13 ± 0.24	1.88 ±0.24
6	Fraction IV (400 mg /Kg)	1.93 ± 0.15	3.50 ± 0.20	2.55 ± 0.17 ^*	2.38 ± 0.13 ^*	2.13 ±0.13 ^*	2.03 ±0.06^*	2.00 ±0.20
7	Fraction V (400 mg /Kg)	1.95 ± 0.25	3.38 ± 0.31	2.99 ± 0.52	2.83 ± 0.48	2.25 ± 0.43 ^*	2.13 ±0.39	2.03 ± 0.30

* Values are significant at P < 0.05 versus control; ** Values are significant at P < 0.01 versus control

Anti-inflammatory activity (Carrageenan-induced edema):

The results are mentioned in Tables 2 and 3, and depicted in Figures 4 and 5. Fractions II and III exhibited significant (P < 0.05) decrease of paw swelling at 60, 90 and 120 min with (61.62%, 70.81% and 79.46%), and (61.11%, 75% and 81.67%) inhibition of inflammation, respectively, whereas fraction IV caused a significant (P < 0.05) reduction of paw edema at 60, 90, 120 and 150 min with 67.88%, 76.68%, 78.89% and 94.82% inhibition of inflammation, respectively, in comparison with the non-treated control group. The inhibitory potencies of fractions II, III and IV at the previously mentioned times were comparable to that of the standard anti-inflammatory drug indomethacin (15 mg/Kg), whereas fraction V showed only a significant decrease in paw swelling at 120 min with 84.62% inhibition of inflammation which was also comparable to that of indomethacin (89.64%). Conversely, fraction I recorded the least anti-inflammatory properties at this tested dose and insignificantly (P > 0.05) reduced the paw edema through the test. Furthermore, a marked but insignificant inhibition of paw swelling was observed for almost all the groups at the test end except for the control group which remained comparatively the highest edematous one.

Antibacterial activity:

The results mentioned in Table 4 showed that the total extracts and fractions of both plants had no inhibitory effects at a concentration of 5 mg/ml, while only a small effect was observed for fractions II, III, IV and V at 10 mg/ml. On the contrary; at 50 mg/ml higher inhibitory effects were observed.

DISCUSSION:

Results of the acute toxicity study of the total ethanolic extract of C. augustum Rox. bulbs suggest a low margin of safety as indicated by the obtained p.o. LD₅₀ in mice. In addition, we also evaluated the analgesic potential of the total extract fractions (I-V) of C. augustum Rox. bulbs using the hot plate algesiometric method. The results collectively demonstrate that some fractions possess analgesic activities in this test at the tested dose (400 mg/Kg). Fractions III and II showed the highest analgesic effects, respectively, followed by the moderately active fractions IV and V, whereas fraction I was the least active or nearly inactive at that given dose. Significant values were observed for fractions II and III throughout most of the study period and were comparable to those of ASA (100 mg/Kg). The results obtained with fractions IV and V probably suggest that the analgesic effect of the former may be relatively greater at that administered dose (400 mg/Kg) but of shorter duration.

The positive results of the previous fractions in the hot plate test suggest their activity against centrally mediated pain,¹⁹ whereas prolongation of the reaction times in it indicates the involvement of supraspinal mechanisms.⁷ Studies on the analgesic effects of Crinums proposed the participation of opioid mechanisms⁷, and the resemblance of Amaryllidaceae alkaloids to morphine and codeine skeletons may account for their analgesic activity.²⁶ Therefore, these findings may explain the higher analgesic activities observed for fractions II and III due to their alkaloidal content in comparison with the other fractions. On the other hand, evaluation of the anti-inflammatory properties of the total extract fractions (I-V) of C. augustum Rox. bulbs revealed marked effects in the carrageenan-induced paw oedema test. The activities of fractions II, III and IV seem to be comparable to that of indomethacin which showed the highest anti-inflammatory effects throughout the test. Accordingly, these results may indicate that fractions II, III and IV contain the main anti-inflammatory principles.

It was reported that irritating compounds can produce pseudo inhibition of carrageenan-induced edema²⁷, but since all the plant fractions were administered orally, their anti-inflammatory properties could not be attributed to such the counter irritant effects. Furthermore, although it was reported that the less polar fraction (I) of the total extract contains flavonoids, sterols, and terpenoids^28-30 which are responsible for the anti-inflammatory and analgesic activities of many plants possessing them³¹, the observed lower effect of fraction I as compared to the other fractions could be attributed to the sub-optimal concentration of such principles in the administered dose.

The comparative antimicrobial screening of C. augustum Rox. and C. asiaticum L. showed that the total extracts of both plants possess inhibitory effects on both S. aureus (Gram +ve) and E. coli (Gram –ve) bacteria, while that on the former was markedly greater. Fractions containing alkaloids (II, III and IV) of both plants were the most effective in comparison with the other fractions especially at 50 mg/ml. It is noteworthy that the inhibitory effects of the total extracts and different fractions of both plants at that tested doses were less than that of ampicillin (500 ppm).

Figure 4. Effect of different fractions of C. augustum Rox. bulbs on the paw swelling in mice

Table 3. % of inflammation at different times after oral administration of different fractions of C. augustum Rox. bulbs in mice

Group No.	Treatment	% of Inflammation
		Pretreatment	30 min	60 min	90 min	120 min	150 min	180 min
1	Control (vehicle)	-	97.37 %	94.74 %	84.21 %	77.89 %	57.89 %	26.32 %
2	Indomethacin (15 mg /Kg)	-	42.48 %	32.12 %	12.43 %	10.36 %	3.63 %	2.59 %
3	Fraction I (400 mg /Kg)	-	66.67 %	52.78 %	51.67 %	44.44 %	22.22 %	8.33 %
4	Fraction II (400 mg /Kg)	-	75.68 %	38.38 %	29.19 %	20.54 %	15.14 %	2.70 %
5	Fraction III (400 mg /Kg)	-	77.78 %	38.89 %	25.00 %	21.11 %	18.33 %	4.44 %
6	Fraction IV (400 mg /Kg)	-	81.34 %	32.12 %	23.32 %	21.11 %	5.18 %	3.63 %
7	Fraction V (400 mg /Kg)	-	73.33 %	53.33 %	45.13 %	15.38 %	9.23 %	4.10 %

Table 4. Results of antimicrobial activities of the total extracts and different fractions of C. augustum Rox. bulbs and C. asiaticum L. leaves*

Crinum species	Test organism	The mean of the inhibition zone (mm)
		Total extract			Fraction I			Fraction II			Fraction III			Fraction IV			Fraction V			Ampicillin
		5 mg/ml	10 mg/ml	50 mg/ml	5 mg/ml	10 mg/ml	50 mg/ml	5 mg/ml	10 mg/ml	50 mg/ml	5 mg/ml	10mg/ml	50 mg/ml	5 mg/ml	10 mg/ml	50 mg/ml	5 mg/ml	10 mg/ml	50 mg/ml	500 ppm
Crinum augustum	S. aureus	-	-	8	-	-	2	-	3	7	-	2	7	-	-	6	-	1	2	10
	E. coli	-	-	3	-	-	-	-	2	4	-	1	3	-	-	2	-	-	-	7
Crinum asiaticum	S. aureus	-	-	7	-	-	3	-	2	6	-	1	6	-	1	5	-	1	3	10
	E. coli	-	-	3	-	-	-	-	-	4	-	1	3	-	-	3	-	-	-	7

* These results are mean of three replicates.

Figure 5. % of inflammation at different times after oral administration of different fractions of C. augustum Rox. bulbs in mice

CONCLUSION:

The positive results of the analgesic, anti-inflammatory and antimicrobial tests enlighten the basis for the folkloric use of both plants in many infectious, pain and inflammatory disorders, and can strongly put forward further investigations of different extracts and individual compounds, especially alkaloids, from Crinums which may play a supportive role in the pharmaceutical field towards development of new drugs of plant origin.

ACKNOWLEDGEMENTS:

We wish to express our thanks to Mr. Remon Roshdy, assistant lecturer of Pharmacology, Department of Pharmacology, Faculty of Medicine, Minia University, Minia, Egypt for kind help with Graph Pad Prism and statistical analyses. Our thanks also due to Mrs. Reham Ali, assistant lecturer of Microbiology, Department of Microbiology, Faculty of Pharmacy, Minia University, Minia, Egypt for kind help during the antimicrobial study.

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Received on 05.10.2011

Accepted on 18.10.2011

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Research Journal of Pharmacognosy and Phytochemistry. 3(6): Nov. - Dec. 2011, 289-296