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Title of Thesis

Institute/University/Department Details
Department of Pharmacology/ University of Karachi
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Keywords (Extracted from title, table of contents and abstract of thesis)
traditional medicine, cyperus rotundus, chromatographic solvent systems, haem-agglutinating activity, c. rotundus

The purpose of this research was to verify its therapeutic effects and margin of safety for human body.

Besides exploring the new pharmacological and biological activities of Cyperus rotundus one of the aspects of present research is to reproduce the standard method of preliminary screening of medicinal plants. This is achieved through the extracting of mixture of pharmacological active compounds and also through their fractions. The fractions are made in different solvent to separate the compounds and then performed different activities on various parameters. Extract of the plant and fractions were 'correlated with the effectiveness in term of percent of response and presented graphically. The graphs represent the comparative best pharmacological and biological activities. The world wide increasing demand for medicines from natural sources has motivated search for drugs with potential hypotensive activity as well.

Aqueous extract of C. rotundus caused a decrease in mean arterial blood pressure in anaesthetized Sprague-Dawley rats in a dose dependent manner. At the dose of 3mg/kg the mean arterial blood pressure was found to reduce by 42.6% from its control. This fall in the mean arterial blood pressure was statistically significant (p<0.OOO5). An increase in the dose of C. rotundus, i.e., 10mg/kg, and 30mglkg showed a less reduction in the mean arterial blood pressure 22.3% and 10.4%, respectively, the reductions in MABP were also statistically significant.

The maximum reduction in the mean arterial blood pressure observed at 3mglkg dose indicates that this dose can be regarded as optimal dosage for hypotensive activity in rats. According to Guyton (1996) changes in systolic and diastolic blood pressure are not usually of same magnitude in different physiological conditions as well as under the influence of various drugs.

On the basis of this study it is suggested that C. rotundus is probably acting both centrally and peripherally to produce changes in blood pressure by altering the peripheral resistance & cardiac mechanics. It is therefore concluded that C. rotundus is an active drug both physiologically and pharmacologically.

The preliminary screening was carried out according to the WHO recommendations, our sample of C. rotundus showed the presence of tannins, saponins, carbohydrates in colour reaction method and the absence of alkaloids, protein and sterols in this sample. Jeong et al. (2000) reported the occurrence of 3 novel sesquiterpene alkaloids, Rotundine A, B and C, from rhizome of Cyperus rotundus.

Haemagglutitlation activity test is a suitable test for determining the toxic effect on different human blood groups (A", B+, AB+, O+, A-, B-, AB-, and 0-). This test was carried out in different concentrations of crude extract i.e. 0.3125, 0.625, 1.25 and 5mglml and significant results were obtained in all groups at higher concentrations. Previously no such activity was carried out on this plant.

Antimicrobial activity tests were carried out on human pathogens bacteria (gram negative such as Morexilla catarhalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphyloccocus aureus and gram positive Acinto bacter) and fungi viz. Candida albicans and Apergillus niger. Excellent, moderate, low and no activity were found in these organisms such as 133.33 % in K. pneumoniae as compared to standard drug, amoxicillin 20mg/ml and 70% ethanol (as fungicide); 90 and 70% moderate inhibition in case of A. niger and S. aureus respectively while low inhibition i.e. 46.66, 37.50 and 33.33 % in E. coli, P. aeruginosa and M catarhalis respectively. No zone of inhibition was observed in Acinto bacter and Candida albicans (Table 5). The antibacterial activity is similar to published data but 90% activity was found against Aspergillus niger which has not been mentioned prior to these studies.

The crude extract of C. rotundus was used for anti-inflammatory activity. The test exhibited a decrease in hind paw volume at the dose 300 and 500 mg/kg administered orally. Significant dose dependent effect was observed at higher dose but it was lesser than aspirin as standard drug on comparison. It was also observed that maximum anti-inflammatory effect started after 1 hour and then started to decrease after 4 hours of administration.

Tail flick method was used for the determination analgesic activity. The temperature and duration were 51 ± 1°C and 0, 1, 2, 3 and 31/2 hours respectively. The tested drug was used in two concentrations i.e. 300 and 500 mg/kg in mice, administered via gastric intubation. The results were compared with control and standard drug (Dioclofenic sodium). Statistically significant activity was recorded during 90-120 minutes after oral administration of drug. The most important significant observation is that this drug has prolonged activity as compared to standard, Diclofenic sodium.

The crude extract and its fractions (ethyl acetate, chloroform, n-butanol, and aqueous) of this plant were analyzed for its spasmogenic and spasmolytic activity in-vitro, on rabbit intestine and the effects were compared with standard drugs i.e. acetylcholine, adrenaline and atropine.

The crude extract of C. rotundus exhibited highly significant (70.16%) relaxing mode of action on smooth muscles of rabbit intestine as compared to control and with standard drug. But when it was fractionated with ethyl-acetate, chloroform, n-butanol and aqueous, fractions except ethyl-acetate fraction which showed spasmolytic action all other showed very strong spasmogenic activity (chloroform 83.87%, n-butanoI 77.11% and aqueous 96.5%). It was also observed that spasmolytic activity was dominant in crude extract and it look like that it has no spasmogenic character in it except initial dose of 1 mg where it showed 5.85% increased action in comparison to control.

Previous reported researches were on rat ileum or rat uterus but our investigation was on isolated intestine of rabbit which showed statistically significant results and these results are in accordance to the anti-diarrhoeal activity of this drug.

There are many parameters to asses the pharmacological actions of a drug and among them assessment of neuro-pharmacological activity is very important. Therefore, it was used in this research to evaluate the drug activity on CNS and in this regard open field, head dip, rearing, traction and swimming induced depression tests were performed. In each test diminished activity was found which is suggestive of passive and sedative action of the drug at higher dose (i.e. 500 mg/kg) as compared to standard drug.

A quick and low cost toxicity test, Brine Shrimp Bioassay (LD50) test, was used to find out toxic action of drug in comparison to Etoposide, a standard drug of toxicity test. The drug showed nontoxic significant effects in 10, 100, 1000 mg/ml concentration (LD50 = 7.4625).

Toxicology of C. rotundus was also carried out in mice. The doses used were 10 mg/kg, 100 mg/kg and 1000 mg/kg. None of the group examined showed significant change in the body weights and mortality. At higher dose 1000 mg/kg, a decreased motor activity, comer sitting, tail erection and palpaberal ptosis in early two hours after dosing but animals returned to normal with in two hours. Toxicity test in male and female rats with 100 and 1000 mg/kg body weight crude extract of C. rotundus exhibited no mortality and none of these animals showed any sign of toxicity except diarrhea but physical behavioral changes were observed in first two hours. After dosing like uncoordinated motor activity, comer sitting and palpaberal ptosis. Non significant decrease was observed in weight of control group I treated rats while 10 % decrease was observed in group II rats treated with 1000 mg/kg and this decrease is statistically significant (p<0.05).

Autopsy revealed that no gross changes were observed in organs like liver, spleen, heart and kidney. No gross change was observed in heart and vessels. Drug did not cause any internal body hemorrhage. In urinary system that is kidney, ureters and urinary bladder were found identical with control. The shape, color, position were found similar to control.

Histopathological examinations of different organs (heart, kidney, liver and spleen) were carried out. After the administration of C. rotundus (1000 mg/kg) in animals no significant result were obtained because histopathological examination showed normal histology and pathology in both control and treated animal.

Our toxicological results showed nontoxic effects in general on body, autopsy and histopathology of organs of treated animals (mice). These results confirm the claims that it is a safe and nontoxic drug.

We have investigated the effects of C. rotundus on blood pressure parameters and heart rate in the anaesthetized normotensive rats.

The blood parameter has not been used before for evaluation and determination of the effects of extract of C. rotundus in Sprague-Dawley rats. The effects of extract of C. ro/undus on different biochemical parameters (glucose, cholesterol, alkaline phosphate, bilirubin, total protein, triglycerides, SOOT, SGPT, r-GT, creatinine, CK, urea, BUN, uric acid, albumin, HDL, LDL and Hb) were observed when 14 consecutive days 1000 mg/kg sample was administered orally in rats. Liver enzymes were found normal and statistically non-significant. A non-significant (p<0.05) increase in serum bilirubin. Y-GT and SGPT was also observed with respect to its control. Extract of C. rotundus produced slight but non-significant decrease in the serum alkaline phosphate (AP) but this increase was also non-significant.

As no major changes were found in biochemical parameter. It may be assumed that this drug is safe. However, further studies have to be carried out on different animal species over a longer duration.

In present gross behavioural changes studies in mice, the crude extract of C. rolundus has decreased the open field and cage crossing activity. The results were statistically significant. This indicated the effect on locomotion and exploration. All psychotropic drugs decrease locomotion and exploratory behavior.

Muscle relaxation automatically decreased exploratory behavior by producing sedation. All hypotensives are sedative to some extent. C. rotundus is also a hypotensive drug (Akperbekova and Guseinov, 1966; Mokkhasmit el al., 1971). The drug showing slight effect on muscle relaxant activity as traction observed by control and C. rolundus extract treated mice showed no significant difference of traction time among control and C. rolundus treated animals.

The relaxing and sedative effect of C. rotundus did not prolong the struggling time as compared to the control animal while antidepressant drug having capability to prolong the struggling time on the basis of present finding it may be suggested that C. rotundus may act as relaxant, sedative and hypnotic but it may not be antidepressant it is also correlated with other behavioral tests like exploration, cage crossing, open field etc. This work may be further expanded to see the effect of C. rotundus on brain biogenic amines especially on adrenaline, dopamine and serotonin in different region of brain. Cyperus rotundus is an herbal medicine and in India is used traditionally for strengthening memory.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
261.36 KB
2 1 Introduction 1
485.88 KB
  1.1 Plant Description 4
  1.2 Literature Search 31
3 2 Experimental 45
224.8 KB
  2.1 General Materials 45
  2.2 Instruments 45
  2.3 Extraction Procedure 48
  2.4 Chemical Tests 49
  2.5 Powder Drug Preparation 50
  2.6 Chromatographic Solvent Systems 51
  2.7 Haem-Agglutinating Activity 51
  2.8 Antibacterial Activity 52
  2.9 Antifungal Activity 53
  2.10 Analgesic Activity 55
  2.11 Anti-Inflammatory Activity 55
  2.13 Evaluation Of Cns Activity 56
  2.14 Smooth Muscles Activity 58
  2.15 Brine Shrimp Bioassay (Ld 50 ) Method 59
  2.16 Gross Behavioral Stud)' In Intact Animals 59
  2.17 Toxicological Studies 64
  2.18 Estimation Of Different Biochemical Parameters 65
  2.19 Histology Of Heart, Liver Spleen & Kidney 66
  2.20 Studies On The Crude Extract Of Cyperus Rotundus On Blood Pressure In Rats 67
4 3 Results 69
290.33 KB
  3.1 Chemical Tests 69
  3.2 Haemagglutinating Activity 71
  3.3 Antimicrobial (Antibacterial & Antifungal) Activity 73
  3.4 Anti-Inflammatory Activity 74
  3.5 Analgesic Activity 76
  3.6 Smooth Muscles Activity 78
  3.7 Evaluation Of Neuropharmacological Activity 87
  3.8 Brine Shrimp Bioassay (Ld 50 ) Method 94
  3.9 Gross Behavioral Study In Intact Animal 95
  3.10 Toxicological Studies 97
  3.11 Effects Of C. Rotundus On Different Biochemical Parameters 99
  3.12 Histopathological Examination Of Heart Results On Blood Pressure In Rats 104
  3.13 Histopathological Examination Of Kidney Results On Blood Pressure In Rats 106
  3.14 Histopathological Examination Of Liver Results On Blood Pressure In Rats 108
  3.15 Histopathological Examination Of Spleen Results On Blood Pressure In Rats 109
  3.16 Results Of The Crude Extract Of C. Rotundus On The Blood Pressure In Rat 111
5 4 Discussion 114
979.73 KB
6 5 Conclusion 130
375.61 KB
7 6 References 133
313.75 KB